Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3' terminus to reduce false-positive signals.
A reverse transcription PCR (RT-PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3' terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 degrees C or 45 degrees C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 degrees C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.
Koo
K
K
Department of Food Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC 27695-7624, USA.
Jaykus
L A
LA
D020413:3' Untranslated Regions; D001409:Bacillus cereus / Q000235:genetics / Q000254:growth & development; D001427:Bacterial Toxins; D020137:Base Pair Mismatch / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D005189:False Positive Reactions; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020413:3' Untranslated Regions; D001427:Bacterial Toxins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; C038705:hlyA protein, Listeria monocytogenes