Multiplex PCR identification of Listeria monocytogenes isolates from milk and milk‐processing environments

Abstract

A multiplex PCR procedure based on genes iap (coding for the invasion-associated 60 kDa protein or p60) and hly (coding for listeriolysin O) of Listeria monocytogenes was to used to investigate the status of its contamination along the major milk-processing environments. Duplex PCR amplified fragments of the iap gene at about 1.45 kb from all strains of major Listeria spp. tested and a 420 bp fragment of hly from L. monocytogenes reference strains. With triplex PCR, all L. monocytogenes strains exhibited a 420 bp fragment of hly as well as a 700 bp fragment of iap instead of its 1.45kb PCR product. The tentative L. monocytogenes isolates (n = 27) out of 566 samples of milk and milk-processing environments in the PALCAM agar from cultures of buffered listeria enrichment broth were further subjected to both API and triplex PCR identification. Both methods identified the same 20 isolates as L. monocytogenes. The triplex PCR procedure detected as low as 3.2 × 10 1 cfu mL −1 of listerial cells even in the presence of L. innocua (10 8 cfu mL −1 ). It could detect 1.4 × 10 2 listerial cells mL −1 directly from milk artificially contaminated with the bacteria. Lower levels of L. monocytogenes cells in milk (1.45 × 10 1 to 1.45 × 10 0 cfu mL −1 ) could be detectable if the inoculated milk samples were cultured for 3-6 h. The bacterium was found not only in raw milk, sewage water and vessel surfaces but also in pasteurized milk.

Activation of Nanoparticles by Biosorption for E. coli Detection in Milk and Apple Juice

“…Under optimal hygienic conditions of milking and cooling equipment the number of germs (plate count) should be under 10,000 germs/mL of milk (as specified in the Food and Agriculture Organization of the United Nations). The other type of contamination comes from the consumption of nonpasteurized drink (like raw milk or apple juice) which could nevertheless pose a health risk [3]. The hazard analysis of critical control points (HACCP) system is a proactive strategy that identifies the critical control points at which these hazards can be managed.…”

lmo0038Is Involved in Acid and Heat Stress Responses and Specific forListeria monocytogenesLineages I and II, andListeria ivanovii

“…Single representative colonies were pure-cultured in BHI, and 1 mL of enriched cultures was taken for DNA extraction LMO0038 IS SPECIFIC TO PATHOGENIC LISTERIA and tested by multiplex PCR. Confirmation tests include, when necessary, sequencing of 16S rRNA, 23S rRNA, and iap as well as serotyping, virulence assay, and independent PCRs targeting L. monocytogenes-specific genes, including hly, mpl, inlB, and lmo0733 (Liu, 2006;Zeng et al, 2006;Chen et al, 2009c).…”

Capacity Building

“…sybr green i has been the most frequently used Dna-binding dye in real-Time PCr (Fairchild, lee, & Maurer, 2006). (2006) hlyA F-gCagTTgCaagCgCTTggagTgaa r-gCaaCgTaTCCTCCagagTgaTCg 420 10 4  10 2 cfu/ml milk Zeng et al (2006) iap F-CaaaCTgCTaaCaCagCTaCT r-TTaTaCgCgaCCgaagCCaa 700 10 4  10 2 cfu/ml milk Zeng et al (2006) iap F -Caa aCT gCT aaC aCa gCT aCT r -gCa CTT gaa TTg CTg TTa TTg 371 3 cfu/g cooked ground beef Klein and Juneja (1997) iap F -gggCTTTaTCCaTaaaaTa r -TTggaagaaCCTTgaTTa 453 10 1 -10 -2 cells/ml or g meat, sausages, cheese Manzano et al (1997) iap F -Caa aCT gCT aaC aCa gCT aCT r -gCa CTT gaa TTg CTg TTa TTg 371 10 1 cfu - Mukhopadhyay and Mukhopadhyay (2007) actA F -gTgaTaaaaTCgaCgaaaaTCC r -CTT gTaaaaCTagaaTCTagCg 400 or 300 4  10 -2 to 4 cfu/g italian soft cheeses longhi et al (2003) actA F -gCTgaTTTaagagaTagaggaaCa r -TTTaTgTggTTaTTTgCTgTC 827 10 1 cfu/25g pork, milk Zhou and Jiao (2005) (Continued ) The convenience and rapidity has made real-Time PCr very pertinent as an alternative to a conventional culture-based or immunobased assay for the detection of L. monocytogenes (norton, 2002).…”