SEQUENCE-SPECIFIC DETECTION OF NUCLEOTIDE SEQUENCES
A method for detecting the presence of a target nucleotide sequence in a sample of DNA is described herein in which a test sample comprising single stranded DNA is exposed to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence. The probe comprises a sequence complementary to the target sequence to be detected and this sequence also includes a recognition sequence for the nicking endonuclease. If the sample contains the target sequence, the probe hybridizes to the target and is cleaved by the nicking endonuclease, which leaves the target intact. Observing the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample of DNA.
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This work was supported in part by contracts W911SR-05-C-0029 and W911 SR-04-C-0036 from the US Department of Defense. The U.S. government may have certain rights in the invention.
BACKGROUND1. Technical Field
The present disclosure relates to methods for the detection of specific nucleotide sequences and reagents and kits for use in practicing the methods.
2. Related Art
Nicking endonuclease enzymes have been previously described. For example, U.S. Pat. No. 6,191,267 discloses recombinant DNA encoding a nicking endonuclease, N.BstNBI, and the production of N.BstNBI restriction endonuclease from the recombinant DNA utilizing PleI modification methylase. U.S. Pat. No. 6,395,523 discloses two methods to engineer nicking endonucleases from existing Type IIs restriction endonucleases, and the production of engineered nicking endonucleases. One approach involves inactivating the dimerization function of a Type IIs restriction enzyme using site-directed mutagenesis approach. Another approach involves replacing the cleavage domain of a Type IIs restriction enzyme with the cleavage domain from the naturally occurring nicking endonuclease, N.BstNBI.
SUMMARYA method for detecting the presence of a target nucleotide sequence in a sample of DNA can comprise exposing a test sample comprising single stranded DNA to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease; and, observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample DNA.
In various embodiments, the method can be multiplexed to detect the presence of a plurality of target nucleotide sequences in a sample of DNA. In alternative embodiments, the method can further comprise producing amplified DNA from a biological sample. Probes can comprise both fluorescent moieties and fluorescence quencher moieties in proximity such that while the probe remains whole, no fluorescence is observed. When such the probes are cleaved, increased fluorescence can be detected, permitting real-time observation of probe cleavage. In alternatives, probes can be attached to a solid surface, for example in a micro array or on the surface of microbeads.
The technology can be applied to the detection of bacterial pathogens in environmental samples, for example a method of detecting Bacillus anthracis is described. Alternatively, the technology can be applied to the detection of viral RNA in crude samples having significant background RNA.
These and other variations are set forth in greater detail below.
We have developed a sensitive method of identifying specific single- or double-stranded DNA sequences and, by extension, other nucleic acids such as RNA that can be converted to DNA. The method involves sequence-specific hybridization of a complementary oligonucleotide probe to a target DNA. When annealed, the oligonucleotide and target create a recognition site for a strand-specific nicking restriction endonuclease. The nicking endonuclease cleaves the oligonucleotide into two pieces, but leaves the target intact. Due to the decreased size of the fragments, their affinity for target DNA is reduced and they dissociate leaving the target free to form a new complex with full-length probe oligonucleotide. The reaction contains excess oligonucleotide (on a mole to mole basis) and so hybridization, cleavage, and dissociation occur many times. The reaction is limited only (at least theoretically) by the availability of oligonucleotide and the stability of the enzyme. Because of the possibility of continuous reaction and turnover of the probe, the assay can be called a streaming probe assay and the probes referred to as streaming probes.
The reaction can be highly specific since it requires complete complementarity between the oligonucleotide and the target at the restriction site to allow restriction cleavage and can be conducted so as to require complete or nearly complete complementarity outside of the enzyme recognition site to allow hybridization. Hybridization temperatures can be adjusted to allow increased or decreased specificity; sequences containing just one mismatch (e.g., single nucleotide polymorphisms (SNPs)) can be distinguished if desired.
Any technique that can determine the presence of different sized or cleaved DNA can be used to measure either the rate or the end point of the streaming assay reaction. For example, in one embodiment, denaturing polyacrylamide gel electrophoresis (PAGE) is a relatively simple technique that can be used to detect cleavage of the probe oligonucleotide. In another embodiment, the method can be made exceptionally sensitive by using fluorescently labeled DNA together with capillary electrophoresis (CE). In yet another embodiment, a real time method can be performed using an oligonucleotide comprising a fluorescent group attached at one end and a quencher on the other end of the oligonucleotide. In this case, cleavage and dissociation of the probe results in increased fluorescence that can be measured in real-time using a fluorescence reader. As one example, we demonstrate that a streaming assay can be used to specifically detect the presence of plasmids, such as Bacillus anthracis pX01 and pX02 plasmids, E. coli genomic DNA and Bacillus subtilis genomic DNA.
The method can be used whenever there is a desire to detect a specific sequence that includes a recognition sequence of a nicking restriction endonuclease that is known or can be engineered from a type IIs restriction endonuclease. For instance, this method is suitable for detecting the presence of specific DNAs in a mixture (microorganism contamination, infection, etc.); and it can be used for SNP analysis and for genotyping. Statistically, one or more unique target sites containing a suitable nicking endonuclease recognition sequence can be expected to be found in most all DNA sequences of sufficient length.
When combined with whole genome amplification (WGA), for example using isothermal multiple displacement amplification (MDA), it is possible to specifically detect genomic DNA amplified from about ten bacterial cells or less. The streaming probe assay can be multiplexed allowing the detection of multiple sequences (multiple genes in one organism or individual genes in multiple organisms). The multiplexing system can be noncompetitive in nature, unlike multiplexing systems that use polymerase chain reaction (PCR), and allows the generation of high throughput quantitative data.
A method utilizing WGA and a streaming probe can have significant advantages over current methods such as PCR. These can include the following. There is no need to purify DNA before amplification. Amplified DNA is generated that can be used directly with the streaming probe. DNA that can be used for thousands of streaming probe reactions is generated at once. Both DNA amplification and detection methods can be nondestructive, i.e., the same DNA can be used for multiple sequential tests including forensic tests.
The methods described herein exploit the particular properties of nicking restriction endonuclease enzymes. When conventional restriction endonucleases bind to their recognition sequences in DNA, they hydrolyze both strands of the DNA duplex at the same time. Two independent hydrolytic reactions proceed in parallel, driven by the presence of two catalytic sites within each enzyme, one for hydrolyzing each DNA strand. That is, restriction enzymes classically recognize a double-stranded DNA binding site and then cleave each strand of the DNA using two independent catalytic cleavage centers. Nicking endonucleases, on the other hand, cut only one strand. Nt.BstNB I is a naturally occurring nicking endonuclease that only cleaves one strand due to its inability to form dimers (4,5). The nicking endonuclease Nt.Alw I was engineered by creating a fusion protein between the DNA binding domain of Alw I and the cleavage/dimerization domain of Nt.BstNB I (6). Three additional nicking endonucleases Nt.BbvC I, Nb.BbvC I and Nb.Bsm I have been created. The methods described herein exploit the single-strand cleavage activities of nicking endonucleases to provide a sensitive assay for detecting the presence of specific sequences in a DNA sample, or any sample that can be converted to DNA containing a nicking site. In principle, the assay will work with any nicking endonuclease.
Several nicking endonucleases are now available from New England Biolabs. For example, N.BstNB I occurs naturally and nicks by virtue of its inability to form dimers. N.Alw I, a derivative of the restriction enzyme Alw I, has been engineered to behave in the same way. Both nick just outside their recognition sequences. N.BbvC IA and N.BbvC IB are alternative derivatives of the heterodimeric restriction enzyme BbvC I, each engineered to possess only one functioning catalytic site. These two enzymes nick within the recognition sequence but on opposite strands. Nb.Bsm I cleaves only one strand of DNA on a double-stranded DNA substrate. Nicking endonucleases are as simple to use as restriction endonucleases.
As disclosed in U.S. Pat. No. 6,395,523, it is possible to engineer known restriction enzymes to hydrolyze only one strand of the duplex, i.e., to produce DNA molecules that are “nicked,” rather than cleaved. Therefore, it is possible to create new specificities as desired from the array of known enzymes and the methods described herein can be generally applied to any sequence for which an appropriate restriction endonuclease exists or can be engineered. The method is not limited to use of Nt.Alw I, Nt.BstNB I, Nb.Bsm I, and Nt.BbvC I, which are exemplified herein.
Thus, a method for detecting the presence of a target nucleotide sequence in a sample of DNA can comprise:
exposing a test sample comprising single stranded DNA to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease; and,
observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample of DNA.
The probe need not be perfectly complementary to the target, except in the recognition sequence. Hybridization conditions can be chosen by the skilled practitioner to provide a desired degree of sequence specific hybridization. In various embodiments, one or more base mismatches can be permitted, or perfect complementarity can be required.
Observing whether or not the probe is cleaved can be accomplished by any technique that can observe the presence of shortened DNA probes or the cleavage of a fluorescently labeled probe, including poly-acrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), and fluorescence resonance energy transfer (FRET). Of these three techniques, CE is the most sensitive. However, FRET analysis can be performed in a real-time streaming assay. Fluorescent labels and quenchers can be placed anywhere in the probe, the only constraint being that they must not inhibit the nicking endonuclease. For instance, two fluorescent labels can be used to increase the signal strength, or probes with different spectral characteristics can be used in multiplexing.
There are other possible ways of detecting the fragments. Other optical detection methods can be used including bioluminescence and phosphorescence techniques, with or without resonance transfer (e.g., BRET and PRET). In addition, lanthanide-based energy transfer (LRET) can be used to observe the separation between appropriate labels. Another possibility is to use Mass Spectroscopy with or without mass spectroscopy tags. Another possible method is Raman Spectroscopy. Indeed, labeling of the probe with a surface enhanced Raman sphere can increase sensitivity many fold. Another way to detect the fragments produced relates to the fact that each cleavage results in a new 3′ hydroxyl and a new 5′ phosphate. The increasing presence of either can be measured and enzymatic activity calculated.
Both single and double stranded DNA can be a target for the assay. Indeed, any DNA molecule that can be made single stranded should work in principle. Furthermore, with some nicking endonucleases, direct detection of RNA will be possible (7). An enzyme that recognizes and cuts RNA/DNA hybrids would work as in
Probes can be of any suitable length as can be chosen by a skilled practitioner with consideration of several factors including the following. Probes will be preferably chosen that are of sufficient length to permit sequence specific hybridization and sufficiently short to permit release of the cleaved probe. The skilled practitioner will recognize that the ideal length will be a function of the melting temperature (Tm) of the full-length probe and the Tm's of the two products. That is, the Tm differential will preferably be sufficient to allow initial hybridization of the probe followed by subsequent dissociation of both probe fragments. Where convenient, these considerations can be circumvented by cycling temperatures between a reaction/annealing phase and a dissociation phase. In exemplary embodiments employing temperature cycling, the reaction/annealing phase can be conducted at about 40° C. to about 50° C., preferably about 43° C. to about 47° C., most preferably about 45° C., and the dissociation phase can be conducted at about 50° C. to about 60° C. or at most the limit of stability for the nicking endonuclease, for example preferably at about 53° C. to about 58° C., e.g., at about 55° C. However, in general it is to be expected that assays can be designed by choosing a probe/target/enzyme combination that permits use of a single temperature at which the whole reaction proceeds efficiently without having to cycle temperatures. Indeed, in preferred embodiments, using nicking enzymes at or near their maximum temperature limit (e.g., 58° C. for Nt.Alw I) yields very fast and sensitive assays.
