PCR Detection of Listeria monocytogenes Using Specific Virulence Factor Genes
To investigate the novel specific genes of Listeria monocytogenes, six genes related with virulence factor of L. monocytogenes were selected from database. Primer sets were constructed from the selected six genes to evaluate the specificity of L. monocytogenes with other Listeria species. The primers were tested with 12 strains of Listeria species: L. monocytogenes (7 strains), L. welshimeri, L. innocua, L. seeligeri, L. ivanovii, and L. grayi. All primer sets showed specificity to L. monocytogenes but not with other Listeria species. These genes could be used as target genes for the detection of L. monocytogenes. Key words: Listeria monocytogenes, polymerase chain reaction (PCR), food-borne pathogen, detection, virulence factor gene. Listeria monocytogenes causes severe infections in humans, including neonatal listeriosis, endocarditis, and meningitis, with a fatality rate as high as 20 to 40%. 1) Therefore, interest in developing a fast, economical, and specific tests for the detection of L. monocytogenes in various types of food has been increasing. However, one of the major problems involved in L. monocytogenes detection is the inability to distinguish L. monocytogenes from other non-pathogenic Listeria species, particularly L. innocua which may predominate in food samples. Conventional methods for detecting L. monocytogenes involve multiple selective enrichment steps followed by biochemical and serotyping. However, these methods are time-consuming and generally require more than 2 days. 2) The advantages of polymerase chain reaction (PCR) with respects to its specificity, reliability, and rapidity helps in the detection of food-borne pathogens. Detection methods of Listeria monocytogenes using PCR have been reported using one or two target genes including hly (listeriolysin O), 3-7) iap (60-kDa secreted invasion-associated protein p60), 8-10) inlA (internalin A), 11,12) and inlB (internalin B). 12) However, this method of detection of Listeria monocytogenes using one target gene has the problems of specificity. The sequence analyses by using hly and iap as target gene of L. monocytogenes, showed strain-specific nucleotide differences among L. monocytogenes strains. 13) These problems can be addressed using several target genes for PCR detection. Analysis of PCR products can offer more precise results in the detection of L. monocytogenes. Therefore, selection of specific genes of L. monocytogenes among Listeria species is needed for PCR detection. The sequences of virulence factor genes such as prfA, plcA, hly, mpl, actA, and plcB in L. monocytogenes have been reported. 14) However, as such there are no reports regarding the specificity of these genes in Listeria species. In this study, specific genes of L. monocytogenes were selected to find unique genes for novel target of PCR. Primers from selected genes were prepared and evaluated in PCR assays for the detecting L. monocytogenes with genomic DNA extracted from pure cultured Listeria species.