Abstract: An imaging flow cytometer includes: a flow channel in which an observation object flows and a length in a width direction is longer than a length in a height direction; an acoustic element configured to apply acoustic waves as standing waves to the flow channel; a light source that irradiates the flow channel with illumination light; an image sensor configured to image at least a line included in a cross section of the observation object crossing a flow line direction which is a direction in which the observation object flows in the flow channel by measuring or imaging the observation object passing through a position irradiated with the illumination light; and circuitry configured to generate an image in which the observation object is scanned in the flow line direction on the basis of a plurality of captured images acquired by the imaging unit imaging the line in a time series.

Abstract: Disclosed herein are methods of identifying biomarkers (such as genes (e.g., RNA or mRNA), proteins, and/or small molecules) that can be used to predict organ or tissue function or dysfunction. In some embodiments, the methods include ex vivo perfusion of the organ or tissue, collection of samples from the organ or tissue (for example, perfusate, fluids produced by the organ (such as bile or urine), or tissue biopsies) and measuring the level of one or more biomarkers in the sample. It is also disclosed herein that an analysis of biomarkers (such as genes (e.g., RNA or mRNA), proteins, and/or small molecules) present in a biological sample from an organ, tissue, or subject can be used to identify whether the organ, tissue, or subject is at risk for (or has) organ dysfunction or organ failure.

Abstract: A diagnostic care coordination system assists a caregiver with treating a patient. The system comprises a diagnostic engine and a care engine. The diagnostic engine is configured to generate a working diagnosis of the patient. The diagnostic engine includes a static diagnosing web containing a plurality of information nodes and a dynamic diagnosing analyzer for presenting to a user a plurality of information requests. The dynamic diagnosing analyzer navigates a user through the plurality of information nodes of the static diagnosing web based upon received responses to said plurality of information requests. Based upon a route through the static diagnosing web as taken by the dynamic diagnosing analyzer, the diagnostic engine generates the working diagnosis. The care engine determines a treatment plan based upon said working diagnosis.

Abstract: Described herein are systems and methods for analyzing biological samples. Including a method for processing an analyte, comprising providing a fluidic device comprising the analyte and one or more polymer precursors; selecting a discrete area within said fluidic device; providing an energy source in optical communication with fluidic device; and selectively supplying a unit of energy generated from the energy source to the fluidic device to generate a polymer matrix within the fluidic device, wherein the polymer matrix is within the discrete area or adjacent to the discrete area.

Abstract: A configurable device arrangement for performing at least one unit operation in a biopharmaceutical process includes a base rack and a plurality of holders for releasable direct or indirect attachment of process components, in particular single-use process components, for the unit operation. The holders are in turn adapted to be releasably attached directly or indirectly to the base rack. The device arrangement further includes a positioning system which defines specific positions for the holders relative to the base rack.

Abstract: Microfluidic channels networks and systems are provided. One network includes a first fluid channel having a first depth dimension; at least a second channel intersecting the first channel at a first intersection; at least a third channel in fluid communication with the first intersection, at least one of the first intersection and the third channel having a depth dimension that is greater than the first depth dimension. Also provided is a flow control system for directing fluids in the network. Systems are additionally provided for flowing disrupted particles into a droplet formation junction, whereby a portion of the disrupted particles or the contents thereof are encapsulated into one or more droplets. Further provided is a method for controlling filling of a microfluidic network by controlling passive valving microfluidic channel network features.

Abstract: Provided herein are methods and composition for immune repertoire sequencing and single cell barcoding. In some aspects, such methods may comprise steps of: (a) forming a plurality of first vessels each comprising: (i) a single cell, and (ii) a single solid support; (b) copying onto the single solid support: (i) a first copy of a first cell polynucleotide from the single cell, and (ii) a second copy of a second cell polynucleotide from the single cell; (c) forming a plurality of second vessels each comprising (i) a single solid support from the plurality of first vessels, and (ii) a barcoded polynucleotide; and (d) amplifying (i) the first copy and the second copy with a first primer set, and (ii) the barcode with a second primer set, wherein a primer of the first primer set is complementary to a primer of the second set; and (e) forming first and second single cell barcoded sequences.

