Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3' terminus to reduce false-positive signals

Abstract

. 2000. A reverse transcription PCR (RT±PCR) method designed to reduce false-positive results due to the co-ampli®cation of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences speci®c to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3 H terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA ampli®cation. Speci®c rRNA was reverse transcribed at low temperature (40 C or 45 C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 C) annealing temperatures is not nearly as ef®cient as ampli®cation using the speci®c primer, ampli®cation of genomic DNA was hindered relative to the ampli®cation of cDNA. The method was readily adapted to the selective ampli®cation of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.

Contrasting Expression of Rhizobial Phytase in Nodules of Two Soybean Cultivars Grown Under Low Phosphorus Availability

“…Furthermore, due to the lower affinity between this mismatched primer and genomic DNA (gDNA), it is a very inefficient primer for gDNA amplification at high annealing temperatures (in this study: 62 • C). Consequently, the mismatched primer can be used to selectively amplify the newly synthesized complementary DNA (cDNA), even in the presence of high quantities of gDNA (Koo and Jaykus, 2000).…”

Towards DNA-Based Technological Advancement in <i>Listeria Monocytogenes</i> Detection

“…RNA is quickly eliminated after the death of an organism. Therefore, reverse transcriptase PCR (RT-PCR), targeting mRNA transcripts of genes like hly, prfA and iap is used for detecting the live LM cells 10,11 . However, the time for running agarose gel to analyse and detect the amplified products is a rate-limiting step for a large number of samples.…”

Methods for the isolation and identification ofListeriaspp. andListeria monocytogenes: a review

“…In the second step the cDNA is used as template for amplification of specific sequences by PCR using target specific oligonucleotide primers and a DNA polymerase to facilitate the reaction. L. monocytogenes has been detected using this method in artificially inoculated meat samples [275] by targeting mRNA transcripts of the hly, prfA and iap genes, in waste samples [276] by targeting the transcripts for rRNA genes, and also for the detection of heat-injured L. monocytogenes by targeting the hly transcript [277].…”