Probes can be labeled with any appropriate labels possessing detectable optical, mass, or resonance signatures and the like for use in any of the techniques described herein and similar measurement methods. In positioning the labels, care is preferably taken to avoid labeling a probe in any position that will substantially impair the functioning of the nicking enzyme.
There are currently several nicking enzymes available. For example, New England Biolabs lists Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPlI in their catalog. Methods of engineering additional specificities have been developed and described. Indeed DNA binding domains other than those from restriction enzymes can be used (8). A temperature resistant nicking endonuclease that would be compatible with PCR can be engineered. One can then perform real-time PCR with a streaming probe and the requirement for an exact sequence match when using these enzymes should reduce false positive rates.
The system is very amenable to multiplexing. In one embodiment, a fluorescent group can be positioned relative to the restriction site in different probes so that different sized fluorescent oligonucleotides are formed from different targets. Alternatively, dyes with different spectral characteristics can be used in probes for different targets.
A streaming assay as described herein works well on DNA amplified by multiple displacement amplification. In exemplary embodiments, amplification occurs first and then the streaming reaction is run. However, it should be relatively simple to perform the two assays simultaneously. The probe can be modified so that it can not act as a primer and is resistant to the 3′ to 5′ exonuclease of the Phi29 polymerase. In preferable embodiments, both reactions run at the same temperature. Conventionally, MDA is performed at 30° C. And, the streaming assay is preferably performed at about 45 to 59° C. However, judicious modification of annealing temperatures and/or buffer conditions and use of alternative polymerase enzymes should permit use of a single temperature.
These methods can be useful in many applications, including: detection and identification of specific organisms in anti-bioterrorism efforts, medical applications for human and animal health, strain/species analysis e.g., ecological studies; molecular biology methods including in situ creation of a signal in a semi-fixed environment such as on a surface or in a gel, creation of a large amount of a specific oligonucleotide from a larger one, sequence-specific activation—e.g., where cleavage product(s) but not the parent (probe) is biologically active, sequence-specific inhibition—where cleavage removes a biological activity of the probe, detection and quantization of levels of DNA or RNA in any system including biological systems or extracts, in vitro assays and the like; and, genomic analyses including SNP/mutation analysis/genotyping. Test samples can include environmental sources such as air (aerosol sampling) water, soil and the like; biological sources can include serum, ascites fluid, cerebrospinal fluid, amniotic fluid, synovial fluid, pleural fluid, saliva, sputum, stool, urine, semen, tissue, biopsies, swabs, and the like from human and non-human sources. The methods can be used to detect sequences in RNA and the samples can comprise RNA, for example including viruses having RNA genomes. In such cases, the methods described herein can comprise preparing DNA from the sample by reverse transcription. The method can be used to detect a wide variety of bacteria, viruses and parasites, such as fungi, protozoa, helminthes, and the like.
Exemplary Protocols for Generating Probes to Detect a DNA Sequence.To generate NESA probes that detect a DNA sequence, a protocol for generating probes can comprise the following steps.
-
- 1. Locate all nicking endonuclease (NE) sites in the sequence of interest, which could be a relatively small piece of DNA or a whole genome. Can restrict search to one NE such as Nt.AlwI or search all.
- 2. Generate oligonucleotide sequences (in silico) that have the following preferred characteristics:
- a. Contains a specific NE site and its surrounding genomic sequence.
- b. NE site positioned within the oligonucleotide so that:
- i. when hybridized to its cognate sequence 2 fragments are generated.
- ii. NE cutting destabilizes the hybridization complex.
- c. Length usually between 16 and 25 bases.
- d. Melting temperature (Tm) is close is close to the optimum for the temperature used for the assay, e.g. for NtAlwI this is approximately 54° C.
- e. For linear probes, a duplex NE site will not be generated from hairpin formation or sequence homodimerization. For stem-loop probes, a duplex will not permit cleavage of stem structure in absence of target sequence.
- f. For linear probes, the sequence lacks stable predicted structure such as hairpins, dimers etc.
- 3. Design size of cleavage products. The NESA reaction generates oligos of defined size. Where detection of cleavage is by observing size of cleaved probes, any method that can monitor the generation of these different fragments can be used to quantify the reaction.
- Examples include but are not limited to:
- a. Sizing using gels such as polyacrylamide gels.
- b. Capillary electrophoresis.
- c. Release of part of the oligo from a solid surface.
- d. Separation of two chemical moieties due to cleavage of the oligo as in FRET analysis.
- Examples include but are not limited to:
- 4. Alternatively, design label arrangement. Although the reaction can be followed using unlabeled oligonucleotides, increased sensitivity can be achieved by labeling with:
- a. One or more fluorescent groups (including fluorescent beads) placed so that they do not inhibit the NE.
- b. Radioactivity.
- c. Raman spectroscopy reporters.
To generate probes that discriminate between two DNA sequences a protocol can comprise the following steps.
-
- 1. Locate all nicking endonuclease (NE) sites in the sequences of interest for one or more NE.
- 2. From each DNA, in silico, generate DNA fragments containing NE sites with enough surrounding sequence to make approximately 16 base sequences. This will generate a group of sequences from each DNA sequence of interest.
- 3. Perform pattern matching between the fragments using a program such as BLAST.
- 4. Discard all sequences that are 100% identical and are found in both DNA sequences.
- 5. The remaining sequences are then designed into probes of length 16 to 25 bases as described above (steps #2 to #5)
- 6. If desired, multiple computer generated probes from each NE site can be evaluated with respect to length, G:C content, Tm, secondary structure and similarity to DNA from other DNA sequences.
- 7. Pattern matching on 16 base fragments speed up the analysis. This can be advantageous when designing a probe specific to one genome that does not match any DNA from any other species. The initial matching is not essential. The length of the initial match can be altered depending on the complexity of the two sequences being studied
Identifying unique probe sequences.
After a set of theoretically unique probe sequences comprising a NE recognition site have been identified. Probes having desirable cleavage patterns can be selected for validation. Probes can be validated in assays using samples comprising common contaminants e.g., bacteria, yeast, human, rodent, and vector material. Probes can be further validated in assays utilizing real-world environmental samples. Validation assays can be performed using any NESA assay conditions described herein.
Theoretically unique probes which do not produce false positives in validation assays are functionally unique.
Fluor-Quench NESA ProbesProbes comprising a fluorescent moiety and a fluorescence quenching moiety can provide real-time observations of probe cleavage and other advantages. The assay and detection can be performed in a single tube or well of a multi-well plate. Real time assays can be less time consuming than end-point assays and offer the possibility of rate quantification as a function of target concentration. Assays can be performed in parallel. The methods can be less expensive. Methods can be performed in a plate reader or qPCR machine, present in most molecular biology labs.
Linear Fluor-Quench NESA Probes contain at least one fluorescent group and at least one quencher. The fluorescent moiety and quencher can be on either side of the cleavage site. Cleavage separates the fluorescent moiety and quencher increasing overall fluorescence. The fluorescent moiety and quencher can be located on the ends of the probe or can be internally linked.
Hairpin Fluor-Quench NESA Probes contain one or more stem loops to bring the fluorescent moiety and quencher close together. Preferably, the probe should have lower background fluorescence relative to a linear probe due to more efficient quenching. Two forms of stem loop are preferred. The nuclease recognition site can be in the stem section but only one strand comprises a complete nicking enzyme recognition sequence so as to avoid background cleavage. The nuclease recognition site can also be positioned in a single stranded loop.
Linear probes can give good signals regardless of the specific arrangement of fluor and quencher. Probes against different targets have can be made to have similar properties. Optimal signal to noise ratio can be obtained when the fluor and quencher were within a few nucleotides of each other. The streaming probe cleavage reaction can occur immediately on addition of enzyme. These probes operate over a wide range of concentrations. Slightly higher sensitivity can be observed with 1 pmol probe compared to 10 pmol.
Hairpin probes can give good signals regardless of the specific arrangement of fluor and quencher. Nicking enzyme recognition sites can be in the stem or loop sections. Endpoint measurement of F/Q probes can increase the signal to noise ratio dramatically.
Surface-Coupled (sc) NESA ProbesProbes coupled to a solid surface can have a number of advantages. If designed so that cleavage removes a fluorescent tag from the surface, just product can be measured by CE, decreasing background. Alternatively, a decrease in bead fluorescence can be monitored. Because the only fluorescence in solution is the product (signal) reactions can be monitored in a fluorometer or qPCR machine.
Many solid surfaces can be used, eg polystyrene beads, surfaces of plastic multi-well dishes, magnetic beads, surface-modified glass slides. Sc-probes containing a fluor and quencher have added advantages (scQ/F probes). The assay and detection can be performed in a single tube or well of a multi-well plate. Real time assays can be performed that are less time consuming than end-point assays and offer the possibility of quantification. Assays can be performed in parallel and are inexpensive. They can be performed in a plate reader or qPCR machine, present in most molecular biology labs. Multiplex is possible. Assays can be set up so that the reaction increases the fluorescence of a solid surface. The use of surface coupled probes allows the production of microarrays.
Standard NESA Probes hybridize specifically to target and contain a nicking endonuclease site such as Nt.AlwI and a fluorescent group attached to the oligonucleotide either at one end or internally. Surface-coupled NESA Probes also have a modification that allows coupling to a solid surface. The sc-probe can be coupled via the 5′, 3′ or an internal residues to a surface as long as the molecule is still cut by a nicking endonuclease. High density coupling can increase the effective concentration of probe. Fluor-Quench sc-NESA Probes, in addition to a fluor and solid surface attachment site, contain one or more quenchers. In some cases the solid surface could act as a quencher e.g. a bead that has inherent quenching activity or a quencher bound to it.
As described and exemplified herein, a method for detecting the presence of an RNA sequence in a sample of biological material, the method comprising: (a) performing a reverse transcription procedure capable of reverse transcribing RNA into a complementary DNA nucleotide, (b) performing a whole genome amplification technique such as multiple displacement amplification to amplify DNA in the sample of biological material to form an amplified sample product; (c) exposing all or part of the amplified sample product to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to a unique sequence known to be present in the transcript of the RNA sequence that also includes a recognition sequence for the nicking endonuclease; and, (d) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of the RNA sequence in the sample of biological material.