Abstract: The present disclosure relates to an automated assay system adapted to receive consumables in the conduct of an assay. The assay system includes at least one of a loading cart, a door support system, a waste storage unit, and a robotic subsystem. The loading cart is configured to organize and transport consumables necessary for the performance of the assay system. The door support system is configured to maintain stability of doors of the automated assay system. The waste storage unit is configured to provide convenient disposal of consumables associated with the automated assay system. The robotic subsystem is configured to transport consumables within the automated assay system.

Abstract: The embodiments of the present disclosure provide a method for analyzing a body fluid proteome. The method comprises: obtaining a sample to be tested I enriched with low-abundance proteins by removing high-abundance proteins in an initial sample A using an affinity technique; obtaining a sample to be tested II enriched with low-abundance proteins by removing high-abundance proteins in an initial sample B using chemical precipitation, wherein the initial sample A and the initial sample B are obtained from a same body fluid sample of a same subject; obtaining a proteome data set I by performing proteomic analysis on the sample to be tested I; obtaining a proteome data set II by performing proteomic analysis on the sample to be tested II; and determining a final quantified proteome data set of the body fluid sample based on the proteome data set I and the proteome data set II.

Abstract: A rotor comprises a support intended to be mounted rotatable around a rotation axis in a centrifuge enclosure. The support having at least one housing for receiving the sample. At least one electrically powered sensor held by the support, configured to measure a property of the sample. A contactless power receiver, configured to receive electrical power without contact from a contactless power transmitter, the contactless power receiver being carried by the support and being jointly rotatable with the support, the contactless power receiver being electrically connected to the at least one sensor to electrically power the at least one sensor during a rotation of the support.

Abstract: Described are methods for identification of likelihood of health outcomes such as the development of a medical condition using health histories from genetically related individuals. Embodiments include: receiving a first set of genetic data associated with the human subject; comparing the first set of genetic data to a plurality of sets of genetic data from a plurality of other individuals; identifying from the comparison a family network comprising individuals genetically related to the human subject as defined by identity by descent; receiving a set of health history data for each individual and each individual in the family network; analyzing the set of health history data to generate a health outcome score for the human subject, the health outcome score being a measure of risk for the human subject to develop a pre-defined health outcome that is associated with the health outcome score; and reporting the health outcome score.

Abstract: A method for identifying lung cancer status in a subject comprising: collecting a biological sample from the subject; detecting the methylation level of a biomarker gene in the biological sample, the biomarker gene being one or more selected from the following genes: BCAT1, CDH1, DCLK1, FOXL2, HOXA9, PTGER4, RARB, RASSF1A, Septin9, and SHOX2; and comparing the detected methylation levels with a normal methylation level of a corresponding biomarker gene in the population to determine the lung cancer status in the subject. Also provided herein is a kit for identifying lung cancer status in a subject.

Abstract: A method for initiating a telemedicine conference on a mobile device is provided. The method comprises receiving diagnostic test results in response to a diagnostic test, determining if the diagnostic test results include a positive result, storing the diagnostic test results on a server disposed on a network, presenting, if the diagnostic test results are positive, a telemedicine initiation option on a screen of the mobile device, determining whether the telemedicine initiation option is selected, sending the diagnostic test results from the server to the telemedicine provider, sending additional medical history information to the telemedicine provider, and initiating a telemedicine conference with the telemedicine provider. Some of these aspects also provide healthcare providers the ability to electronically send prescriptions and provide users the ability to use a mobile application to send prescriptions to pharmacies to be filled.

