成都市冷藏即食预包装熟肉制品中单核细胞增生李斯特菌定量检测及污染风险研究

Research on quantitative and contamination level of Listeria monocytogenes from pre-packaged refrigerated ready-to-eat meat in Chengdu City

目的为探明了解冷藏即食预包装熟肉制品中单核细胞增生李斯特菌(单增李斯特菌)污染情况及潜在风险,本研究针对短保质期的冷藏即食预包装熟肉制品开展单增李斯特菌定量污染风险调查。方法2022年5~8月,从成都市采集样品64份,依据采用传统培养分离鉴定和RT-PCR分子检测方法进行单增李斯特菌的定性和定量检测,分离菌株提取DNA后进行二代全基因组测序并开展相关流行病学特征分析。结果检测结果发现64份即食熟肉样品中,6份样品检出单增李斯特菌,检出份数为6/64。6份阳性样品均由某同一工厂生产,其中5份为卤肉三拼样品。定量检测结果表明,卤肉三拼的单增李斯特菌污染水平为30~200 CFU/g;单增李斯特菌阳性的猪肉样品污染水平为<10 CFU/g。阳性样品中的6株分离菌株经全基因组测序分析鉴定为单增李斯特菌,均含有LIPI-1毒力岛相关基因,多序列位点分型发现主要的序列型(ST)为ST3(n=2)和ST121(n=2),其次是ST8和ST9。单核苷酸多态性(SNP)结果分析发现同一工厂分离到的单增李斯特菌菌株LM22071911和LM22080802之间检测到23个SNP差异,单增李斯特菌株LM22062706和LM22080806之间检测到13个SNP差异,说明在该工厂加工环节存在两种克隆群的单增李斯特菌直接传播的风险。结论成都市部分市售冷藏即食预包装熟肉制品污染多种李斯特菌,需加强源头控制。

Objective In order to find out the contamination situation and potential risk of Listeria monocytogenes in refrigerated ready-to-eat prepackaged foods.This study carried out a quantitative contamination risk survey of L.monocytogenes for ready-to-eat cooked meat products with short shelf life.Methods We collected 64 samples of ready-to-eat cooked meat from Chengdu,and carried out qualitative and quantitative detection of L.monocytogenes according to traditional culture method and RT-PCR molecular detection method.Additionally,the second generation of the whole genome sequencing was performed after DNA extraction of the isolated strains,and relevant epidemiological characteristics were analyzed.Results The results showed that L.monocytogenes were isolated from 6 samples of cooked meat(6/64).All 6 L.monocytogenes positive samples were produced by the same manufactory,of which 5 were three platters of marinated meat.The results of quantitative detection showed that the contamination level of L.monocytogenes in three platters of marinated meat was 30-200 CFU/g,and the contamination level of cooked pork samples with positive L.monocytogenes was lower as<10 CFU/g.Six isolates were identified as L.monocytogenes harboring LIPI-3 virulence island related genes by whole genome sequencing analysis.In silico MLST analysis found that the main ST types were ST3(n=2)and ST121(n=2),followed by ST8 and ST9.Single nucleotide polymorphisms(SNP)results showed that 23 SNP difference was detected between L.monocytogenes strains LM22071911 and LM22080802,and 13 SNP differences were detected between L.monocytogenes strains LM22062706 and LM22080806.All four strains above were isolated from the same factory,indicating that there was a risk of direct transmission of two clonal groups of L.monocytogenes in the processing of the factory.Conclusion Some refrigerated ready-to-eat prepackaged foods sold in Chengdu are facing the contamination by L.monocytogenes,and it is necessary to strengthen the control in processing.