鲜切果蔬中四种病原微生物多重PCR检测技术

摘要/Abstract

摘要： 研发了可同时检测鲜切果蔬中的单增李斯特菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌O157:H7的多重PCR检测方法。根据单增李斯特菌inlA基因、鼠伤寒沙门氏菌invA基因、大肠杆菌O157:H7 wzy、金黄色葡萄球菌的nuc基因设计及筛选出4对引物。对多重PCR反应体系及条件进行优化。结果表明：25 μL反应体系，各组分终浓度分别0.8 × PCR buffer，MgCl2为3.0 mM，dNTPs 0.25 mM，inlA、invA、wzy、nuc引物浓度分别为0.2 μM、0.4 μM、0.3 μM、0.4 μM，exTaq DNA 聚合酶为1.0 U，退火温度为56.6 °C，72 °C延伸为30 s。该反应体系对单增李斯特菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌O157:H7的检出限分别为3.5 × 106 cfu/mL，4.8 × 105 cfu/mL，1.6 × 105 cfu/mL，2.4 × 105 cfu/mL。将优化的多重PCR方法对不同接种量富集后验证，结果表明，经过9 h富集后，该方法检出限为1 cfu/mL。该方法在鲜切莴苣、鲜切黄瓜、鲜切木瓜、鲜切哈密瓜中应用同样可扩增出四条目标菌。因此，利用所建立的多重PCR方法对鲜切果蔬中侵染的病原菌检出限可达到1 cfu/g。该方法相较于传统的培养检测方法可节约大量的劳力、试剂、时间等，检测时间由原来的5-7天，缩短至9-11 h，对于企业或分析检验中心大批量样品的监测具有指导意义。

关键词: 多重PCR, 快速检测, 食源性病原微生物, 鲜切果蔬

Abstract: The objective of this study was to establish multiplex PCR detection method for Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli O157:H7 on the fresh-cut vegetables and fruits.Four pairs of specific primers were designed according to the inlA gene of L. monocytogenes, the invA gene of Salmonella typhimurium, the nuc of Staphylococcus aureus and the wzy gene of E. coli O157:H7. the result showed that 25 μL PCR system including 0.8 × PCR buffer, MgCl2 is 3.0 mM, dNTPs is 0.25 mM, inlA primer, invA primer, wzy primer, nuc primer is respectively 0.2 μM, 0.4 μM, 0.3 μM, 0.4 μM, exTaq DNA is 1.0 U. Annealing temperature at 56.6 ℃ for 30 s. The detection limite of multiplex PCR was 3.5 × 106 cfu/mL (L. monocytogenes), 4.8 × 105 cfu/mL (Salmonella typhimurium)，1.6 × 105 cfu/mL (Staphylococcus aureus)，2.4 × 105 cfu/mL (E. coli O157:H7). The different inoculation concentrations was determined using multiplex PCR, detection limit is 1 cfu/mL after 9 h enrichment. It is effectivity to applying for fresh-cut lettuce, fresh-cut cucumber, fresh-cut papaya, fresh-cut cantaloupe. Hence, the detection limit of multiplex PCR is 1 cfu/g for pathogen microorganism inoculated into fresh-cut fruits and vegetables. This method could be save labor, reagent and time compare with traditional culture method. The detection time is changing from 5-7 days to 9-11 h. This method can lay a technology foundation to detect and control the pathogenic microorganism from sample for company and analysis and detection center

Key words: Multiplex PCR, rapid detection, foodborne pathogen, fresh-cut fruits and vegetables

中图分类号:

TS207.4

冯可胡文忠姜爱丽萨仁高娃徐永平司琦马新秀. 鲜切果蔬中四种病原微生物多重PCR检测技术[J]. 食品科学, 0, (): 0-0.

Wen-zhong HU JIANG Ai-Li. Multiplex PCR Detection Method for Four Foodborne Pathogen on Fresh-cut Fruits and Vegetables[J]. FOOD SCIENCE, 0, (): 0-0.

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