Quantitative Assay for the Simultaneous Detection and Speciation of Bacterial Infections

Abstract

An adaptation of the real-time PCR assay allows for highly sensitive detection of any eubacterial species with simultaneous speciation. The assay relies on a ‘multiprobe’ design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair, and it is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step can be used to effectively decontaminate or remove background DNA. The real-time system reliably identifies 14 common bacterial species with a detection limit of 50 fg.

1 . A method for detecting and determining species source of eubacterial DNA in a sample, comprising:
 amplifying template DNA in a sample using a real-time polymerase chain reaction (PCR), wherein the PCR employs primers and at least two fluorogenic probes, wherein the primers if in the presence of a  S. aureus  16S rRNA gene amplify a segment of the gene comprising a conserved region and a first divergent region, wherein the conserved region comprises at least 18 contiguous nucleotides which are at least 80% identical among at least 10 eubacterial species wherein the first divergent region comprises at least 10 contiguous nucleotides and differs by at least 3 nucleotides from a second divergent region found in  Bradyrhizobium japonicum  16S rRNA gene; wherein each of the fluorogenic probes comprises a reporter dye and a quencher dye, a first of the two fluorogenic probes hybridizing to the conserved region and the second of the two fluorogenic probes hybridizing to a third divergent region of a first species of eubacteria, wherein the reporter dyes of the first and the second probes have non-overlapping emission spectra;   monitoring fluorescence emissions of the reporter dyes;   determining presence of eubacteria in the sample if emissions characteristic of the reporter dye of the first probe are detected; and   determining presence of the first species of eubacteria in the sample if emissions characteristic of the reporter dye on the second probe are detected.   
     
     
         2 . The method of  claim 1  wherein a third fluorogenic probe is employed in the real-time PCR, wherein the third fluorogenic probe hybridizes to a fourth divergent region of 16S rRNA gene in a second species of eubacteria and wherein the reporter dye of the third fluorogenic probe has a non-overlapping emission spectrum from the reporter dyes of the first and second probes. 
     
     
         3 . The method of  claim 2  wherein a fourth fluorogenic probe is employed in the real-time PCR, wherein the fourth fluorogenic probe hybridizes to a fifth divergent region of 16S rRNA gene in a third species of eubacteria and wherein the reporter dye of the fourth fluorogenic probe has a non-overlapping emission spectrum from the reporter dyes of the first, second, and third probes. 
     
     
         4 . The method of  claim 1  wherein the segment of  S. aureus  16S rRNA gene comprises nucleotides 890 to 1051. 
     
     
         5 . The method of  claim 1  wherein the conserved region of  S. aureus  16S rRNA gene comprises nucleotides 1002 to 1024. 
     
     
         6 . The method of  claim 1  wherein the first divergent region comprises nucleotides 945 to 978 of  S. aureus  16S rRNA gene. 
     
     
         7 . The method of  claim 1  wherein the sample is a treated blood sample. 
     
     
         8 . The method of  claim 7  wherein the blood sample is from a patient suspected of systemic bacteremia. 
     
     
         9 . The method of  claim 7  wherein the sample was treated to extract DNA from cells. 
     
     
         10 . The method of  claim 1  wherein the sample is urine. 
     
     
         11 . The method of  claim 1  wherein the sample is cerebrospinal fluid. 
     
     
         12 . The method of  claim 1  wherein the primers comprise primers p890F and p1033R (SEQ ID NO: 1 and 2, respectively.) 
     
     
         13 . The method of  claim 1  wherein the segment amplified is at least 125 bp. 
     
     
         14 . The method of  claim 1  wherein the segment amplified is at least 150 bp. 
     
     
         15 . The method of  claim 1  wherein the segment amplified is at least 160 bp. 
     
