Investigation on prevalence of Listeria spp. and Listeria monocytogenes in animal-derived foods by multiplex PCR assay targeting novel genes

Chilled and frozen animal-derived food can be contaminated by Listeria spp., emerging foodborne pathogens in food industry. The objective of this study was to mine novel target genes by comparative genomics approach for multiplex PCR detection and differentiation of Listeria monocytogenes and other Listeria spp. in food. Multiplex PCR assay targeting the genetic markers LMOf2365_2721, AX25_00730, lin1814, int, lwe1673, and Oxidoreductase gene, resulted in the amplification of DNA fragments of 583 bp, 703 bp, 421 bp, 994 bp, 345 bp, and 201 bp from L. monocytogenes, Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi, respectively. The detection limits of the multiplex assays were as low as 89 fg/μL genomic DNA and 910 CFU/mL of bacterial culture. The prevalence of Listeria spp. was determined using the developed multiplex PCR assay and standard microbiological method in a total of 200 food samples collected from different supermarkets and traditional agri-product markets in Nanjing, China. A total of 28 samples were found to be positive for the presence of Listeria, including 10.9% (6/55) of livestock meat samples, 22% (11/50) of poultry samples, 15% (6/40) of shellfish samples, 13.3% (4/30) of octopus samples and 4% (1/25) of freshwater fish samples. Of these, 13 isolates were classified as L. monocytogenes, 11 were classified as L. innocua, 2 were classified as L. ivanovii and 3 were classified as L. welshimeri. These results demonstrate that the multiplex PCR assay based on novel target genes is able to rapidly detect the Listeria spp. in 12 h with high accuracy and sensitivity, which may be used in the future for detection of Listeria spp. in animal-derived food products.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

A multiplex-conventional PCR assay for bovine,ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific

A multiplex PCR assay was developed for rapid and reliable identification of bovine, ovine, caprine and fish species in feedstuffs simultaneously. The method merges the use of bovine, ovine, caprine and fish primers that amplify fragments (ovine; 119 bp, caprine; 142 bp, fish; 224 bp and bovine; 271 bp) of the mitochondrial t.glu gene forward and cyt b reverse, 12S rRNA, 12S rRNA, and ATPase subunit 8 genes respectively, and a universal 18S rRNA primers that amplifies a 99 bp from eukaryotic DNA. To evaluate the effect of heat treatment, a severe sterilization condition (133 °C at 300 kPa for 20 min) was applied. Multiplex analysis of the reference feedstuff samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this multiplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for bovine, ovine, caprine and fish species in feedstuffs.