This method can be performed where the RNA sequence is a viral RNA pathogen genome. The method can also be performed in multiplex to permit detection of one or more different RNA and/or DNA genomes by exposing all or part of the amplified sample product to a DNA probe and a nicking endonuclease comprises simultaneously exposing all or part of the amplified sample product to a plurality of DNA probes directed to a plurality of different pathogens and/or pathogen strains, wherein each probe comprises a sequence complementary to a unique sequence known to be present in the transcript of an RNA genome of a pathogen that also includes a recognition sequence for the nicking endonuclease or a unique sequence known to be present in a DNA genome of a pathogen that also includes a recognition sequence for the nicking endonuclease.
As noted above, the sample of biological material can comprise a plurality of unpurified biological contaminants. For example, the sample can be an unpurified environmental air sample washed from a collection device such as an air filter or a liquid sample. The sample can be concentrated and/or buffered, but need not be purified or filtered.
A method for detecting the presence of a target nucleotide sequence in a sample of DNA can alternatively comprise (a) exposing a test sample comprising single stranded DNA to a nicking endonuclease and a substrate surface onto which a DNA probe is affixed under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease; and, (b) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample DNA.
Such surface coupled probes provide convenient packaging, storage, and detection. The substrate surface onto which a DNA probe can comprise the surface of a plastic or glass bead. The substrate surface onto which a DNA probe is affixed can also comprise a surface of a well, for example in a multiple well plate. Detection can be by observing the release of a fragment of cleaved probe from the surface. In such a method, the probe can comprise a fluorescent tag that is released from the substrate surface if the probe is cleaved by the nicking endonuclease and observing whether the probe is cleaved by the nicking endonuclease comprises detecting the presence of fluorescent tag released from the surface.
Prior to the assay, the substrate surface onto which the DNA probe is affixed can be kept desiccated. Assays using surface coupled probes can be multiplexed using a plurality of substrate surfaces, each substrate surface comprising a different DNA probe. For example a bead can contain one or more probes, and one or more beads can be used in one NESA reaction. Alternatively, a multiwell plate can be constructed with one or more probes in one or more wells. For example, sixteen wells containing three probes each permit simultaneous screening of 48 samples.
In another aspect, a method for detecting the presence of a target nucleotide sequence in a sample of DNA can comprise: (a) exposing a test sample comprising single stranded DNA to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease, a fluorescent tag, and a fluorescence quencher, the tag and quencher being situated on different sides of the recognition sequence for the nicking endonuclease, a first stem portion of the probe being capable of hybridizing to a second stem portion of the probe, the first and second stem portions being separated by a loop portion, the tag and quencher being located in the probe such that the quencher is effective to quench fluorescent emissions of the tag when the stem portions are hybridized to each other; and, (b) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of fluorescent emissions of the fluorescent tag indicates the presence of the target nucleotide sequence in the sample DNA.
The probe can be designed such that the recognition sequence for the nicking endonuclease is located in the loop portion, or the recognition sequence for the nicking endonuclease can be located in one stem portion where the other stem portion includes a mismatch so that the probe does not comprise a duplex recognition sequence for the nicking endonuclease.
A DNA probe can include a sequence complementary to a unique sequence of a target DNA molecule that also includes a recognition sequence for a nicking endonuclease, a fluorescent tag, and a fluorescence quencher, the tag and quencher being located on different sides of the recognition sequence for the nicking endonuclease, a first stem portion of the probe being capable of hybridizing to a second stem portion of the probe unless the probe is cleaved at a cut site of the nicking endonuclease, the first and second stem portions being separated by a loop portion, the tag and quencher being located in the probe such that the quencher is effective to quench fluorescent emissions of the tag when the stem portions are hybridized to each other. Again, the recognition sequence for the nicking endonuclease can be located in the loop portion or the recognition sequence for the nicking endonuclease can be located in one stem portion and the other stem portion includes a mismatch so that the probe does not comprise a duplex recognition sequence for the nicking endonuclease.
A substrate for surface coupled probes can comprise a surface onto which a DNA probe is affixed where the probe comprises a sequence complementary to a unique sequence of a target molecule sequence that includes a recognition sequence for a nicking endonuclease. The substrate can be a plastic or glass bead, or a multiwell plate where one or more of the wells comprising one or more different DNA probes, each different probe comprising a sequence complementary to a unique sequence of a target molecule sequence that includes a recognition sequence for a nicking endonuclease. The probe can comprise a fluorescent tag that is released from the substrate surface if the probe is cleaved by a nicking endonuclease. The substrate surface onto which the DNA probe is affixed can be stored desiccated, for example when beads or a mutiwell plate is a part of a kit for performing a NESA assay. A kit can comprise a plurality of substrates, e.g. plates, beads, and the like, different substrates comprising one or more different probes.
The following examples serve to further illustrate various aspects and embodiments of the methods described herein. These examples should not be considered limiting in any way.
EXAMPLES Materials and MethodsThe following materials and methods are used in the examples below unless otherwise indicated.
Genomic DNA. Genomic E. coli and B. subtilis genomic DNAs were supplied by Molecular Staging Incorporated and were generated using MDA (9) from 100 E. coli cells using their REPLI-g® kit. Real-time polymerase chain reaction (PCR) was used to assess the purity of the genomic DNAs (not shown). B. subtilis DNA was also generated in house using Qiagen's REPLI-g® kit. Genomic DNA from all other organisms was generated using Qiagen's REPLI-g® kit. The identity of the genomic DNA was confirmed by PCR-sequencing.
PCR amplified DNA for use in streaming assay reactions. The 16 S genes from E. coli and B. subtilis were amplified by PCR using the primers 16Sf (1) and 16Sr (2). Single stranded 16S DNA was prepared using single primer (16Sr) PCR and the amplicon. PCR primers were designed on sequences within the E. coli (E-oligonucleotides) or B. subtilis (B-oligonucleotides) 16S RNA genes. PCR reactions were set up using the DyNAzyme PCR reaction kit (MJ Research) with 25 pmoles primers and with 30 cycles (1 min @ 94° C., 1 min @ 55° C., 1 min @ 72° C.) proceeded by a 10 min @ 95° C. denaturation step and followed by a 8 min @ 72° C. extension step.
Oligonucleotides. Oligonucleotides used in these examples are shown in Table 1. Nicking endonuclease sites are in bold, mutations from the wild-type sequence are in lower case. E probes; E. coli based; B probes, Bacillus subtilis based; c probes, complement of a probe sequence.
Nicking Endonucleases. Table 2 summarizes the nicking endonucleases used in these examples. All nicking endonucleases were obtained from New England BioLabs. Reaction conditions were as suggested by the manufacturer. The amount of enzyme, target DNA, and oligonucleotide probe used, and the length, temperature and volume of the reaction, varied from experiment to experiment and are given in the text and/or figure legends.
PAGE Samples were separated on a 20% polyacrylamide, 7 M urea gel using a standard procedure (3).
Fluorescence assays using a FRET probe. Oligonucleotides were constructed that had a 5′ fluorescence quencher (Iowa black, IDT) and a 3′ fluorescent group, Alexa 488. Probe streaming reactions were performed in a multi-well plate and analyzed on a SpectraMax Gemini EM, Molecular Devices' fluorescence plate reader using an excitation wavelength of 484 nm and an emission wavelength of 525 nm. The relative fluorescence units shown represent the actual readings minus background fluorescence.
Fluorescence assays using Capillary Gel electrophoresis. Oligonucleotides were constructed that had either a 5 or a 3′ fluorescein group. Two instruments were used for this analysis, either a Beckman P/ACE MDQ LIF or an AB1 3130XL. Electrokinetic loading was used in all cases. For the Beckman P/ACE MDQ LIF instrument, the distance from the loading point to the detector was either 20 cm or 10 cm depending on the experiment and the eCAP ssDNA 100-R kit from Beckman was used with voltages between 9,000 and 30,000 volts and loading times of 2 to 10 seconds. In analyses using the ABI 3130XL, the POP6 polymer was used on a 36 cm capillary using ABI's Fragment Analysis Protocol.
Example 1 FRET-based Streaming AssayIn the streaming assay, as in the non-streaming assay, the oligonucleotide probe is cleaved into two shorter products. There are a number of ways of measuring this cleavage. One way is to measure the change in fluorescence resonance energy transfer between a donor and an acceptor fluorescent moieties, or a fluorescent moiety and a quencher, arranged on opposite side of the cleavage site on a probe, e.g., a FRET assay.
Usually, when a fluorescent molecule is activated by a certain wavelength of light it emits light (fluoresces) at a longer wavelength and this emitted light can be measured using a fluorometer. In FRET, when an acceptor or quencher is present in close proximity to a fluorescent molecule (i.e., a donor), rather than fluoresce, the energy is absorbed by the acceptor or quencher that can or can not (i.e., a dark quencher) emit light at an even longer wavelength.
By arranging a fluor moiety and a quencher moiety in each of the products of parent probe cleavage, i.e., on opposite sides of the probe cleavage site, the parent probe will be quenched due to the proximity of the fluor and quencher but not quenched in the cleaved probe products. That is, cleavage of the probe by a restriction endonuclease physically separates the quencher from the fluor, thereby reducing quenching and causing a measurable increase in fluorescence. This kind of assay is illustrated in
Target DNA is first denatured by heating at 95° C. for 10 min in the presence of a molar excess of an oligonucleotide probe (
Theoretically, in the presence of active enzyme the reaction should repeat continuously until near completion. The overall sensitivity of the assay is therefore largely a factor of how much fluorescent probe is available for cleavage and the background (quenched) level of fluorescence.
Example 1.1 Detection of the E. coli 16S rRNA Gene in pcr-Amplified DNA Using the Fret-Based Streaming AssayAn oligonucleotide probe was developed that hybridizes to E. coli 16S DNA and is cleaved by Nt.Alw I. This probe has reduced binding affinity for B. subtilis 16S DNA due to 3 nucleotide differences between the 16 S DNA of the 2 species (Tablet,
The utility of this probe was tested using PCR-amplified 16S DNA from E. coli and B. subtilis. The amplification was performed using a set of universal primers (
As can be seen in
Reactions were set up using the E. coli 16S amplicon and increasing amounts of 16S B. subtilis genomic (i.e., nonspecific) DNA (
For some applications a simple, low cost assay is most appropriate. To demonstrate such an embodiment of the method, a reaction was performed using a probe comprising a fluorescein residue at the 5′ end and without a quencher residue (
The 5mer probe cleavage product is clearly visible in lane 2 where the sample comprised 100 fmoles E. coli complement oligonucleotide (E1c) but not in lane 3 where the sample comprised 100 fmoles B. subtilis complement oligonucleotide (B1c). The 11 mer cleavage product is not seen because it does not retain the fluorescent label.