Abstract: The present invention relates to pancreatic cancers and the use of biomarkers in biological samples for the diagnosis of such conditions, in particular pancreatic ductal adenocarcinoma. The biomarker panel comprises LYVE1, REG1 and TFF1. Methods of treatment are also provided, as well as kits useful in the diagnosis and treatment of pancreatic ductal adenocarcinoma. The present invention is particularly useful in detecting early-stage PDAC.

Abstract: The disclosure herein relates to systems, methods, and devices for medical image analysis, diagnosis, risk stratification, decision making and/or disease tracking. In some embodiments, the systems, devices, and methods described herein are configured to analyze non-invasive medical images of a subject to automatically and/or dynamically identify one or more features, such as plaque and vessels, and/or derive one or more quantified plaque parameters, such as radiodensity, radiodensity composition, volume, radiodensity heterogeneity, geometry, location, perform computational fluid dynamics analysis, facilitate assessment of risk of heart disease and coronary artery disease, enhance drug development, determine a CAD risk factor goal, provide atherosclerosis and vascular morphology characterization, and determine indication of myocardial risk, and/or the like.

Abstract: An apparatus and method for examining an organism surface includes a pliable member including a contact layer, a plurality of channels, and a reaction layer, wherein the plurality of channels are disposed between the contact layer and the reaction layer; each of the channels having a size in the range of micrometers and formed of biocompatibility materials, and the contact layer including a plurality of spherical projections having a size in the range of micrometers and formed of biocompatible materials, and the reaction layer including examining molecules and a reaction unit.

Abstract: The present disclosure is generally directed to an apparatus for using a feedback loop to optimize meals, may include at least a processor; and a memory communicatively connected to the processor, the memory containing instructions configuring the at least a processor to retrieve nutrition data from a database. The processor may be configured to generate an optimization score, wherein generating the optimization score may include training an optimization machine-learning model, wherein the optimization machine-learning model is trained with optimization training data, inputting a nutrient quantity to the optimization machine-learning model to output a target nutrient score, and generating an optimization score as a function of the nutrition data and the target nutrient score.

Abstract: An optofluidic diagnostic system and methods for rapid analyte detections. The system comprises an optofluidic sensor array, a test plate and an optical detection cartridge. The sensor array supports one or more distinct sensor units, each having a reactor section designed to temporarily enter a series of different kinds of wells in the test plate. One kind of well is a sample reservoir that holds reagent solution to be transferred into the reactor section. Another kind of well is a drainage chamber that removes reagent solution from the reactor section. A third kind of well is a colorant reservoir that holds a colorant reagent transferable into a reactor section. Finally, the sensor unit is transferred to the optical detection cartridge where it is placed into an isolation booth during the optical detection process so that its flat observation face is stationed in a viewing window opposite an optical detector lens.

Abstract: This document provides methods and materials for identifying metastatic malignant skin lesions (e.g., malignant pigmented skin lesions). For example, methods and materials for using quantitative PCR results and correction protocols to reduce the impact of basal keratinocyte contamination on the analysis of test sample results to identify metastatic malignant skin lesions are provided. This document also provides methods and materials for treating skin cancer. For example, methods and materials for identifying a mammal (e.g., a human) having a pre-metastatic skin lesion (e.g., pre-metastatic melanoma) and treating that mammal with pentamidine (4,4?-[pentane-1,5-diylbis(oxy)]dibenzenecarboximidamide) are provided.

Abstract: Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3? end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3? of the positional domain.

Abstract: Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3? end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm2. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3? of the positional domain.

Abstract: The present disclosure provides methods and systems for nucleic acid preparation and/or analysis. Nucleic acids may be derived from one or more cells. Nucleic acid preparation may comprise generating nucleic acid molecules of varying lengths. Nucleic acid analysis may comprise identifying nucleic acid sequence information with nucleosome position information.

Abstract: The disclosure provides methods of identifying a human subject as a candidate for treating or inhibiting a liver disease by inhibiting HSD17B13. The disclosure also provides methods of treating a subject who is PNPLA3 Ile148Met+ by administering an inhibitor of HSD17B13. The disclosure also provides method of detecting a PNPLA3 Ile148Met variant and functional HSD17B13 in a subject. The disclosure also provides method of identifying a subject having a protective effect against liver disease. The disclosure also provides inhibitors of HSD17B13 for use in the treatment of a liver disease.

