Reverse Transcription PCR

Detection of pathogens in contaminated food products by PCR can result in false positive data due to amplification of DNA from nonviable cells. Most bacterial mRNA have very short half-life due to rapid degradation by endogenous RNases. As a consequence, nonviable cells that are disrupted in cellular transcription will rapidly lose their cellular mRNA. Studies were undertaken to develop a sensitive method for the detection of viable L. mono-cytogenes based on amplification of mRNA by reverse transcription PCR (RT-PCR). In a first attempt, Klein and Junega[19] isolated total RNA from L. monocytogenes samples and, following DNase treatment, amplified the RNA by RT-PCR with recombinant Thermus thermophi-lus DNA polymerase and primers specific for the iap gene. Amplicon detection was then accomplished by Southern hybridization to a digoxigenin-labeled gene probe. Viable L. monocytogenes in a cooked meat sample that was originally inoculated with ca. 3 CFU/g could be detected. A modification of the fluorogenic 5' nuclease PCR assay of Bassler et al.[5] to detect mRNA as a monitor of viable L. monocytogenes has also been developed.[20] This assay, after a DNase treatment of the sample, amplifies hlyA transcript by using the Tth DNA polymerase. The test, with primers that included the 3'-end of the transcript, was proven to be an accurate indicator of viability as measured by colony-forming unit determination.1-20-1 Koo and Jaykus have also adapted their quantitative PCR method with fluorogenic asymmetric probes for the amplification of hlyA or iap transcripts. Because of a special design of the RT-PCR primer set, they used no DNase treatment of the RNA extract prior to RT. The method utilized the AMV reverse transcriptase and Taq DNA polymerase in place of the Tth DNA polymerase for cDNA synthesis and both cDNA amplification and 5' hydrolysis of the fluorogenic probe, respectively.1-6-1 Based on a different concept, an isothermal nucleic acid sequence-based amplification assay (NASBA) was devised to detect L. monocytogenes by targeting hlyA mRNA. The generated amplicons, which incorporated biotin-labeled UTP, were then hybridized with an immobilized capture probe and detected by colorimetry.[21] Other investigators reported the development of a NASBA system amplifying Listeria 16S rRNA sequences. The latter system utilizes a hybridization method to specifically detect L. monocytogenes amplimers.[21] The specificity of the method was not tested toward L. grayi, and is not amenable to the detection of viable bacteria as, contrary to mRNA, rRNA is an extremely stable molecule.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment