An Optimised Quantitative Real-Time PCR Assay for Listeria Monocytogenes Including and Internal Amplification Control

Abstract

The assessment of results obtained by PCR is a critical issue for its implementation as a routine tool in food microbiology diagnostics. As an analytical technique, PCR is exposed to inhibitors that can produce false negative results or underestimation in quantitative analysis. In this study, we illustrate the design, development and optimization of a duplex real-time PCR (RTi-PCR) assay for the quantitative detection of Listeria monocytogenes based on the co-amplification of a L. monocytogenes-specific gene (hly) sequence (Rodriguez-Lazaro et al., 2004a) and an internal amplification control (IAC) to evaluate PCR performance. A PCR IAC is a non-target DNA that is co-amplified with the same set of primers that target sequence. The assay was 100% specific as it allowed unambiguously detection of 49 L. monocytogenes isolates of different serotypes and sources and 104 strains of other species were negative. The detection and quantification limits were 8 and 30 cfu respectively, and PCR linearity and efficiency were R=0.997 and 0.80 respectively. Interestingly, the assay was capable of detecting underestimation of L. monocytogenes in reactions containing inhibitory culture media (e.g. Fraser and Half Fraser media) and foodstuffs (e.g. raw salmon or raw pork meat). Specific L. monocytogenes detection could also be performed using agargrown colonies and by conventional PCR. In conclusion, we present a simple and rapid duplex RTi-PCR assay for the quantitative detection of L. monocytogenes, which can also assess potential underestimations or false negative results.