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

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes , foodborne pathogen. This 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 the hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB , and mpl genes showed weak (<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. [ABSTRACT FROM AUTHOR]