Example 3 A Highly Sensitive Capillary Electrophoresis (CE) AssayCapillary electrophoresis (CE) can be used to detect probe cleavage and can provide a more sensitive alternative to the FRET assays of Example 1. CE separates charged molecules by their size and has long been used to separate DNA fragments. The Beckman P/ACE MDQ LIF system is programmable and has the ability to detect very small oligonucleotides. However, any suitable CE system can be used. The CE assay was set up in the same way as the denaturing acrylamide gel assay shown in
A CE-based assay was also used to examine the effects of varying the levels of target and probe.
The 5mer product eluted at about 18.4 minutes and was only seen when E. coli 16S amplicon was present. At levels of 100 and 10 pmoles of target and probe, nearly all the E1 probe was cleaved. As levels were reduced further there was a reduced signal but still clearly visible signal with just 100 fmoles of target and probe. The initial reaction was performed in 200 μl, and a 1 μl sample of this reaction was diluted into 100 μl before electrokinetic injection. It is expected that only a fraction of the material present in the sample enters the capillary tube. Thus the 5mer peak seen with the original 100 fmoles probe and target reaction, reflects the signal obtained from far less than 500 attomoles target. This shows a remarkable sensitivity especially given that the probe and target were in equimolar amounts rather than the probe being in excess. We also determined the effects of decreasing the amount of target while retaining a constant level of probe (100 pmoles). As can be seen in
In
The sensitivity of the assay demonstrated in
The streaming assay can be used to distinguish between closely related DNA sequences as shown in
As can be seen in
CE instruments capable of separating oligonucleotides that differ in size by one nucleotide are available. Such instruments, for example the Beckman P/ACE, can be used to perform a multiplex assay where probes that yield different lengths of cleavage product are used against multiple targets in one reaction. In such a multiplex reaction, each probe can be designed such that the cleavage site for the nicking endonuclease produces a unique sized cleavage product.
The Beckman P/ACE, is a single capillary, one-color machine so its multiplex ability is limited to distinguishing sizes. Another instrument, the ABI 3130XL, has capacity for 16 capillaries and can handle fluors of four different colors. With such a device, multiplexing assays can include probes that are labeled with different colored fluorescent moieties.
The results of a four-plex assay are shown in
In the presence of the four target DNAs, four distinct signals were generated. These data demonstrate the capability of multiplexing. Since the instrument is capable of using fluors with four different colors, a multiplex assay of 16 probes is an obvious extension. Indeed, the resolving power of the capillaries is such that multiplex assays with more than 16 probes are possible (we estimate, based on the resolving power of the capillaries, that a 40-plex is possible). These data, taken together with the above examples, also show the generality of the streaming probe, because the feasibility of the assay with three different organisms (including E. coli) using both chromosomal and plasmid sequences has been demonstrated.
Example 4 Use of a Combination of MDA and Streaming Probe to Detect Approximately 10 BacteriaOne of the goals in applications such as detection of bio-warfare agents, is to detect vanishingly small numbers of organisms. To demonstrate that the combination of MDA and streaming probe can be sensitive enough to detect low levels of bacteria, a serial dilution of a culture of B. subtilis was performed and the samples split into two. One half was used to quantitate the number of bacteria present; the other half was used to perform MDA.
As can be seen in
The use of temperature cycling: One possible rate limiting effect is the dissociation of the probe fragments from the target after the probe has undergone endonucleolytic cleavage. Indeed, as the concentration of cleaved probe increases with time, there could be a significant inhibition of the process. Initially our assays were set up at 45° C. However, cycling between two temperatures, a reaction temperature (45° C.) and a dissociation temperature 55° C. might lead to an increased rate of reaction. The results of this strategy are shown in
Use of excess probe: Another possible rate-limiting step is the annealing of the probe to the target. This can be mitigated by using high concentrations of probe (pmoles) to drive the reaction forward. Interestingly, as much as practically all of the probe can be seen to be cut in assays containing as little as 1 fmole target, indicating a probe turnover of at least 1000.
Use of other nicking endonucleases: To demonstrate the generality of the method using other nicking endonucleases, a probe, E2 (
These data show that the assay is not dependent on one nicking endonuclease but that other nicking endonucleases can be used so long as they cleave just one DNA strand.
Detection of single strand targets: One-sided PCR was used to create a single-stranded E. coli 16 S DNA. 100 fmole (65 ng) 16 S E. coli or B. subtilis single-stranded DNA was incubated with 100 pmole 16 S E. coli specific FRET probe E1, denatured at 95° C. for 10 minutes and incubated with 50 units N.Alw 1 in a total volume of 200 μl. and cycled between 45° C. (1 min) and 55° C. for 10 sec. At the indicated times, fluorescence was determined. Inset: 100 pmole probe was incubated with 50 units of E. coli DNAse I at 37° C. Fluorescence was determined at the indicated times. As can be seen in
F, FAM; IB, iowa black; |, cut site; blue text, recognition site; red text, fluorescent attachment site; F, fluorescein; FAM, 6-carboxy-fluoroscein. All sequences written 5′ to 3′. The targets sequences are underlined in set 1, and are the complement of the probes in sets 2, 3 and 4 with the exception of the T attached to the fluorescein
Standard 20 μl NESA reactions were set up in 96-well qPCR plates containing 10 pmol probe, 200 fmol target oligo. Reactions were incubated at 58° C. for 5′, 2 μl Nt.AlwI added, and fluorescence measured every minute at 58° C. Results are illustrated in
Real time measurements were taken. Standard 20 μl NESA reactions were set up in 96-well qPCR plates containing 10 pmol probe, 200 fmol target oligo, 2 μl Nt.AlwI. Reactions were incubated at 58° C. and fluorescence measured every minute. Results are illustrated in
Results are illustrated in
A linear F/Q probe nicking enzyme streaming assay was compared with an assay employing capillary electrophoresis. For F/Q assay, standard 20 μl NESA reactions were set up in 96-well qPCR plates containing 10 pmol probe. Reactions were incubated at 58° C. for 5′, 2 μl Nt.AlwI added, and fluorescence measured every minute at 58° C. For the CE assay, standard 10 μl NESA reactions were set up with 1 pmol probe. Reactions were incubated at 58° C. and analyzed by CE. Results are illustrated in
Hairpin F/Q Probes can give very high signal to noise ratios at reduced temperatures. A 20 μl NESA reaction was set up using standard conditions. After 5 min @ 58° C., 2 μl Nt.AlwI was added and the reaction continued at 58° C. The enzyme was heat killed and cooled to 25° C. For the Melt curve, the temperature was then increased 1° C. every 10 sec. Superb signal to background is obtained below 40° C. (background is virtually zero) Results are illustrated in
Coupling of 3′-amino modified oligos to Qiagen LiquiChip carboxylated microbeads. A 1 ml suspension of LiquiChip beads was vortexed for 2 min. Aliquots of 125 μl were transferred to microfuge tubes, centrifuged for 3 min at 10,000 g, and the supernatant discarded. The pellet beads were resuspended in 50 μl of 0.1 M MES pH 4.5, and vortexed for 30 sec. One nmol (10 μl of 100 pmol/μl solution) of 3′-Amino modified oligos previously resuspended in 0.1 M MES pH 4.5 were added and vortexed for 30 sec. 10 μl of freshly prepared EDC ([N-(dimethylaminopropyl)-N′-ethylcarbodiimide] Fluka, St Louis, Mo.) were added and the mixture vortexed for 10 sec. Coupling reactions were placed in a light-tight box and agitated every 30 min for 2 hours at 24° C. Coupling reactions were then centrifuged for 3 min at 10,000 g and the supernatant discarded. 1 ml of the wash buffer (PBS pH 7.2, 0.02% Tween-20) was added to each reaction. Pellets were resuspended using a pipette and then centrifuged for 3 min at 10,000 g. The supernatant was removed with a pipette and discarded. The washing step was repeated twice. A final rinse of the washed pellet was performed by the addition of 150 μl TE pH 8.0, centrifugation for 3 min at 10,000 g, and removal of the supernatant. The TE equilibrated/washed coupled beads were resuspended in 50 μl TE pH 8.0. These surface-coupled probes (sc-probes) were stored up to four months at 4° C.
Coupling of 3′-amino modified oligos to Maleic Anhydride Coated Polystyrene Plates. Ten μl (100 pmol/μl) of 3′-Amino modified oligos previously resuspended in 0.1 M MES pH 4.5) were added to each well of a reacti-bind amine-binding, maleic anhydride activated plate (clear, 8-well strips) (Thermo Fisher Scientific Inc. Waltham, Mass.). Forty μl of PBS pH 7.4 were added to each well, and mixed by gently pipetting. Wells were sealed with OptiClear (B) film (Biorad, Hercules, Calif.) and incubated in a light tight box for 4 hours at 24° C. Plates were agitated gently every 30 min during incubation. After 4 hours, the supernatants were discarded, and 250 μl of blocking buffer (TE, pH 8.0) were added to each well. Plates were sealed and incubated in the dark with occasional agitation for 1 hour. Following blocking, the supernatants were discarded and the wells washed twice with 250 μl of wash buffer (PBS with 0.05% Tween-20, pH 7.2). Plates were washed twice again with 250 μl of blocking buffer, discarding the buffer each time. To each well, 100 μl of blocking buffer were added and the plates sealed. Plates were stored at 4° C. in the dark. One set of plates was used to test if they could be stored dry. Buffer was completely removed and the plates stored at 4° C. for 48 hours.
Quantifying coupling efficiency of bead coupled probes. To determine the concentration of the beads only, the bead-bound probes were vortexed for 5 sec and diluted 100-fold using MES pH 4.5 and their concentration determined using a hemocytometer. Next, 3 μl of undiluted resuspended probe-beads were added to 7 μl containing 1 μl of 10× DNase I buffer (Promega, Madison, Wis.), 1 μl of DNase 1 (Promega), 5 μl of H2O and incubated at 37° C. for 10 min. 1 μl of stop solution (20 mM EGTA pH 8.0) was added and the reaction incubated at 65° C. for 10 min. Following this DNAse inactivation step, 19 μl of H2O were added to the reaction. Uncoupled beads, and free probe were also DNase treated for use as background controls. Fluorescence was measured using a fluorescent plate reader (excitation 495 nm, emission 520 nm). Sample fluorescence was plotted against a standard curve prepared from serial dilutions of DNAse-treated uncoupled probe. The number of fluorescent oligonucleotides attached to each bead was then calculated for each coupling reaction.
NESA reactions using surface coupled probes (sc-probes). Each NESA reaction contained 1 μl (1 pmol to 10 amol) of target oligonucleotide (the complementary sequence of the probe), 1 μl Restriction Endonuclease Buffer-2 (NEB), 3 μl sc-probes, and 4 μl H2O. The 9-μl mixture was heated to 95° C. for 10 min, 4° C. for 10 sec, then 58° C. for 5 min. Once the reaction had reached 58° C., 1 μl Nt.AlwI (NEB, Ipswich, Mass.) was added followed by incubation at 58° C. for 60 min and then 80° C. for 20 min to stop the reaction.
