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-positi ve results due to the co-amplification of contaminating genomic DNA is repo rted. Feasibility of the method was evaluated using 16S rRnrA sequences spe cific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-ba ses containing three consecutive mismatched bases near its 3' terminus (pri mer B16RT), was used for reverse transcription and in subsequent cDNA ampli fication. Specific rRNA was reverse transcribed at low temperature (40 degr ees C or 45 degrees C) in the presence of primer B16RT. As subsequent PCR u sing primer B16RT at high (62 degrees C) annealing temperatures is not near ly as efficient as amplification using the specific primer, amplification o f genomic DNA was hindered relative to the amplification of cDNA. The metho d was readily adapted to the selective amplification of mRNA of the Listeri a monocytogenes listeriolysin O (hly) gene.