Comprehensive genomic analysis of the potential limitations of several published PCR primers targeting prfA-virulence gene cluster in Listeria species.

Abstract

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes, foodborne pathogen. The present study conducted in silico genomic analysis to investigate the specificity and binding efficacy of four published pairs of PCR primers targeting Listeria prfA-virulence gene cluster (pVGC) based on Listeria sequences available. We first performed comprehensive genomic analyses of the pVGC, the main pathogenicity island in Listeria spp. In total, 2961 prfA, 642 plcB, 629 mpl, and 1181 hlyA gene sequences were retrieved from the NCBI database. Multiple sequence alignments and phylogenetic trees were generated using unique (non-identical or not-shared) sequences of each represented genes, targeting four pairs of PCR primers published previously, namely 202 prfA, 82 plcB, 150 mpl and 176 hlyA unique gene sequences. Only hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB and mpl genes showed weak (less than 50%) matching results. In addition, nucleotide variations were observed at the 3' end of the primers, indicating non-binding to the targets could potentially cause false-negative results. Thus, we propose designing degenerate primers or multiple PCR primers based on as many isolates as possible, to minimize the false-negative risk and reach the aim of low tolerable limits of detection.