食源性致病菌快速检测试剂盒的研制  

Study on Rapid Detection Kit of Food-borne Pathogenic Bacterias by the Multiplex PCR

作　　者：王文娟[1,2] 周明东[1,2] 姜彦君[1] 赵宏坤[1] 

机构地区：[1]青岛农业大学食品科学与工程学院山东省肉类食品质量安全工程技术研究中心,山东青岛266109 [2]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国食品学报》2012年第2期144-150,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家"十二五"科技支撑项目(2012BAK17B05)

摘　　要：目的:研制一种快速检测沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特菌的三重PCR试剂盒,并在山东泰安地区禽肉市场采样检测。方法:以沙门氏菌的invA基因、单增李斯特菌溶血素hlyA基因和金黄色葡萄球菌耐热核酸酶Nuc基因序列作为目的基因片段金黄色葡萄球菌、沙门菌和单核细胞增生李斯特菌的特异性抗原基因序列,分别设计1对引物,优化各种工作条件,建立一种多重PCR诊断试剂盒。结果:该试剂盒特异性好,敏感性高,沙门氏菌检出极限为4.7cfu/mL,单核细胞增生李斯特菌检出极限17cfu/mL,金黄色葡萄球菌检出极限4.5cfu/mL,可在5h之内得到检测结果。在山东泰安地区取样58份,其中25份样品中检出沙门氏菌阳性,带染率43.1%;6份样品中检出单增李斯特菌阳性,带染率为10.3%;3份样品中检出金黄色葡萄球菌阳性,带染率为5.2%。结论:建立的试剂盒能同时检测上述3种病原菌,可用于食品及其原料的生物安全监测,也可用于兽医临床诊断。Objective： To develope a kit for detection of Salmonella spp., Listeria monocytogenes and Sta. aureus by multiplex PCR. Methods： Characteristic genes such as invA, hlyA and Nuc were chosen as the target genes for duplex PCR system. Many factors of duplex PCR were optimized. Results： The specificity was satisfactory and the sensitivity was up to 4.7 cfu/mL of Salmonella spp., 17 cfu/mL of Listeria monocytogenes and 4.5 cfu/mL of Sta. aureus in the system. The whole test can be completed in 5 h. Salmonella was detected in 43.1% （25/58） of the samples, whereas L. monocytogenes and Sta. aureus were identified in 10.3%（6/58） and 5.2%（3/58）, respectively. Conclusion： The detection kit has the superiority in theory and practical application, and also examines three pathogens once. It can be used for food and raw materials＇ bio-safety monitoring and also can be used in clinical diagnosis.

关 键 词：多重PCR 食品检测 沙门氏菌 单增李斯特氏杆菌 金黄色葡萄球菌 

分 类 号：TS207.4[轻工技术与工程—食品科学]