Abstract: Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

Abstract: Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

Abstract: The invention relates to a method for detecting a dengue infection in a patient blood sample, comprising the steps: a) Performing an analysis of prespecified parameters of blood platelets and prespecified types of blood cells in the sample and determining parameter values for the prespecified parameters of the platelets and the prespecified types of cells; b) Obtaining sample parameters from the values determined in step a); and c) Evaluating the sample parameters in relation to a prespecified criterion, wherein, if the criterion is fulfilled, a dengue infection is present.

Abstract: Described herein are systems and methods for analyzing biological samples. Including a method for processing an analyte, comprising providing a fluidic device comprising the analyte and one or more polymer precursors; selecting a discrete area within said fluidic device; providing an energy source in optical communication with fluidic device; and selectively supplying a unit of energy generated from the energy source to the fluidic device to generate a polymer matrix within the fluidic device, wherein the polymer matrix is within the discrete area or adjacent to the discrete area.

Abstract: A cosmetic composition including exosomes derived from Galactomyces as an active ingredient is provided for skin regeneration, skin elasticity improvement or skin wrinkle reduction. The cosmetic composition has excellent effects on skin regeneration, skin elasticity improvement and/or skin wrinkle reduction.

Abstract: The invention relates to methods for determining whether a subject is afflicted with a motor neuron disease, the method comprising conducting an analysis of cerebrospinal fluid and/or plasma, measuring the level of one or more sterol/oxysterol analytes, and comparing these to reference values. Further, the invention relates to methods of identifying agents suitable for the treatment of MND, and monitoring the progress of the disease.

Abstract: A micro-total analysis system and a method thereof are provided. The micro-total analysis system includes: a microfluidic device, configured to accommodate a liquid to be detected; an optical unit, configured to form a first light irradiated to the microfluidic device; and a detection unit, configured to detect the liquid to be detected and output a detection signal to obtain detection information.

Abstract: Provided are a microfluidic apparatus, a method and system for introducing a substance into a cell. The microfluidic apparatus includes a cavity channel, a bulk wave generating device and a surface acoustic wave generating device; a microstructure is arranged on an inner wall of the cavity channel, and the microstructure is constructed for forming a bubble by a solution at the microstructure when the solution is injected into the cavity channel; the bulk wave generating device is configured to generate a bulk wave, the bulk wave enables the bubble to resonate for generating a flow field; and the surface acoustic wave generating device is configured to generate a surface acoustic wave and control a position of at least one particle in the solution.

Abstract: The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect one or more of Follistatin-related protein 3, Basigin, Cathepsin B, and Tenascin as diagnostic and prognostic bio-marker assays in renal injuries.

Abstract: In one aspect of the invention, the fluidic device includes a fluidic chip includes a body having a first surface and an opposite, second surface, one or more channels formed in the body in fluidic communications with input ports and output ports for transferring one or more fluids between the input ports and the output ports, and a fluidic chip registration means formed on the first surface for aligning the fluidic chip with a support structure; and an actuator configured to engage with the one or more channels at the second surface of the body for selectively and individually transferring the one or more fluids through the one or more channels from at least one of the input ports to at least one of the output ports at desired flowrates.

Abstract: In situ-generated microfluidic capture structures incorporating a solidified polymer network, methods of preparation and use, compositions and kits therefor are described. Microfluidic capture structures may be advantageously used for assays performed within the microfluidic environment, providing flexibility in assaying micro-objects such as biological cells. Assay reagents and analytes may be incorporated within the microfluidic capture structures.