NESA reactions using sc-probes on plates. Target oligonucleotides (the complementary sequences of the probes) were first denatured for 2 min at 95° C. (5 μl complement (1 pmol/μl), 2.5 μl of 10× NEBuffer-2 (NEB), and 17.5 μl of H2O). Each reaction was added to a well of a plate preheated to 58° C., the plate sealed, and incubated for 5 min at 58° C. 25 μl of preheated diluted Nt.AlwI (2.5 μl 2.5 μl 10× NEBuffer-2 (NEB), 17.5 μl H2O) were then added and incubation continued at 58° C. for 60 min. The reaction was then inactivated by incubating at 80° C. for 20 min. Two μl of each reaction were prepared for CE analysis as described.
Capillary gel electrophoresis (CE). Samples were analyzed using an Applied Biosystems 3130×1 Genetic Analyzer with electrokinetic injection as described previously (6). In brief, NESA reactions centrifuged at 2000 g for 5 min at 24° C. Two μl of the supernatant were diluted 100-fold (20-fold with water and then 5-fold with formamide and then 10 μl were used for injection. A set of Hex™-labeled standards (5, 17, 21, 30, and 50 nucleotides in length) was run with each sample to aid in peak identification.
Real time NESA (rtNESA) using sc-probes. A 10-μl reaction mixture containing 1 μl of diluted complement (1 pmol to 10 amol), 1 μl NEBuffer-2, 3 μl sc-probes, 5 μl H2O was heated to 95° C. for 10 min, 4° C. for 10 sec, then 58° C. for 5 min. Ten μl of diluted Nt.AlwI (2 μl Nt.AlwI, 1 μl of NEBuffer-2, 7 μl of H2O) were added and the mixture incubated for a further 60 min at 58° C. Fluorescence was determined every minute of the 60 min incubation using 5 s reads and excitation/emission wavelengths of 495 nm/520 nm. All rtNESA experiments were run and analyzed using the BioRad iQ5 RT-PCR Detection System. The efficiency of coupling probes to polystyrene beads (4 μm diameter) was investigated. On average, 0.6×106 probes were coupled to each bead. Each bead is supposed to have 108 binding sites, approx 0.5% coupling efficiency. There can be about 3,000 beads per μl. 3 μl sc-probes used per reaction=1.8×109 probe molecules per reaction (˜3 fmol)
For capillary electrophoresis the limit of detection can be between 100 and 10 amol (10-17 M) for sc-Probes on polystyrene beads. Samples can be injected for at least 360 s with little increase in background. Samples can be used directly, bead removal is not necessary.
For F/Q sc-Probes on polystyrene beads, real time assays can perform well. Kinetics of sc-probes and uncoupled probes are very similar. There can generally be no increase in background fluorescence during the reaction. for sc-Probes on multi-well plates, very strong signals could be measured by CE. Sc-probe plates are stable desiccated for at least 48 hours and work at least as well as “wet” plates. Desiccated plates are likely a way for long term storage of sc-probes. Results are illustrated in
These probes have the same base sequence and have been aligned with spaces where necessary. F, FAM; IB, iowa black; AmM, amide linkage, BHQ, black hole quencher 1. Data using the probes in bold are shown in these examples. The other probes gave similar results (data not shown).
(References cited in this example are numbered in accordance with the listing at the end of this example.)
Introduction Although culture methods have been used for many years to identify microorganisms, they are not so helpful with very slow growing organisms, with those that are difficult or impossible to grow in vitro, and those organisms that do not survive harvesting methods such as filtration. For these reasons, molecular biological methods have been developed that allow the rapid identification of bacteria (7, 10, 16, 17, 19). The leading technique for this is undoubtedly the polymerase chain reaction (PCR), especially quantitative PCR (qPCR), that can not only identify an organism but can also determine relative bacterial load (15, 22). Although PCR is exquisitely sensitive and can be multiplexed, albeit with a usual decreased sensitivity, there is a caveat to using PCR as a detection method. PCR detection from complex samples, such as environmental or clinical samples, often requires purification of the DNA to remove inhibitors of the PCR reaction such as heme, humic acids, chelating agents, and metals (3, 4, 12, 21). These inhibitors can give rise to false negatives that could be devastating in the case of medical diagnostics and catastrophic in the case of environmental surveillance for biowarfare agents. Unfortunately the isolation and purification processes necessary to obtain pure DNA are often time consuming and difficult with field studies, while the efficiency of recovery from such methods can vary and affect the sensitivity of PCR detection (18). We have recently described a technique called nicking endonuclease signal amplification (NESA) that uses fluorescent oligonucleotide probes in isothermic cycles of hybridization, cleavage, and dissociation to detect the presence of low levels of specific genomic DNA (5). In this report, we show that a combination of multiple displacement amplification and NESA can be used to detect genomic DNA in crude environmental samples without the need for DNA with samples that are refractory to PCR. We show the general utility of the system by developing NESA probes specific for B. anthracis chromosomal DNA as well as the B. anthracis plasmids pX01 and pX02 that are required for virulence. These probes were combined in a multiplex assay that distinguishes between strains of B. anthracis.
Materials and Methods Oligonucleotides, Genomic DNA, Environmental Samples. Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, Iowa). All probes were fluorescently labeled at the 5′ position with 6-carboxyfluorescein (FAM™) and were purified by ion exchange HPLC. Five oligonucleotide size standards (5, 17, 20, 30 and 50 bases) were synthesized with hexachlorofluorescein (HEX™) at their 5′ ends. Genomic DNAs were obtained from Zyagen, San Diego, Calif. (Bovine, Cat, Dog, Monkey, Mouse, Sheep, Porcine, Equine, Human); Lawrence Livermore National Laboratory (Chicken, Drosophila melanogaster, Rabbit, Rat, Flea, Mosquito); Johns Hopkins Bloomberg School of Public Health (Tick, Ixodes scapularis); BEI, Manassas, Va., (B. anthracis strains Ames BEI 02005259004, Vollum BEI D2005276004, GT68 BEI D2005255003, Δ Sterne BEI D2004322001, GT41 BEI D2004056001, BACI055 BEI D2005027001, GT28 BEI D2006030002A1, Sterne BEI D2005075001, GT3 BEI D2004050007; Yersinia pestis strains F361/66 South America 1966 BEI 02005041002, India 1898 BEI D2005056003, 342 15-91 Russia 1960 BEI D2005060001, 338 ZE942122 Zimbabwe 1994 BEI D2005042001, 346 16-34 Vietnam 1970 BEI D2005056002); The Naval Medical Research Center, Silver Spring, Md. (B. anthracis Ames); ATCC, Manassas, Va. (Mycobacterium smegmatis, Thermotoga maritima, Borrelia burgdorferi, Chlorobium tepidum, Bacteroides fragilis, Shewanella oneidensis, Pseudomonas aeruginosa, Staphylococcus lugdunesis, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus schleiferi, Bacillus thuringiensis, Methanosarcina mazei, Spiroplasma citri, Sulfolobus solfataricus, Lactobacillus plantarum, Pyrococcus furiosus, Streptomyces avidinii, Yersinia enterocolitica, Neisseria meningitidis, Paenibacillus sp., Helicobacter pylori, Deinococcus radiourans, Clostridium aceto-butylicum, Bacillus cereus, Rhodobacter Saccharomyces cerevisiae, Candida albicans); U.S. Army Edgewood Chemical Biological Center (ECBC), Aberdeen Proving Ground, Md. (B. anthracis strains. Ames, ANR1, NNR-.1, NNR1-.1, VNR1-.1, Sterne; Yersinia pestis strains 9108101, A1122, Amal, 9800419, 9808723, Harbin; Staphylococcus aureus enterotoxin B positive strains ATCC 13566, 14458, 19095, 27664, 51651, 51811, 51811; Bacillus cereus ATCC 14579, Bacillus subtilis ATCC 27370, Bacillus thuringiensis BGSC 4AZ1, Bacillus thuringiensis BGSC 4G1); Molecular Staging Inc. (E. coli, B. subtilis). Genomic DNAs from pathogenic organisms were guaranteed not to contain infectious agents by the suppliers and licenses/permits were obtained from the appropriate agencies for purchasing and shipping. The Georgetown University's Institutional Biosafety Committee approved use of the genomic DNAs. BioWatch filter pieces were obtained from Lawrence Livermore National Laboratory. Crude extracts of these environmental filters were made by placing the filters in 2 ml screw cap vials along with 300 μl each of 2 types of acid-washed glass beads, particle size=106 μm (Sigma G4649) and 425-600 μm (Sigma G8772), and 600 μl TE pH 8.0. Tubes were shaken in a mini bead beater (Biospec Products, Inc #3110BX) at 4800 rpm for 3 min. Vials were microfuged at 13,200 rpm for 5 min and 500 μl of the aqueous phase was then transferred to a fresh tube. These crude samples had some colored particulate matter in them and some were viscous. These extracts were used in experiments to compare the ability of NESA and PCR to detect bacteria in crude environmental samples. Two sets of purified DNA were obtained from Lawrence Livermore National Laboratory to be used in the screening of probes. One set consisted of 96 pools of DNA representing 2000 BioWatch filters. The other set consisted of DNAs isolated using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc, Carlsbad, Calif.) from 54 soil samples from geographically diverse regions of the United States.
Whole Genome Amplification of Genomic DNA. Genomic DNAs (10 ng) were amplified by multiple displacement amplification (MDA) (5) using the REPLI-g Kit from Qiagen (Valencia, Calif.) according to the manufacturer's instructions. Reactions were incubated at 30° C. for 16 hours, followed by heat inactivation of the polymerase at 65° C. for 3 minutes. MDA DNA was quantified using the PicoGreen assay using the manufacturer's protocol (Invitrogen), and specificity of amplification by PCR analysis.
Amplification of E. coli genomic DNA in the presence of BioWatch extracts. BioWatch samples were mixed by pipetting up and down to insure resuspension of particulate matter in the samples immediately prior to transfer to a 96-well PCR plate. Approximately, 100,000 E. coli cells, in a suspension of 0.5 μl PBS, were added to each sample. Following the addition of the cells to the sample, the protocol for “Whole Genome Amplification from Blood or Cells” (REPLI-g Handbook, Qiagen, January 2005) was followed with the following exception: the volume of nuclease-free water in the master mix was reduced from 27 μl to 24.5 μl to account for the additional volume from the BioWatch samples. MDA reaction products were diluted 40 fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). One μl of diluted MDA reaction was quantified using a Quant-iT PicoGreen dsDNA Reagent Kit (Invitrogen) according to the manufacturer's instructions. PicoGreen assays were read on a Tecan GENios microplate reader. BioWatch samples were tested for the ability to interfere with the PicoGreen assay by directly adding un-amplified BioWatch sample to a PicoGreen assay mix; no interference with quantification was observed.
