Development of PCR-based detection method specific for Listeria monocytogenes in raw and ready-to-eat processed products

The isolation of Listeria spp. by enrichment in Listeria selective broth, pour plating and streak plating on selective agar plates was done. The samples consisted of 12 processed foods, 8 seafoods, one freshwater fish and four meat products. Out of a total of 339 colonies that produced black discoloration on Fraser agar, 163 was found to be gram positive. However, only 85 among these were Catalase positive. Out of the 85 isolates, only 20 showed haemolytic activity. Finally only two isolates were motile and assigned as putative Listeria monocytogenes. These two were added to the previously isolated 10 putative L. monocytogenes isolates. All 12 isolates, plus 6 reference strains were subjected to API-Listeria kit identification. Out of the 18 tests conducted, 7 showed positive identification for L. monocytogenes (one isolate gave only 96% identity but the others were above 98%). The rest were shown to be Listeria innocua (3), L.grayi (3), Listeria spp. (1) and those that show unacceptable profile (4). Genomic DNA was extracted from the putative Listeria isolates. Each genomic DNA was subjected to PCR using lis primer in which Lm2a, Lm2b, Lm3 and 027a2 showed positive bands. The latter isolates were then tested with Randomly Amplified Polymorphic DNA (RAPD) primer OPT07. Lm2a and Lm3 showed similar bands, while 027a2 has a different band pattern. When five primer sets, obtained from the literature were tested as to their specificity for L. monocytogenes detection, only two primer sets (LL1/LL4 and iapA/iapB) showed good discrimination from the other Listeria spp. The unique molecular marker for L. monocytogenes was identified through RAPD-Polymerase Chain Reaction (PCR) using primer with code 066. The molecular marker was cloned, hybridized and sequenced. Sequence analysis presented 100% homology with L. monocytogenes, proving that the isolated bacterial culture was L. monocytogenes in character. Primers were designed from the generated forward and reverse sequences of the molecular marker. The primers designed have melting temperatures of at most 60 deg C and GC contents from 45-60% which complies with the standard parameters for primer designing. Each forward and reverse primer produce a hairpin and an average of 12 self-dimers. The primers designed were initially tested during actual runs of PCR. Also, the primers can be used to develop a specific PCR-based identification kit for detection of L. monocytogenes in many food samples. The enrichment technique in Listeria broth was found adequate. Raw food samples were directly subjected to enrichment and cells that grew were washed three times with distilled water prior to cell lysis. The liquid extracts of ready-to-eat samples may contain chemical preservatives were first centrifuged at 10,000 rpm for 5 min and washed two times with sterile water prior to enrichment. Cell lysis with 1N NaOH at a final concentration of 0.1 N NaOH and five minute boiling over water bath of cells obtained in Eppendorf tube were found effective for a successful PCR reaction. A study was conducted to determine the presence of the pathogenic bacterium L. monocytogenes in selected raw and processed meat products using: (1) conventional method which involves growth in selected medium, isolation, confirmatory biochemical and molecular tests; (2) PCR using the L.monocytogenes DAS kit (BIOTECH-UPLB) and 3) Vitek Immuno Diagnostic Assay System (VIDAS) using the Enzyme Linked Flourescent Assay technique (ELFA) in some samples. The methods employed proved to be discriminatory enough in the detection and identification of Listeria from non-Listeria isolates.