Abstract: An optical readout method for detecting a precipitate (e.g., a precipitate generated from the LAMP reaction) contained within a droplet includes generating a plurality of droplets, at least some which have a precipitate contained therein. The droplets are imaged using a brightfield imaging device. The image is subject to image processing using image processing software executed on a computing device. Image processing isolates individual droplets in the image and performs feature detection within the isolated droplets. Keypoints and information related thereto are extracted from the detected features within the isolated droplets. The keypoints are subject to a clustering operation to generate a plurality of visual “words.” The word frequency obtained for each droplet is input into a trained machine learning droplet classifier, wherein the trained machine learning droplet classifier classifies each droplet as positive for the precipitate or negative for the precipitate.

Abstract: A system for capturing a biomolecule in a single cell includes: a two-dimensional array having single cell capture holes on one surface of a substrate, and biomolecule capture areas inside the substrate each comprising a biomolecule capture member for capturing a biomolecule extracted from individual cells respectively captured by the single cell capture holes; a flow channel for flowing a sample containing cells to be assayed, from a 1st direction parallel to the surface of the substrate; a structure on the surface of the substrate and opposed to the 1st direction on the downstream side of each of the single cell capture holes; a 1st application means applying a 1st flow, and a 1st control means; and a 2nd application means applying a 2nd flow orthogonal to the one surface of the substrate toward each biomolecule capture area from each corresponding single cell capture hole, and a 2nd control means.

Abstract: A sample group forming section 24 classifies samples derived from microorganisms into groups according to empirical information showing the species or strain of each sample. A differential analysis section 27 performs a differential analysis using a peak matrix created based on the result of the grouping. An operator enters group rearrangement conditions concerning the drug resistance of microorganisms. Under the entered conditions, a sample group rearranging/rearrangement-cancelling section 25 rearranges the already formed groups by selecting or merging groups using another kind of previously registered empirical information which shows the drug resistance of each group. The differential analysis section 27 performs a differential analysis using a peak matrix newly created based on the result of the rearrangement of the groups. Thus, differential analysis results concerning the resistance to different drugs can be sequentially acquired as the group rearrangement condition is successively changed.

Abstract: Methods and devices for isolating microbial cells from a sample, extracting eukaryotic DNA from a sample, and identifying the microbial species in the sample are disclosed herein.

Abstract: Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.

Abstract: Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.

Abstract: Systems and methods are disclosed to detect single-nucleotide variations (SNVs) from somatic sources in a cell-free biological sample of a subject by generating training data with class labels; in computer memory, generating a machine learning unit comprising one output for each of adenine (A), cytosine (C), guanine (G), and thymine (T) calls; training the machine learning unit; and applying the machine learning unit to detect the SNVs from somatic sources in the cell-free biological sample of the subject, wherein the cell-free biological sample comprises a mixture of nucleic acid molecules from somatic and germline sources.

Abstract: A method and system are disclosed for detecting microbial pathogens in a sample suspected of containing the pathogens. The method includes combining loop-mediated isothermal amplification (LAMP) reagents and a polymer gel, such as a hydrogel, together with the sample to form a mixture. The gel polymerizes over a short time to immobilize the viral particles within the mixture. If target DNA/RNA are present in the sample, amplicons are produced. The target microorganisms are detected by visually detecting the presence or absence of the amplicons. The target microorganism concentrations may be determined based on the number of fluorescent amplicon dots after the reaction using a smartphone or a fluorescent microscope. The method may be employed for rapidly and inexpensively quantifying microbial pathogens in environmental water samples with high sensitivity.

Abstract: A method of preparing a conditionally active polypeptide from a parent polypeptide, comprising steps of evolving a DNA encoding the parent polypeptide by increasing a net charge of the parent polypeptide using one or more techniques selected from increasing a total number of codons of charged amino acid residues in the DNA and decreasing a total number of codons of uncharged amino acid residues in the DNA to create mutant DNAs; expressing the mutant DNAs to obtain mutant polypeptides; and selecting the conditionally active polypeptide from the mutant polypeptides which exhibits a decrease in activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition. The conditionally active polypeptide, pharmaceutical compositions containing same, nanoparticle and drug conjugates thereof and uses thereof are also provided.