Locus Representation of BioWatch MDA Samples. MDA reaction products were diluted 4000-fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 1 μl of diluted MDA sample was added to a PCR reaction (0.5 units Platinum Taq polymerase (Invitrogen), 0.3 μM each of forward and reverse primers, 0.25 μM TaqMan probe, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 1 mM dNTPs, 1× Rox reference dye (Invitrogen)). Reactions were performed and quantified with an Applied Biosystems 7300 Real Time PCR system under the following conditions: 10 min at 95° C., followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min. The sequences of the PCR primers and TaqMan probes for the ExoV and OmpA loci have been published previously (20).
Nicking Endonuclease Signal Amplification (NESA). NESA reactions were performed as previously described (11). In brief, 1 pmole fluorescently labeled probe, 1 μl NEBuffer 2 (New England BioLabs), 1 to 4 μl MDA genomic DNA and dH2O to a total volume of 9 μl were added to thin-walled PCR tubes or microplates. Samples were incubated at 95° C. for 10 min to denature the genomic DNA and then equilibrated at 58° C. for 5 min. One μl (10 U) of Nt.AlwI (New England BioLabs) was added and the reaction incubated at 58° C. for 1 h. The enzyme was denatured by heating the samples to 80° C. for 20 min. Reactions were stored at 4° C. until analysis by capillary electrophoresis (CE) or at −20° C. for long-term storage. Multiplex assays for B. anthracis were set up with 1 pmole each of a chromosomal, pXO1 and pXO2 probe. The reaction was kept at 10 μl by reducing the volume of water added. In the case of large-scale screening, the NESA reactions were processed using a Beckman Biomek® FX robotic workstation.
Capillary gel electrophoresis. NESA reactions were analyzed using an Applied Biosystems 3130xl Genetic Analyzer (Foster City, Calif.) using a 16 capillary array; electrokinetic injection was used in all cases. The distance from the loading point to the detector was 35 cm. POP-6™ polymer from Applied Biosystems was used with an injection voltage of 1.2 kV and a loading time of 18 seconds. Run voltage was 15 kV for 10 minutes; oven temperature was set at 60° C. Prior to loading on the ABI 3130xl, the reactions were diluted 100-fold: a 20-fold dilution with water followed by a 5-fold dilution with formamide. Final loading volume was 10 μl. A set of 5 Hex™-labeled standards was run with each sample to aid in peak identification, which was performed using GeneMapper® software (Applied Biosystems).
Results. A combination of MDA and NESA reproducibly detects bacterial contamination in crude (BioWatch) environmental samples. Analysis of the DNA in environmental samples is usually constrained by low levels of DNA and the presence of inhibitors of enzymatic reactions (23). We designed our assay to largely overcome both of these constraints by first amplifying DNA nonspecifically using MDA (14) and then detecting specific sequences with NESA (
qPCR is sensitive to inhibitors in the Environmental Extracts PCR analysis is known to be particularly sensitive to the presence of contaminants. We thus suspected that the crude environmental samples we were using would act as poor templates for PCR. PCR reactions were set up in which 10% of the PCR reaction consisted of the environmental extract, however these reactions all failed to give a signal (not shown) and so the experiment was repeated with 4% extract (
Development of a multiplex assay for B. anthracis The sensitivity of the MDA/NESA assay and its resistance to environmental contaminants makes it an attractive assay system to detect bacterial contamination. To demonstrate the utility of the assay, we set out to develop a system for designing and screening NESA probes capable of identifying specific microorganisms. Our initial goal in this was to develop probes that could identify individual strains of B. anthracis.
Probe design—Bioinformatics For the purposes of the bioinformatics screen, the Nt.AlwI site was placed 2 bases from the 5′ end of the probe (
Probe Screening Candidate probes were first tested against three near neighbors of the B. anthracis genome, B. cereus, B. subtilis, and B. thuringiensis; any candidate probes showing positive results were eliminated from subsequent screening in order to eliminate false positive detection in assays for B. anthracis. In total, all 133 probes were tested against 14 strains from the 3 near neighbors. Surviving probes were then tested against the eukaryote and prokaryote MDA panels, including various strains of B. anthracis MDA. Probes showing positive results for the B. anthracis strains only and no cross-reactivity with the background panels were then tested against the 96 MDA pools of 4000 environmental aerosol samples, and subsequently the 54 MDA soil samples. All remaining probes were then retested against the B. anthracis strains, prokaryote and eukaryote panels.
Discrimination between strains of B. anthracis Our large scale screening analysis identified a set of probes that could detect B. anthracis chromosomal and plasmid sequences but that did not react with any of the DNAs in our screening panels including over 2000 environmental samples. To test whether these probes could be used to genotype unknown samples correctly, a blinded experiment was performed. DNA samples from a set of B. anthracis strains and related Bacillus species were coded, amplified, and subjected to NESA at ECBC. These reactions were then analyzed by CE at Georgetown University Medical School, and the results decoded by ECBC. All three related Bacillus species tested gave a positive result (
Parallel and multiplex NESA For analysis of a large number of probes against one or more organisms, a system that is amenable to parallel, and/or multiplex analysis is essential. For instance, in environmental sampling for pathogens, a whole host of different targets could be envisioned (1). During our large scale screening, analysis was performed with 96 reactions in parallel using 96-well plates and a Beckman Biomek FX robotic liquid handling station. The MDA reaction produces a vast excess of DNA enabling over 100 NESA reactions from a 50-μl MDA reaction and this can be scaled up easily. Although 500 ng of MDA DNA was used in these screening assays, less than 100 ng of MDA DNA is sufficient for NESA (11). To increase the efficiency of the assay even more we developed a multiplex assay using FAM16 labeled probes specific for B. anthracis chromosomal and plasmid (pXO1 and pXO2) sequences that could be distinguished from each other by size using capillary electrophoresis. These probes were used in NESA reactions in the presence of MDA amplified genomic DNA from B. anthracis and related Bacillus species. Capillary electrophoresis readily distinguished cleaved and uncleaved probe both for pXO1 and pX02 and chromosomal DNA in the same assay (
Discussion We have demonstrated that the combination of MDA and NESA can be used to develop fast, sensitive and specific multiplex assays for organism identification. This combination of techniques provides an alternative to PCR, which is sensitive to common environmental inhibitors.
Sensitivity The theoretical sensitivity of our combined MDA/NESA assay is one genome equivalent since it has been shown that MDA can be performed on one bacterium (13, 20). Detection levels of one bacterium are likely to be hard to obtain routinely but we have previously demonstrated the ability to detect 7 cfu of B. subtilis in crude samples (11).
Assay time The slowest step in the assay is the MDA reaction that is routinely run overnight as recommended by the manufacturer. Preliminary results using the new ultrafast kit from Qiagen indicate that a standard MDA reaction yields sufficient DNA for NESA within 90 min. Data provided by Qiagen indicate that sufficient DNA should be produced within 45 min, but we have yet to confirm this. Further increases in speed can be possible by supplementing the reaction with additional enzyme (unpublished) or by using semi-specific primers for the region of the genome under investigation. The sensitivity is also dependent on the type of target DNA since plasmid DNA is usually amplified better in MDA than genomic DNA (6). Plasmid DNA can also give a stronger signal compared to chromosomal DNA due to the higher copy numbers of many plasmids compared to chromosomal DNA. In the case of B. anthracis, probes against pXO1 and pXO2 usually gave stronger signals than chromosomal probes.
NESA reactions were run for 1 h here, but have been shown previously to be nearly complete within 30 min (11). Thus a 30 min NESA should be sufficient in all cases except where maximum sensitivity is required. Following NESA, CE separation is complete within 20 min. The use of a 16-capillary machine and a triplex assay would put the through rate at 48 probes in 20 min.
Multiplex Capability We have demonstrated that it is possible to multiplex NESA reactions using size discrimination to detect B. anthracis chromosomal, and plasmid (pXO1 and pXO2) DNA in the same assay. Multiplex assays can be designed using different fluors (20), or by placing the fluor on either the 5′ (as in this paper) or the 3′ end (unpublished) of the probe. Although discrimination by size on CE often has a resolution of less than one base, the small size of the fluorescent oligos used in NESA results in migration that depends on base composition as well as overall length. Multiplex assays must be optimized with regard to the migration pattern of the cleaved probes since fluorescent oligos of different sizes can run at the same position, oligos of the same length can separate into two distinct peaks and oligos of the same length and base composition can resolve into separate peaks when labeled with different fluors (20). For instance, the pXO1 and chromosomal probes are cleaved into fluorescent oligonucleotides of identical length yet they resolve on CE (
Bioinformatics of Probe Selection We designed our bioinformatics pipeline to first identify short regions (24mers) that contained Nt.AlwI sites in B. anthracis and then selected against those regions that contained an identical 16 base region, anchored by the Nt.AlwI site, in GenBank. This screening procedure was computationally intense and resulted in a relatively small number of potentially acceptable targets (approximately 2.5%). However, remarkably 17% of these regions yielded probes with absolute specificity in the screening assays conducted suggesting that probe development against other targets is feasible. Indeed, preliminary results with Y. pestis chromosomal probes indicate a similar success rate. It should be noted that each Nt.AlwI site can yield multiple probes so that the 136 unique probes analyzed are only a small fraction of what is available. In recent unpublished work, we have successfully made probes against reverse transcribed MS2 bacteriophage (3.85 kb) and Dengue virus genomic RNA (˜10.7 Kb) indicating that the MDA/NESA technique can be used on small genomes.
The probe design is shown in
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(References cited in this example are listed at the end of this example.)
Sequence-specific genomic assays for the detection and typing of microorganisms using procedures such as PCR are common. However, in the case of RNA genomes, their small size and the requirement to first copy the RNA into cDNA often limits their sensitivity. We have developed a new assay for organisms with RNA genomes that uses a combination of reverse transcription, multiple displacement amplification (MDA) and nicking endonuclease signal amplification (NESA). This assay, which we term rNESA, achieves high sensitivity, differentiates between viral serotypes, and is insensitive to common environmental and biological contaminants. Using the small RNA virus surrogate MS2 bacteriophage, we were routinely able to detect 100 attograms of genomic RNA with rNESA. At 10 attograms (5 genomes) the NESA assay detected MS2 approximately 40% of the time. With the same samples, a published rtPCR assay had a detection threshold of 10 fg genomic RNA (5,000 genomes). The presence of a large excess of either human mRNA, or environmental contaminants, had no effect on the specificity of the assay and only a small effect on sensitivity. The utility of the assay for larger RNA viruses was shown by the development of a rNESA multiplex assay specific for the four serotypes of the human pathogenic Dengue virus. These probes had absolute specificity between the Dengue virus serotypes and no cross reactivity with human RNA or with two other members of the Flaviviridae family, West Nile virus and St Louis Encephalitis virus. The Dengue assay detected 2 fg Dengue virus RNA (˜345 genome equivalents).
