Patent ID: 8877464

Claim:
A method for simultaneously detecting and quantifying a microbial nucleic acid in a biological sample, the method comprising: a) providing a reaction mixture comprising: a control nucleic acid, one or more primer pairs that hybridize to distinct sequence portions of the microbial nucleic acid and to distinct sequence portions of the control nucleic acid, and two or more probes that hybridize to each of the sequences amplified by the one or more primer pairs, wherein the microbial nucleic acid and the control nucleic acid hybridize to different probes; b) adding the biological sample to the reaction mixture; c) performing one or more cycling steps, wherein each cycling step comprises: an amplifying step comprising producing one or more amplification products derived from the microbial nucleic acid if present in the sample and producing an amplification product derived from the control nucleic acid, and a hybridizing step comprising hybridizing the amplification products with the two or more probes, wherein the two or more probes are each labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety and each of the two or more probes carries a different fluorescent moiety dye; and d) qualitatively detecting and quantitatively measuring fluorescent signals generated in step c), wherein the fluorescent signals generated by the control nucleic acid and the microbial nucleic acid are proportional to their concentration and are indicative of the detection and quantification of the microbial nucleic acid, wherein the fluorescent signal from the control nucleic acid is indicative of an amplification occurring in the amplifying step even in the absence of the fluorescent signals from the microbial nucleic acid and wherein the fluorescent signal from the control nucleic acid is analyzed at a first fluorescence threshold to obtain a quantitative result and at a second fluorescence threshold to obtain qualitative result; thereby simultaneously detecting and quantifying the microbial nucleic acid in the biological sample.