Abstract: The invention relates to a method of predicting therapeutic responses to TNF blockers before anti-TNF therapy comprising analyzing immune parameters to selected stimuli in patients before therapy and its use for anti-TNF therapy. The invention relates also to a method of determining a predictive biomarker of response to anti-TNF therapy and to the use of the predictive biomarker obtained by the method.

Abstract: Compositions, methods and kits are described for identifying biomolecules (e.g., proteins and nucleic acids) expressed in a biological sample that are associated with the presence, development, or progression of a disease (such as cancer), or more generally determination of the etiology or risk factors associated with a disease. Sample types analyzed by the disclosed methods include but are not limited to archival tissue blocks that have been preserved in a fixative, tissue biopsy samples, tissue microarrays, and so forth. The methods disclosed herein correlate expression profiles of biomolecules with various disease types, and allow for the determination of relative survival rates; in some embodiments, the methods permit determination of survival rates for a subject with cancer. In other embodiments, the disclosure relates to methods for evaluating therapeutic regimes for the treatment, such as treatment of cancer.

Abstract: Systems, methods, and devices for forming an array of emulsions. An exemplary device comprises a frame and at least one or a plurality of separate microfluidic modules mounted to the frame and each configured to form an array of emulsions. In some embodiments, each module may be mounted by snap-fit attachment. The device also may include the same sealing member bonded to a top side of each module and hermetically sealing each of the modules. Another exemplary microfluidic device for forming an array of emulsions comprises a stack of layers bonded together. The stack may comprise a port layer forming a plurality of ports. Each port may have a top rim formed by a protrusion that encircles the central axis of the port. The rims may be coplanar with one another to facilitate bonding of a sealing member to each rim.

Abstract: An apparatus includes a tester to detect a biological signature of a biological sample, a processor, and a memory operably coupled to the processor. The memory stores instructions to cause the processor to receive an indication of the biological signature from the tester, and to generate, using a smart contract and through communication with a distributed ledger, a cryptographic token including a digital identifier based on the biological signature. The cryptographic token is transmitted to a remote processor for verification of the biological sample, in response to receiving the cryptographic token. The tester can detect the biological signature within a predetermined test duration that is less than a DNA sequencing duration associated with the biological sample, and the biological signature has a data precision sufficient to uniquely identify the biological sample from a plurality of biological samples.

Abstract: Embodiments of the invention include a system and method of using biomarkers in the diagnosis of lung cancer. A subject can be screened for lung cancer based on expression of specific mRNAs, miRNAs or the detection of a nucleic acid, protein, peptide or other biological molecule in blood, serum or plasma. Embodiments include 29 specific miRNAs for use as biomarkers to screen or distinguish healthy individuals from individuals affected with the disease, including a lung cancer, along with 81 additional nucleic acids, proteins or peptides for use as biomarkers to screen or distinguish healthy individuals from individuals affected with the disease, including a lung cancer. Levels of more than one of the mRNAs, miRNAs or proteins can be scored and compared to one or more threshold values to diagnose or determine the prognosis of lung cancer. Embodiments also include a kit for screening healthy subjects from subjects affected with the disease.

Abstract: Sequencing methods, devices, and systems are described. Arrays of nanoscale electronic elements comprising two electrodes separated by an insulating layer are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the insulating region. A sequencing reaction mixture comprising nucleotide analogs having impedance labels is introduced to the array of nanoscale electronic elements under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by identifying the types of labels of the nucleotide analogs that are incorporated into the growing strand using measured impedance.

Abstract: The description provides two-stage methods of nucleic acid amplification and detection reactions, which are useful for rapid pathogen detection or disease diagnosis. In particular, the description provides a method comprising a first-stage slow rate amplification reaction followed by a plurality of second-stage fast rate amplification reactions that are simultaneously monitored in real-time, and wherein a rapid rate of amplification is indicative of the presence of a site of interest.