Detection and identification of microorganisms is critical in the areas of medical diagnostics, biodefense and environmental biology. Many detection methods have been used including selective culture in vitro, animal inoculation, mass spectrometry, antibody-dependant assays and assays that use DNA sequence-specific hybridization (Ivnitski et al. 2003; Lim et al. 2005; Peruski and Peruski 2003; Rotz and Hughes 2004). Of the DNA-based techniques, PCR is perhaps most often used because of its high specificity, speed, and its adaptability to new targets. RNA viruses, however, are an exception because there is an additional requirement to first convert the RNA to DNA. This first step, which uses reverse transcriptase, is inefficient and limits the sensitivity of the overall assay (Bustin and Nolan 2004). In qPCR, a relatively small target DNA sequence is amplified using a gene-specific primer pair in sequential rounds of denaturation, annealing, and DNA synthesis using a heat tolerant DNA polymerase. The product of the reaction is detected using fluorescent probes. That is, amplification and detection essentially occur at the same time. We recently separated these two steps by amplifying DNA nonspecifically using MDA and then detecting the presence of specific sequences using NESA (Kiesling et al. 2007). In NESA, a single stranded fluorescent probe containing a nicking endonuclease recognition sequence anneals to the target sequence forming a double stranded recognition and cleavage site. The nicking endonuclease then cleaves the probe but leaves the target intact. The small probe pieces dissociate from the target spontaneously allowing multiple rounds of hybridization and cleavage to occur. The cleaved probe can be detected in a number of ways although we usually use capillary electrophoresis. We have also used FRET-based probes where fluor and quencher are separated upon DNA cleavage (unpublished). A recent paper used cleavage of molecular beacons in their NESA reaction (Li et al. 2008). In this paper, we describe an MDA-NESA protocol (rNESA) that allows detection of RNA viral genomes with a sensitivity that outperforms rtPCR. Remarkably, the assay can also be performed directly on environmental samples such as extracts of BioWatch filters that are refractory to PCR analysis. BioWatch is a US government program that monitors air for biological threat agents using filtration (Report 2003). We demonstrate the power of rNESA by developing a multiplex assay against the four Dengue virus serotypes. Probes against each of the four Dengue serotypes were specific for the respective serotype and did not cross react with other closely related RNA viruses. In addition, the Dengue virus rNESA assays were able to detect low levels of Dengue RNA even in the presence of a large excess of nonspecific (human) RNA.
ResultsDetection of genomic RNA using reverse transcriptase and NESA For the initial experiments, we used MS2 bacteriophage genomic RNA as a target. MS2 is often used as an RNA virus surrogate since there is no risk of infection and it can be handled at biosafety level I (O'Connell et al. 2006). MS2 first strand cDNA was reverse transcribed as detailed in methods. This cDNA was then used in a series of NESA reactions using NESA probes designed to hybridize to either the cDNA (MS2-S) or to the RNA strand itself (MS2-1). As expected, the MS2-S probe recognized the target cDNA and was cleaved by Nt.AlwI as shown by the production of cleaved probe (
rNESA is resistant to inhibitors found in crude environmental samples Detection of genomic DNA or RNA in environmental samples usually requires purification of the genomic material in order to remove contaminants that inhibit enzymatic reactions. In the case of PCR, inhibitors can cause loss of specific signal resulting in false negatives and/or the inappropriate amplification of non-target DNA resulting in false positives (Bustin and Nolan 2004; Lantz et al. 2000; Radstrom et al. 2004). Both scenarios are unacceptable in programs, such as BioWatch (Report 2003), that are designed to detect the release of biowarfare agents in American cities. To compare the effects of contaminants found in crude BioWatch samples on rtPCR and rNESA, a series of MS2 rtPCR and rNESA reactions was set up containing 100 fg MS2 genomic RNA and 10% (v/v) of BioWatch sample. All 14 independent BioWatch extracts inhibited the amplification of MS2 genomic RNA by rtPCR and six of these extracts caused the rtPCR assay to fail completely (
Sensitivity in a complex background (selectivity) One concern is that a large excess of non-target RNA could decrease rNESA's ability to efficiently amplify and detect some minute quantity of target RNA. To test selectivity, decreasing levels of MS2 genomic RNA (100 fg to 100 ag) were added to rNESA reactions containing 10 or 100 pg of purified total human RNA (
rNESA probes specific for Dengue virus serotypes To demonstrate that rNESA can be used to detect pathogenic RNA viruses we set out to develop probes specific for each of the 4 Dengue virus serotypes (Chambers et al. 1990). The sequence of the 4 serotype probes and their relationship to the 4 viral serotypes is shown in (
Development of a multiplex Dengue virus assay Initial experiments with the four serotype-specific probes showed that although the signal peaks resolve by capillary electrophoresis (CE), they are not well separated and had some overlap. To obtain signals that are fully separated on CE, two new probes (3T and 4T) were generated that had additional non base pairing residues (T's) at their 3′ ends (Table 1) and thus yield larger probe fragments (
Discussion We have described a new technique that can be used to develop specific assays for the detection of organisms with RNA genomes. The technique, which is a combination of reverse transcription, MDA and NESA, retains the properties of the individual techniques. For instance the inhibitor-resistant phi29 DNA polymerase in MDA allows detection in dirty backgrounds and fast, nonspecific whole genome amplification while the moderate temperature resistance of Nt.AlwI (up to 60° C.) allows the development of specific DNA probes. Remarkably, the assay is more sensitive than rtPCR both for the small (3.579 kb) virus substitute, MS2 and for the larger (˜10.7 kb) Dengue virus.
Sensitivity We have shown that rNESA can detect 100 ag of MS2 RNA routinely and 10 ag about 40% of the time. 10 ag is equivalent to approximately 5 genomes. A surprising finding was that rNESA was more sensitive than published rtPCR MS2 assays (Dreier et al. 2005; O'Connell et al. 2006; Rolfe et al. 2007). This is probably due to the nature of the cDNA synthesized in the reverse transcriptase step. Reverse transcription is dependent on an initial DNA primer that initiates cDNA synthesis. Since the ability of primers to initiate productive cDNA synthesis is highly dependent on the location of hybridization due to folding of the RNA, random primers are often used to sample many initiation sites (Bustin and Nolan 2004). Although this results in relatively efficient cDNA synthesis, most cDNA molecules are not full length. This reduces the ability of PCR to amplify the cDNA because some cDNA molecules will not contain sequences having both forward and reverse priming sites. These sequences will not act as templates for PCR and thus limits of detection will decrease. This is not the case for rNESA because neither amplification of the cDNA with MDA, nor amplification of the signal with NESA, requires a primer pair. It is possible that the NESA target sequence (approximately 20 bases) can be split in two resulting in loss of signal but this will occur far less frequently than separation of the forward and reverse primers of rtPCR that can be 100 or more nucleotides apart. For the same reason, NESA can be less sensitive to degradation of the RNA during analysis. The Dengue virus assay was not as sensitive as the MS2 assay. We do not know if this is related to the larger size of the Dengue virus. However, the Dengue rNESA assay was still approximately 10-fold more sensitive than the qPCR assay.
Specificity We have previously shown that NESA probes can be used to distinguish between bacterial species (Kiesling et al. 2007). Here, we show that it is possible to develop specific probes against the closely related serotypes of Dengue virus. These probes not only distinguish between Dengue serotypes but they also do not interact with West Nile virus and St. Louis Encephalitis virus that are also members of the Flavivirus genus. Both the Dengue and MS2 probes are also refractive to total human RNA. We are currently developing probes that are specific for groups of organisms. For instance we are developing a probe that recognizes all Dengue virus serotypes and another that recognizes many of the members of the Flavivirus genus.
Selectivity We show that rNESA selectively detects 100 ag of MS2 genomic RNA (˜50 genomes) in the presence of 10 pg total human RNA. 10 pg of human RNA is the approximate amount of RNA in 2 to 10 cells, thus the assay can theoretically detect the equivalent of 5 viral genomes per human cell. With 100 pg of human RNA there was a decrease in sensitivity of the assay presumably due to competition between the human RNA and MS2 RNA for reverse transcriptase. It would be interesting to see if the use of MS2-specific reverse transcriptase primers results in increased sensitivity in the presence of such a large excess of human RNA.
Application to environmental monitoring and medical diagnostics In both of these applications, samples are often contaminated with potential inhibitors. Heme in blood samples, for instance, is a well-known inhibitor of PCR as is humic acid from soil in environmental assays (Radstrom et al. 2004). For this reason, samples often undergo extensive purification before enzymatic analysis. We have shown that crude environmental samples that are refractory to rtPCR analysis can be assayed with rNESA with little loss of sensitivity and no drop out. That is, rNESA is particularly resistant to enzymatic inhibitors and thus the genomic RNA does not need to be extensively purified.
Dengue Multiplex Assay The Dengue multiplex assay we have described discriminates between all four dengue serotypes, does not interact with the related viruses SLE and WNV, and is applicable to samples containing human RNA. The assay should thus be useful to detect acute Dengue virus infection since detectable viral RNA coincides with the onset of viraemia. Saxena In our rNESA Dengue multiplex assay we can detect 345 genome equivalents. Saxena et al. (Saxena et al. 2008) recently described a multiplex assay for Dengue serotypes with a sensitivity of 2,500 copies. However, it is hard to directly compare the two assays since the assay described by Saxena et al (Saxena et al. 2008) is based upon in vitro transcription of a small region of the virus compared to our full-length assays, and supporting evidence for their sensitivity data is not shown. The apparent increased sensitivity of the rNESA multiplex assay over this rtPCR multiplex assay warrants further investigation using clinical samples. Indeed, the ability to use crude RNA samples, and the easily automated MDA and NESA reactions (unpublished) would make this an attractive diagnostic assay.
Non-viral RNA targets Although the work presented in this paper targets viral RNA, the technique we describe should be amenable to any RNA target that can be reverse transcribed. Indeed, the sensitivity of the assay is such that detection of mammalian mRNA targets should be possible.
MethodsOligonucleotides, genomic RNA and Nt.Alw1 Oligonucleotides (Table 1) were from Integrated DNA Technologies (Coralville, Iowa,) except the β-actin PCR primers (Stratagene). Probes contained a FAM group at either the 5′ or 3′ end and were purified by RNAse-free ion exchange HPLC. Oligonucleotide size standards were synthesized with hexachlorofluorescein (HEX™) at their 5′ ends. MS2 genomic RNA was obtained from Roche Applied Science (Indianapolis, Ind.). Dengue virus serotype-3, West Nile virus, and St Louis Encephalitis virus genomic RNAs were obtained from BEI resources (Manassas, Va.). Human total RNA was from Stratagene (Cedar Creek, Tex.). Dengue viral Serotypes 1, 2, and 4 genomic RNAs were synthesized by in vitro transcription using pRS424 plasmids containing full-length genomic cDNA inserts (Polo et al. 1997; Puri et al. 2000). Briefly, the plasmids were linearized immediately after the last stop codon using SacI. Linearized plasmids were gel purified using Qiagen Qiaex II Gel Extraction Kit. 2 pg of purified linear plasmid was used with the Ambion SP6 Megascript Kit. The in vitro transcription reactions were incubated for 4 hrs following the manufacturers instructions. The in vitro transcribed RNA was purified using Qiagen RNeasy Mini Kit. Purified RNA was run on a 0.7% agarose gel for determination of correct size and lack of degradation. RNA concentrations were determined by A260 and with the Invitrogen Molecular Probes Quant-it RiboGreen RNA Assay Kit. Serotypes of the four Dengue virus RNA samples were confirmed using serotype specific primers in rtPCR (Saxena et al. 2008).
