Internally Controlled Multiplex Detection and Quantification of Microbial Nucleic Acids
First Claim
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1. A method for detecting and quantifying a microbial nucleic acid in a biological sample, comprising:
- a) providing a reaction mixture, comprising;
one or more quantitative standard nucleic acids,two or more primer pairs, each pair being specific for different sequence portions of the microbial nucleic acid from a single microorganism, andone or more primer pairs specific for the one or more quantitative standard nucleic acids,b) adding the biological sample to the reaction mixture,c) performing one or more cycling steps, wherein each cycling step comprises an amplifying step, wherein the amplifying step comprises producing two or more different amplification products derived from the microbial nucleic acid, if present in the biological sample, and producing one or more amplification products derived from the one or more quantitative standard nucleic acids,d) detecting and quantitating detectable signal or signals generated by the amplification products derived from the microbial nucleic acid, wherein the presence or absence of the detectable signal or signals generated by the amplification products derived from the microbial nucleic acid is indicative of the presence or absence of the microbial nucleic acid in the biological sample,e) detecting and quantitating detectable signal or signals generated by the amplification products derived from the one or more quantitative standard nucleic acids, andf) determining the quantity of the microbial nucleic acid in the biological sample by comparison of the amount of the detectable signal or signals generated by the amplification products derived from the microbial nucleic acid to the amount of the detectable signal or signals generated by the one or more amplification products derived from the one or more quantitative standard nucleic acids.
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Abstract
The present invention relates to new methods and uses for the detection and quantification of microbial nucleic acids employing an internal quantitative reference. Preferred methods are based on the amplification of nucleic acids, preferably the polymerase chain reaction. Further provided are kits comprising components for performing said methods and uses. Moreover, an analytical system for advantageously performing the method according to the invention is disclosed.
18 Citations
14 Claims
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1. A method for detecting and quantifying a microbial nucleic acid in a biological sample, comprising:
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a) providing a reaction mixture, comprising; one or more quantitative standard nucleic acids, two or more primer pairs, each pair being specific for different sequence portions of the microbial nucleic acid from a single microorganism, and one or more primer pairs specific for the one or more quantitative standard nucleic acids, b) adding the biological sample to the reaction mixture, c) performing one or more cycling steps, wherein each cycling step comprises an amplifying step, wherein the amplifying step comprises producing two or more different amplification products derived from the microbial nucleic acid, if present in the biological sample, and producing one or more amplification products derived from the one or more quantitative standard nucleic acids, d) detecting and quantitating detectable signal or signals generated by the amplification products derived from the microbial nucleic acid, wherein the presence or absence of the detectable signal or signals generated by the amplification products derived from the microbial nucleic acid is indicative of the presence or absence of the microbial nucleic acid in the biological sample, e) detecting and quantitating detectable signal or signals generated by the amplification products derived from the one or more quantitative standard nucleic acids, and f) determining the quantity of the microbial nucleic acid in the biological sample by comparison of the amount of the detectable signal or signals generated by the amplification products derived from the microbial nucleic acid to the amount of the detectable signal or signals generated by the one or more amplification products derived from the one or more quantitative standard nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. The analytical system of claim 14, further comprising a transfer module for transferring the biological sample from the sample preparation module to the reaction receptacle.
Specification