     
         16 . A method for detecting and determining species source of eubacterial DNA in a sample, comprising:
 filtering a real-time PCR reaction mixture to remove double stranded DNA contaminants having a length of ≧125 by to form a filtrate, wherein the PCR reaction mixture comprises primers and at least two fluorogenic probes, wherein the primers if in the presence of a  S. aureus  16S rRNA gene amplify a segment of the gene comprising a conserved region and a first divergent region, wherein the conserved region comprises at least 18 contiguous nucleotides which are at least 80% identical among at least 10 eubacterial species, wherein the first divergent region comprises at least 10 contiguous nucleotides and differs by at least 3 nucleotides from a second divergent region of  Bradyrhizobium  japonicum 16S rRNA gene, wherein each of the probes comprises a reporter dye and a quencher dye, a first of the two fluorogenic probes hybridizing to the conserved region and the second of the two fluorogenic probes hybridizing to a third divergent region of a first eubacterial species, wherein the reporter dyes of the first and the second probes have non-overlapping emission spectra;   adding a sample comprising template DNA to the filtrate;   amplifying template DNA in the filtrate;   monitoring fluorescence emissions of the reporter dyes;   determining presence of eubacteria in the sample if emissions characteristic of the reporter dye of the first probe are detected; and   determining presence of the first species of eubacteria in the sample if emissions characteristic of the reporter dye on the second probe are detected.   
     
     
         17 . The method of  claim 16  wherein a third fluorogenic probe is present in the real-time PCR reaction mixture, wherein the third fluorogenic probe hybridizes to a fourth divergent region of a 16S rRNA gene in a second species of eubacteria and wherein the reporter dye of the third fluorogenic probe has a non-overlapping emission spectrum from the first and second probes. 
     
     
         18 . The method of  claim 17  wherein a fourth fluorogenic probe is present in the real-time PCR reaction mixture, wherein the fourth fluorogenic probe hybridizes to a fifth divergent region of a 16S rRNA gene in a third species of eubacteria and wherein the reporter dye of the fourth fluorogenic probe has a non-overlapping emission spectrum from the first, second, and third probes. 
     
     
         19 . The method of  claim 16  wherein the segment comprises nucleotides 890 to 1051 of  S. aureus  16S rRNA gene. 
     
     
         20 . The method of  claim 16  wherein the conserved region comprises nucleotides 1002 to 1024 of  S. aureus  16S rRNA gene. 
     
     
         21 . The method of  claim 16  wherein the first divergent region comprises nucleotides 945 to 978 of  S. aureus  16S rRNA gene. 
     
     
         22 . The method of  claim 16  wherein the sample is a treated blood sample. 
     
     
         23 . The method of  claim 22  wherein the blood sample is from a patient suspected of systemic bacteremia. 
     
     
         24 . The method of  claim 22  wherein the sample was treated to extract DNA therefrom. 
     
     
         25 . The method of  claim 16  wherein the sample is urine. 
     
     
         26 . The method of  claim 16  wherein the sample is cerebrospinal fluid. 
     
     
         27 . The method of  claim 16  wherein the primers comprise primers p890F and p1033R (SEQ ID NO: 1 and 2, respectively.) 
     
     
         28 . The method of  claim 16  wherein the segment amplified is at least 125 bp. 
     
     
         29 . The method of  claim 16  wherein the segment amplified is at least 150 bp. 
     
     
         30 . The method of  claim 16  wherein the segment amplified is at least 160 bp. 
     
     
         31 . The method of  claim 16  wherein the conserved region is at least 80% identical among at least 14 eubacterial species. 
     
     
         32 . The method of  claim 1  wherein the conserved region is at least 80% identical among at least 14 eubacterial species. 
     
     
         33 . The method of  claim 1  wherein the first divergent region comprises at least 15 contiguous nucleotides. 
     
     
         34 . The method of  claim 1  wherein the first divergent region comprises at least 20 contiguous nucleotides. 
     
     
         35 . The method of  claim 1  wherein the first divergent region comprises at least 25 contiguous nucleotides. 
     
     
         36 . The method of  claim 1  wherein the first divergent region comprises at least 30 contiguous nucleotides. 
     
     
         37 . The method of  claim 16  wherein the first divergent region comprises at least 15 contiguous nucleotides. 
     
     
         38 . The method of  claim 16  wherein the first divergent region comprises at least 20 contiguous nucleotides. 
     
     
         39 . The method of  claim 16  wherein the first divergent region comprises at least 25 contiguous nucleotides. 
     
     
         40 . The method of  claim 16  wherein the first divergent region comprises at least 30 contiguous nucleotides. 
     
     
         41 . The method of  claim 1  wherein the first divergent region differs by at least 4 nucleotides from the second divergent region. 
     
     
         42 . The method of  claim 16  wherein the first divergent region differs by at least 4 nucleotides from the second divergent region.