Reverse Transcription RNA was reverse transcribed using the Invitrogen SuperScript III first strand synthesis kit following manufacturers protocol with slight adjustments. Briefly 2 μl of the RNA was added to the 8 μl annealing reaction containing 15 μM random hexamers (New England BioLabs), and 1 μl of the annealing buffer, incubated at 65° C. for 5 min, and placed on ice for 2 min. 2 μl of the reverse transcriptase and 10 μl of the 2× reaction buffer were added to the 8 μl annealing reaction while on ice. The 20-μl reaction was heated to 25° C. for 10 min, 45° C. for 50 min, followed by 95° C. for 5 min. 10 μl of the 20-μl reaction were used in the proceeding ligation reactions.
Ligation and MDA Ligation and MDA were performed using the Qiagen Whole Transcriptome Kit following the manufacturers instructions except that an additional enzyme inactivation step (95° C. for 5 min) was added following the completion of the ligation reaction. The MDA reaction was performed for eight hours
NESA Reaction 2 μl of the 50-μl MDA reaction were used in each 10-μl NESA reaction. The NESA reaction contained 1 μl Restriction Endonuclease Buffer-2 (NEB), 1 μl (1 pmol) probe, 2 μl of target MDA, 5 μl H2O. The 9-μl mixture was heated to 95° C. for 10 min, 4° C. for 10 sec, then 58° C. for 5 min. Once the reaction had reached 58° C., 1 μl Nt.AlwI (NEB, Ipswich, Mass.) was added followed by incubation at 58° C. for 60 min and then 80° C. for 20 min to stop the reaction.
Capillary gel electrophoresis Samples were analyzed using an Applied Biosystems 3130xl Genetic Analyzer with electrokinetic injection as described previously (Kiesling et al. 2007). In brief, NESA reactions were diluted 100-fold with formamide and then 10 μl were used for injection. A set of Hex™-labeled standards (5, 17, 21, 30, and 50mer) was run with each sample to aid in peak identification.
rtPCR and Melting Curve Analysis 2 μl of RNA was mixed with 20 μM of each forward and reverse primer, 25 μl 2× BioRad iScript One-Step Sybr Green Mix, 2 μl of Reverse Transcriptase, and RNAse-free H2O for a total reaction volume of 50 μl. Thermocycling parameters were as follows for the following RNA templates: MS2 RNA: 48° C. for 10 min, 95° C. for 5 min, 40 cycles of 95° C. for 10 sec, 62° C. for 30 sec 72° C. for 30 sec, Dengue viral RNA: 50° C. for 10 min 95° C. for 5 min 40 cycles 95° C. for 10 sec 55° C. for 30 sec 72° C. for 30 sec, Human RNA: 94° C. for 5 min 60° C. for 5 min 72° C. for 1.5 min 35 cycles of 94° C. for 45 sec 60° C. for 45 sec 72° C. for 1.5 min followed by final extension of 72° C. for 10 min. All completed rtPCR runs were followed by a melting curve analysis consisting of the following steps: 95° C. for 1 min 55° C. for 1 min, followed by 80 cycles starting at 55° C. for 10 sec increasing in 0.5° C. increments each cycle. All rtPCR experiments were run and analyzed using the BioRad iQ5 RT-PCR Detection System with iQ5 Optical System Software v1.1.
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While the methods and articles described herein have been described with reference to specific embodiments, this application is intended to cover those various changes and substitutions that can be made by those of ordinary skill in the art.
REFERENCESThe following publications, as well as all others referenced in the disclosure, are incorporated herein by reference in their entirety:
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Claims
1-26. (canceled)
27. A substrate comprising a surface onto which a DNA probe is affixed, wherein the DNA probe comprises a sequence complementary to a unique sequence of a target molecule sequence that includes a recognition sequence for a nicking endonuclease.
28. The substrate of claim 27, wherein the substrate is a plastic or glass bead.
29. The substrate of claim 27, wherein the substrate is a plate comprising a plurality of wells, the plurality of the wells comprising one or more different DNA probes, each different probe comprising a sequence complementary to a unique sequence of a target molecule sequence that includes a recognition sequence for a nicking endonuclease.
30. The substrate of claim 27, wherein the probe comprises a fluorescent tag that is released from the substrate surface if the probe is cleaved by a nicking endonuclease.
31. A kit comprising a plurality of desiccated substrates according to claim 27, different substrates comprising one or more different probes.
32. A method for detecting the presence of a target nucleotide sequence in a sample of DNA, the method comprising: (a) exposing a test sample comprising single stranded DNA to a nicking endonuclease and a substrate surface according to claim 27 under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease; and,
- (b) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample DNA.
33. The method of claim 32, wherein the substrate surface onto which the DNA probe is affixed comprises a surface of a plastic or glass bead.
34. The method of claim 32, wherein the substrate surface onto which the DNA probe is affixed comprises a surface of a well in a plate.
35. The method of claim 32, wherein the probe comprises a fluorescent tag that is released from the substrate surface if the probe is cleaved by the nicking endonuclease and the step of observing whether the probe is cleaved by the nicking endonuclease comprises detecting the presence of fluorescent tag released from the substrate surface.
36. The method of claim 32, wherein prior to exposing the test sample comprising single stranded DNA to the nicking endonuclease and the substrate surface onto which the DNA probe is affixed, the substrate surface onto which the DNA probe is affixed is desiccated.
37. The method of claim 32, wherein exposing the test sample comprising single stranded DNA to the nicking endonuclease and the substrate surface onto which a DNA probe is affixed comprises exposing the test sample to a plurality of different beads, each comprising a substrate surface, each different substrate surface comprising a different DNA probe.
38. A DNA probe comprising a sequence complementary to a unique sequence of a target DNA molecule that also includes a recognition sequence for a nicking endonuclease, a fluorescent tag, and a fluorescence quencher, the tag and quencher being located on different sides of the recognition sequence for the nicking endonuclease, a first stem portion of the probe being capable of hybridizing to a second stem portion of the probe unless the probe is cleaved at a cut site of the nicking endonuclease, the first and second stem portions being separated by a loop portion, the tag and quencher being located in the probe such that the quencher is effective to quench fluorescent emissions of the tag when the stem portions are hybridized to each other.
39. The probe of claim 38, wherein the recognition sequence for the nicking endonuclease is located in the loop portion.
40. The probe of claim 38, wherein the recognition sequence for the nicking endonuclease is located in one stem portion and the other stem portion includes a mismatch so that the probe does not comprise a duplex recognition sequence for the nicking endonuclease.
41. A method for detecting the presence of a target nucleotide sequence in a sample of DNA; the method comprising:
- (a) exposing a test sample comprising single stranded DNA to a DNA probe according to claim 38 and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence, wherein the probe comprises a sequence complementary to the target sequence that also includes a recognition sequence for the nicking endonuclease, a fluorescent tag, and a fluorescence quencher, the tag and quencher being situated on different sides of the recognition sequence for the nicking endonuclease, a first stern portion of the probe being capable of hybridizing to a second stem portion of the probe, the first and second stem portions being separated by a loop portion, the tag and quencher being located in the probe such that the quencher is effective to quench fluorescent emissions of the tag when the stem portions are hybridized to each other; and,
- (b) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of fluorescent emissions of the fluorescent tag indicates the presence of the target nucleotide sequence in the sample DNA.
42. The method of claim 41, wherein the recognition sequence for the nicking endonuclease is located in the loop portion.
43. The method of claim 41, wherein the recognition sequence for the nicking endonuclease is located in one stem portion and the other stem portion includes a mismatch so that the probe does not comprise a duplex recognition sequence for the nicking endonuclease.
44. A method for detecting the presence of an RNA pathogen genome and optionally a DNA pathogen genome in a sample of biological material wherein the sample comprises a plurality of unpurified contaminants, the method comprising:
- (a) performing a reverse transcription procedure capable of reverse transcribing an RNA molecule in said sample of biological material into a complementary DNA molecule,
- (b) performing multiple displacement amplification to amplify DNA in the sample of biological material to form an amplified sample product;
- (c) simultaneously exposing all or part of the amplified sample product to a plurality of DNA probes directed to a plurality of different pathogens and/or pathogen strains, wherein each probe comprises a sequence complementary to a unique sequence known to be present in a transcript of a pathogen genome or a unique sequence known to be present in a DNA genome of a pathogen that also includes a recognition sequence for the nicking endonuclease; and,
- (d) observing whether the probe is cleaved by the nicking endonuclease, wherein the presence of probe cleaved by the nicking endonuclease indicates the presence of a pathogen in the sample of biological material.
45. A kit for detection and/or identification of a dengue virus comprising one or more NESA probes comprising sequences selected from among: 5′-CGTACTAGGATCACAAGAAGGA-3′, 5′-TTGGATCATAGGGTATTGGATCTA-3′, 5′-GTTGTCCTTGGATCGCAAGAGGGA-3′, 5′-GTTGTCCTTGGATCGCAAGAGGGATT-3′, 5′-AGTGCTGGGATCTCAGGAAGGA-3′ and 5′-AGTGCTGGGATCTCAGGAAGGATTTT-3′. . .
46. A method for making one or more NESA probes for the detection of a pathogen genome, the method comprising:
- (a) identifying nucleotide, sequences of about 16-25 nucleotides in a pathogen genome that include a nicking endonuclease recognition sequence,
- (b) discarding identified sequences that are not unique to the pathogen genome to produce a listing of unique identified sequences,
- (c) synthesizing one or more probes comprising unique identified sequences; and,
- (d) contacting one or more samples of biological material with said one or more synthesized probes and the nicking endonuclease in the presence of one or more common contaminants, thereby validating the synthesized probe or probes.
Type: Application
Filed: Feb 23, 2010
Publication Date: Mar 15, 2012
Applicant: Georgetown University (Washington, DC)
Inventors: Mark Danielsen (Germantown, MD), Joel Credle (Washington, DC), Eugene A. Davidson (Boynton Beach, FL), Kenneth L. Dretchen (North Potomac, MD)
Application Number: 13/202,887
International Classification: C40B 30/04 (20060101); C12Q 1/70 (20060101); C12Q 1/68 (20060101); C07H 1/00 (20060101); C07H 21/04 (20060101); C40B 40/06 (20060101);
