叠氮丙啶-实时荧光聚合酶链式反应-高通量测序技术鉴定冷链食品中多种病原菌

Identification of multiple pathogens in cold chain foods by propidium monoazide-real time fluorescent polymerase chain reaction-high throughput sequencing technology

中文关键词:  PMA-qPCR  冷链食品  沙门氏菌  金黄色葡萄球菌  蜡样芽胞杆菌  副溶血性弧菌  单核细胞增生李斯特氏菌  生物进化树  溯源分析

英文关键词:propidium monoazide-real time fluorescent polymerase chain reaction technology  high throughput sequencing  molecular phylogenetic tree  cold chain food  pathogens

中文摘要:

      目的 应用叠氮丙啶-实时荧光聚合酶链式反应-高通量测序技术鉴定冷链食品中多种病原菌, 构建病原菌分子进化树。方法 以冷链食品中的沙门氏菌、金黄色葡萄球菌、蜡样芽胞杆菌、副溶血性弧菌、单核细胞增生李斯特氏菌5种病原菌作为研究对象, 应用叠氮丙啶-实时荧光聚合酶链式反应技术作为冷链食品中病原菌检测初筛手段, 应用微生物培养法以及生化鉴定仪器法进行方法比对与结果验证, 运用高通量测序以及分子进化树构建作为冷链食品中所分离病原菌的种属地位确证方法。结果 叠氮丙啶-实时荧光聚合酶链式反应技术成功扩增了冷链食品中生活状态病原菌的特征性核酸片段, 排除了死亡细菌以及阴性对照菌的干扰, 病原菌检出限可达到1×103 CFU/mL, 一次反应可检测42份试样, 可以在18 h内完成检测工作。在冷链食品中病原菌抽样检测调查中, 随机采集的751份冷链食品, 共检出62株病原菌, 病原菌总体检出率为8.3% (62/751)。通过后续的16S rRNA测序以及葡萄球菌属、弧菌属以及李斯特菌属分子进化树的构建, 成功溯源了金黄色葡萄球菌的污染来源并完成病原菌种属定位。结论 本方法特异性好、灵敏度高、检测通量高, 为冷链食品及相关食品中病原菌的精确检测与溯源分析提供新的思路与方法。

英文摘要:

      Objective To identify a variety of pathogenic bacteria in cold chain food by using propidium monoazide-real time fluorescent polymerase chain reaction-high throughput sequencing technology, and to construct molecular evolutionary tree of pathogenic bacteria. Methods The 5 kinds of pathogens in cold chain food, including Salmonella, Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus and Listeria monocytogenes, were taken as the research objects, and the propidium monoazide-real time fluorescent polymerase chain reaction technology was used as the primary screening method for the detection of pathogens in cold chain food. Microbial culture method and biochemical identification instrument method were used to compare the methods and verify the results. High-throughput sequencing and molecular evolutionary tree construction were used to confirm the species status of the isolated pathogens in cold chain food. Results The characteristic nucleic acid fragments of living pathogens in cold chain foods were successfully amplified by propidium monoazide-real time fluorescent polymerase chain reaction technology, eliminated the interference of dead bacteria and negative control bacteria. The limit of detection of pathogenic bacteria could reach 1×103 CFU/mL, 42 samples could be detected in one reaction, and the detection work could be completed within 18 h. In the sampling investigation of pathogens in cold chain food, 62 strains of pathogens were detected in 751 samples randomly collected, and the overall detection rate was 8.3% (62/751). Through subsequent 16S rRNA sequencing and the construction of molecular evolutionary trees of Staphylococcus, Vibrio and Listeria, the contamination sources of Staphylococcus aureus were successfully traced and the pathogenic bacteria species were located. Conclusion This method has good specificity, high sensitivity and high throughput, and provides a new idea and method for accurate detection and traceability analysis of pathogens in cold chain foods and related foods.