Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing

Abstract

A method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

1 . A method for identifying an organism based on amplified genetic sequences, comprising the steps of:
 (1) reverse transcribing RNA to DNA within a sample with a viral reverse transcriptase;   (2) initially conducting PCR using at least one outer primer having a sequence within the class:
 SEQ ID NO: 1 (AAYGGGTGAGTAACACGT) for Gram positive bacteria or, 
 SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT) for Gram negative bacteria, and 
   at least one primer having a sequence within the class:
 SEQ ID NO: 5 (CCCGRGAACGTATTCACSG), 
   wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C   (3) subsequently conducting nested PCR, using inner primers, comprising at least one primer having a sequence within the class:
 SEQ ID NO: 6 (CTACGGGAGGCWGCAGTRRGGAAT), and 
 at least one primer having a sequence within the class: 
 SEQ ID NO: 7 (WGGGTATCTAATCCTRTTTGMTCCCCW); and 
   (4) identifying the organism based on an amplified genetic sequence linked to the inner primers.   
     
     
         2 . The method according to  claim 1 , wherein the organism is unidentifiable by PCR or nested PCR if the sample is not subjected to reverse transcription. 
     
     
         3 . The method according to  claim 1 , wherein said PCR is conducted using all primers having sequences within SEQ ID NO: 1 or SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. 
     
     
         4 . The method according to  claim 1 , wherein said outer primer and inner primers comprise primers adapted to selectively amplify to a 16S ribosomal RNA sequence of the organism. 
     
     
         5 . A method for identifying an organism in a sample, the sample having inadequate DNA material characteristic of the organism to conduct the identification using PCR processes, comprising:
 (a) reverse transcribing RNA material within the sample to DNA using a Reverse Transcriptase (RNA-dependent DNA polymerase) enzyme;   (b) conducting PCR using sense and antisense primers for a first highly conserved genetic sequence generic for the class of organism using a DNA Polymerase (DNA-dependent DNA polymerase) enzyme;   (c) conducting nested PCR using sense and antisense primers for a second highly conserved genetic sequence, within the first highly conserved genetic sequence using a DNA Polymerase (DNA-dependent DNA polymerase) enzyme; and   (d) identifying the organism based on an identification of at least one characteristic amplified sequence linked to at least one of the second highly conserved sequences.   
     
     
         6 . The method according to  claim 5 , wherein said first and second highly conserved genetic sequences each comprise a part of DNA sequence corresponding to a 16S ribosomal RNA sequence of the organism. 
     
     
         7 . The method according to  claim 5 , wherein at least one of the primers are part of a degenerate set, further comprising the step of employing a plurality of primers within the class definition of the degenerate set, wherein the degenerate set specifically targets variations within one of the first and second highly conserved sequences substantially without amplifying DNA unrelated to the first or second highly conserved sequences. 
     
     
         8 . The method according to  claim 5 , wherein PCR is conducted using primers comprising:
 at least one primer within the class:
 SEQ ID NO: 1 (AAYGGGTGAGTAACACGT) and 
 SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT); and 
   at least one primer having a sequence within the class:
 SEQ ID NO: 5 (CCCGRGAACGTATTCACSG), 
   wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C   
     
     
         9 . The method according to  claim 5 , wherein nested PCR is conducted using:
 at least one primer having a sequence within the class:
 SEQ ID NO: 6 (CTACGGGAGGCWGCAGTRRGGAAT), and 
   at least one primer having a sequence within the class:
 SEQ ID NO: 7 (WGGGTATCTAATCCTRTTTGMTCCCCW), 
   wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C.   
     
     
         10 . The method according to  claim 5 , wherein said DNA Polymerase (DNA-dependent DNA polymerase) enzyme comprises a temperature resistant, DNA-dependent DNA Polymersase. 
     
     
         11 . The method according to  claim 5 , wherein:
 the PCR is conducted using all primer sequences falling within the class of at least one of:   SEQ ID NO: 1 (AAYGGGTGAGTAACACGT) and   SEQ ID NO: 8 (RAYGGGTGAGTAAYGYMT); and   and all primer sequences falling within the class:   SEQ ID NO: 5 (CCCGRGAACGTATTCACSG),   wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C.   
     
     
         12 . The method according to  claim 5 , wherein the nested PCR is conducted using:
 all primer sequences falling within the class:
 SEQ ID NO: 6 (CTACGGGAGGCWGCAGTRRGGAAT); and 
   all primer sequences falling within the class:
 SEQ ID NO: 7 (WGGGTATCTAATCCTRTTTGMTCCCCW), 
   wherein R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C.   
     
     
         13 . The method according to  claim 5 , wherein the organism is unidentifiable by PCR or nested PCR if the sample is not subjected to reverse transcription. 
     
     
         14 . The method according to  claim 5 , wherein said PCR step employs primers adapted to amplify a plurality of classes of organisms. 
     
     
         15 . A method for identifying an organism, comprising:
 (a) reverse transcribing RNA material in a sample to DNA with a reverse transcriptase (RNA-dependent DNA polymerase);   (b) conducting PCR using sense and antisense primers for a first highly conserved 16S bacterial ribosomal sequence;   (c) conducting nested PCR using sense and antisense primers for a second highly conserved 16S bacterial ribosomal sequence, within a PCR transcript derived from use of the PCR sense and antisense primers for amplifying the first highly conserved 16S bacterial ribosomal sequence; and   (d) identifying the organism based on unconserved amplified 16S bacterial ribosomal sequences within a nested PCR transcript derived from use of the nested PCR sense and antisense primers for amplifying the second highly conserved 16S bacterial ribosomal sequence,   wherein the organism is detectable even when organismal DNA in the sample is inadequate for PCR or nested PCR based identification.   
     
     
         16 . The method according to  claim 15 , wherein at least one of the primers are part of a degenerate set, further comprising the step of employing a plurality of primers within the specifications of the degenerate set. 
     
     
         17 . The method according to  claim 15 , wherein at least one of the primers are part of a degenerate set, further comprising the step of employing all primers within the specifications of the degenerate set.