| Salmonella, Escherichia coli (E. coli) O157:H7, Campylobacter jejuni (Cj) and Listeria monocytohenes (Lm) are currently four of the most important foodborne pathogens worldwide. With the purpose of investigating the prevalence of foodborne pathogens in retail fresh meats, the serotypes and drug resistant status of Salmonella isolates, the contamination of these four foodborne pathogens in retail fresh meats collected from 4 cities in Guangxi province, the serotypes and drug resistance of Salmonella isolates were detected and evaluated in the study. A rapid detection method--LAMP for Salmonella was also developed. The study was aim to provide technical supports for the hazard analysis of foodborne pathogens in retail fresh meats and the rapid detection of Salmonella.Totally 788 retail meat samples including beef, pork, chicken and duck, collected from the wet markets and supermarkets in the four cities Nanning, Yulin, Qinzhou and Baise during 2014-2015 were used to isolate and identify the four pathogens referred to the Chinese National Standard Method and the developed PCR methods. Serotyping and antimicrobial resistance against 16 kinds of drugs of the 111 Salmonella strains isolated from the samples were assayed.16 sets of PCR primers were designed to amplify 16 different drug-resistance genes of 5 different kinds of antibiotics including chloramphenicols, β-Lactams, sulfonamides, aminoglycosides and tetracyclines. The consistency between the detection results of phenotype and gene of drug-resistance was also further analyzed. LAMP primer sets targeting the gene invA were designed for the development of a rapid detection of Salmonella. LA-320C was used for real-time monitoring and calcein/MnCl2 were added for visualization of the amplification reaction. Specificity, sensitivity of the developed LAMP method and its application detection on food samples were evaluated.The results showed that Salmonella ranked the highest contamination in the total meats by 14.1%(111/788), followed by E. coli 0157:H7 (9.0%, 71/788), Cj (6.2%,49/788) and Lm (1.3%,10/788). A total of forty-two serotypes were identified among 111 Salmonella isolates, and Salmonella derby (25.2%,28/111), Salmonella agona (18.0%,20/111) and Salmonella enteritidis (7.2%,8/111) were the dominant and leave 3 untyped. Salmonella isolates showed the highest resistance to SIZ (66.7%), followed by TET (63.9%), while isolates showed the most sensitive to CIP.61.3% of the isolates were resistance to at least 3 antibiotics and 6 strains even showed resistance to at least 10 antibiotics. The resistance of Salmonella showed geologically different, and isolates from Qinzhou showed the highest resistance. Totally 10 drug-resistance genes were detected from 111 isolates and the dominant gene of chloramphenicols, (3-Lactams, sulfonamides, aminoglycosides and tetracyclines were flo, blaTEM-1 sulⅡ, strB, and tetA respectively. The consistency rates between drug-resistance gene and drug-resistance phenotype of chloramphenicols, β-Lactams, sulfonamides, aminoglycosides and tetracyclines was 78.1%,96.2%,81.1%,70.7% and 94.4% respectively. The LAMP method was found 100% specific based on the detection of 32 Salmonella strains and 6 non-Salmonella bacteria. The detection minimal limit of LAMP was 8X102 copies/μL and was 1000 times more sensitive than the regular PCR method. The consistency rate of the detection results between the LAMP method and the Chinese National Standard Method of Salmonella detection on the 100 meat samples was 94.4%, and the detection duration of the LAMP method were shortened by 48 hours when compared with the latter.The results of the investigation revealed that retail meats in the four cities were contaminated by 4 kinds of foodborne pathogens with different levels and Salmonella was the most dominant pathogen. Drug-resistance status of Salmonella isolates was severe and the successfully developed LAMP was a specific, rapid and sensitive method for the detection of Salmonella. |
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