Qualitative and quantitative detection of microbial nucleic acids

Abstract

The present invention relates to new methods and uses for the qualitative and quantitative detection of microbial nucleic acids using at least a first control nucleic acid, or a first and a second control nucleic acid in different concentrations. The method is based on amplification of nucleic acids, for example the polymerase chain reaction. Further provided are kits comprising components for performing said methods and uses.

1 . A kit for simultaneously detecting and quantifying a microbial nucleic acid in a biological sample, the kit comprising
 a control nucleic acid which is an internal quantitative standard nucleic acid and a qualitative internal 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,   at least a first probe labeled with a first donor fluorescent moiety and a first corresponding acceptor fluorescent moiety that hybridizes to the microbial nucleic acid, and   at least a second probe labeled with a second donor fluorescent moiety and a second corresponding acceptor fluorescent moiety that hybridizes to the control nucleic acid,   wherein the fluorescent moieties provide fluorescent signals for simultaneous qualitative and quantification of the microbial nucleic acid.   
     
     
         2 . A kit for simultaneously detecting and quantifying a microbial nucleic acid in a biological sample, the kit comprising
 a first and a second control nucleic acid, wherein the first control nucleic acid is a quantitative standard nucleic acid and the second control nucleic acid is a qualitative internal control,   one or more primer pairs that hybridize to distinct sequence portions of the microbial nucleic acid and to distinct sequence portions of the first and second control nucleic acid,   at least a first probe labeled with a first donor fluorescent moiety and a first corresponding acceptor fluorescent moiety that hybridizes to the microbial nucleic acid,   at least a second probe labeled with a second donor fluorescent moiety and a second corresponding acceptor fluorescent moiety and a third corresponding acceptor fluorescent moiety that hybridizes to the control nucleic acid,   wherein the fluorescent moieties provide fluorescent signals for simultaneous qualitative and quantification of the microbial nucleic acid, and   wherein the first and second control nucleic acids are provided within one control reagent.   
     
     
         3 . The kit of  claim 2 , wherein the first control nucleic acid is present in a concentration of 20-5000 times the limit of detection of the microbial nucleic acid, and wherein the second control nucleic acid is present in a concentration of 1-25 times the limit of detection of the microbial nucleic acid. 
     
     
         4 . The kit of  claim 2 , wherein the first and the second control nucleic acid are provided within one control reagent. 
     
     
         5 . The kit of  claim 2 , wherein the first and second control nucleic acid are amplified by the same primer pair but hybridize to different probes. 
     
     
         6 . An analytical system for simultaneously detecting and quantifying a microbial nucleic acid in a biological sample, the system comprising:
 a sample preparation module comprising a lysis buffer and a vessel for isolating and purifying the microbial nucleic acid, and   an amplification and detection module comprising a reaction receptacle in which the methods performed comprise:   a) providing a reaction mixture comprising:   a first and a second control nucleic acid in different concentrations, wherein the first control nucleic acid is a quantitative standard nucleic acid and the second control nucleic acid is a qualitative internal control,   one or more primer pairs that hybridize to distinct sequence portions of the microbial nucleic acid and to distinct sequence portions of the first and second control nucleic acid, and   three or more probes that hybridize to teach of the sequences amplified by the one or more primer pairs, wherein the microbial nucleic acid and the first and second control nucleic acid each 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 first control nucleic acid and an amplification product derived from the second control nucleic acid, and   a hybridizing step comprising hybridizing the amplification products with the three or more probes, wherein the three or more probes are each labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety and each of the three 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 first 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 second 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 a first fluorescent signal from the first control nucleic acid is analyzed to obtain a quantitative result and a second fluorescent signal from the second control nucleic acid is analyzed to obtain a qualitative results;   thereby simultaneously detecting and quantifying a microbial nucleic acid in a biological sample.   
     
     
         7 . The analytical system of  claim 6 , additionally comprising a transfer module for transferring the biological sample from the sample preparation module to the reaction receptacle. 
     
     
         8 . The analytical system of  claim 6 , wherein the methods performed further comprise: in step d) determining the quantity of the microbial nucleic acid in the biological sample by comparison of the fluorescent signals generated by the microbial nucleic acid and the first control nucleic acid. 
     
     
         9 . The analytical system of  claim 6 , wherein the methods performed further comprise: in step d) detecting the microbial nucleic acid in the biological sample by comparison of the fluorescent signals generated by the microbial nucleic acid and the second control nucleic acid. 
     
     
         10 . An analytical system for simultaneously detecting and quantifying a microbial nucleic acid in a biological sample, the system comprising:
 a sample preparation module comprising a lysis buffer and a vessel for isolating and purifying the microbial nucleic acid, and   an amplification and detection module comprising a reaction receptacle in which the methods performed comprise:   a) providing a reaction mixture comprising: a control nucleic acid which is an internal quantitative standard nucleic acid and a qualitative internal 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 a qualitative result, and   wherein the second fluorescence threshold is higher than the first fluorescence threshold; thereby simultaneously detecting and quantifying the microbial nucleic acid in the biological sample.   
     
     
         11 . The analytical system of  claim 10  additionally comprising a transfer module for transferring the biological sample from the sample preparation module to the reaction receptacle. 
     
     
         12 . The analytical system of  claim 10 , wherein the methods performed further comprise: in step d) determining the quantity of the microbial nucleic acid in the biological sample by comparison of the fluorescent signals generated by the microbial nucleic acid and the control nucleic acid. 
     
     
         13 . The analytical system of  claim 10 , wherein the methods performed further comprise: in step d) detecting the microbial nucleic acid in the biological sample by comparison of the fluorescent signals generated by the microbial nucleic acid and the control nucleic acid.