Abstract: The present invention refers to a method and a kit for performing temperature calibration in high resolution melting PCR experiments. The present invention further refers to a method for optimal calibration allowing read-out of identical or similar melting temperatures for target and calibrator. The present invention further refers to an apparatus for performing the method and a computer program for executing the method.

Abstract: The present invention provides a fluid transfer control method, the method based on measurements of intensities of dyes within the fluid to be transferred. In more detail, the present invention makes use of control dyes and quencher molecules for the fluid transfer controls.

Abstract: The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

Abstract: The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

Abstract: An analysis method for quantitative PCR experiments including multiple genes of interest, multiple samples and multiple conditions is provided. For calculating the relative expression status of multiple target genes from multiple samples under multiple different conditions, a calculation method based on up to three different parameters is performed. In addition to normalization against one or multiple reference gene(s) and normalization against a reference sample (calibrator), a third normalization step against a base value is performed in a target-, sample- and condition-specific manner.

Abstract: The invention is directed to a system for performing multi-color real time PCT, comprising a flexible real time PCT instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixture which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

Abstract: The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

Abstract: The invention is directed to a system for performing multi-color real time PCR, comprising a flexible real time PCR instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixture which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

Abstract: The present invention provides a fluid transfer control method, the method based on measurements of intensities of dyes within the fluid to be transferred. In more detail, the present invention makes use of control dyes and quencher molecules for the fluid transfer controls.

Abstract: The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

Abstract: The present invention concerns a method for determining the efficiency of the amplification of a target nucleic acid comprising the following steps: (i) preparation of a dilution series of the target nucleic acid, (ii) amplifying the target nucleic acid under defined reaction conditions, the amplification being measured in real-time (iii) setting a defined signal threshold value, (iv) determining the cycle number at which the signal threshold value is exceeded for various dilutions, (v) determining the amplification efficiency as a function of the amount of original target nucleic acid. The present invention also concerns a method for the quantification of a target nucleic acid in a sample in which the efficiency of the amplification reaction is determined in this manner and is taken into account in the quantification.

Abstract: The present invention is directed to hybridization probes hybridizing adjacently to another at a target nucleic acid sequence, wherein one member of said hybridization probes comprises (i) a nucleotide sequence entity which is substantially complementary to the sequence of the target nucleic acid, (ii) a fluorescent entity being either a FRET donor entity or a FRET acceptor entity, and (iii) a spacer entity connecting the nucleotide sequence entity and the fluorescent entity.

Abstract: The invention is directed to a system for performing multi-color real time PCT, comprising a flexible real time PCT instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixtuer which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

Abstract: The present invention concerns a method for determining the efficiency of the amplification of a target nucleic acid comprising the following steps: (i) preparation of a dilution series of the target nucleic acid, (ii) amplifying the target nucleic acid under defined reaction conditions, the amplification being measured in real-time (iii) setting a defined signal threshold value, (iv) determining the cycle number at which the signal threshold value is exceeded for various dilutions, (v) determining the amplification efficiency as a function of the amount of original target nucleic acid. The present invention also concerns a method for the quantification of a target nucleic acid in a sample in which the efficiency of the amplification reaction is determined in this manner and is taken into account in the quantification.

Abstract: The present invention is directed to a method for identification of a pathogenic organism from a predetermined group of pathogens, comprising (i) isolating a clinical sample containing at least partially purified nucleic acid, (ii) subjecting at least a first aliquot of said clinical specimen to at least one amplification and detection reaction in one reaction vessel comprising (iia) an amplification step using at least a first set of amplification primers capable of amplifying a preselected nucleic acid sequence region from several or all members of said predetermined group of pathogens, (iib) a detection step using at least 2, 3 or multiple hybridization reagents, said reagents together being capable of specifically detecting a pre-selected nucleic acid sequence region from all members of said group of pathogens, said detection step (iib) comprising steps monitoring hybridization of each of said hybridization reagents at a pre-selected temperature, said hybridization being indicative for at least the genus

Abstract: The present invention is directed to a method and a kit for amplifying and detecting a target nucleic acid, wherein the composition containing reagents to perform and monitor nucleic acid amplification in real time comprises at least a first hybridization probe labeled with a first fluorescent entity and a pyrophosphatase.

Abstract: The invention is directed to a pair of FRET hybridization probes consisting of a first oligonucleotide carrying a FRET donor entity and a second oligonucleotide carrying a FRET acceptor entity, wherein said first oligonucleotide carrying the donor fluorescent entity is carrying at least one second entity, said second entity being a compound which is capable of quenching fluorescence emission of said donor fluorescent entity. In addition, the invention is directed to methods of using the same.

Abstract: The present invention concerns a labelling reagent in which the label is bound via an amide bond and a linker to a residue of the molecule which is essentially characterized in that the N atom of the amide bond and the label are linked together directly by a covalent bond. In particular these are phosphoramidites or reactive supports suitable for nucleic acid synthesis. The invention also concerns processes for the production of such supports from suitable precursors.

Abstract: Compositions, reaction mixtures, and kits, as well as methods for their use, comprising a pair of FRET hybridization probes hybridizing adjacently to a target nucleic acid sequence, each hybridization probe comprising (i) a nucleotide sequence entity which is substantially complementary to the sequence of the target nucleic acid, (ii) a fluorescent entity, being either a FRET donor entity or a FRET acceptor entity, and (iii) a spacer entity connecting the nucleotide sequence entity and the fluorescent entity. The intensity of fluorescence emission from the FRET donor entity and from the FRET acceptor entity is not substantially affected by quenching activity of nucleotide residues present in the sequence of the target nucleic acid or in the nucleotide sequence entities of the hybridization probes.

Abstract: The present invention concerns a method for determining the efficiency of the amplification of a target nucleic acid comprising the following steps: (i) preparation of a dilution series of the target nucleic acid, (ii) amplifying the target nucleic acid under defined reaction conditions, the amplification being measured in real-time (iii) setting a defined signal threshold value, (iv) determining the cycle number at which the signal threshold value is exceeded for various dilutions, (v) determining the amplification efficiency as a function of the amount of original target nucleic acid. The present invention also concerns a method for the quantification of a target nucleic acid in a sample in which the efficiency of the amplification reaction is determined in this manner and is taken into account in the quantification.