空肠弯曲菌快速检测试剂盒研制及AOAC验证  

The Development and Validation of Campylobacter jejuni Rapid Detection Kit According to AOAC Method Validation Guidelines

作　　者：申进玲 蒋原 赵丽娜 薛峰[2] 杨捷琳 祝长青 邵景东 韩伟 Shen Jinling;Jiang Yuan;Zhao Lina;Xue Feng;Yang Jielin;Zhu Changqing;Shao Jingdong;Han Wei(Technical Center of Animal Plant and Food Inspection and Quarantine,Shanghai Customs,Shanghai 200135;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095;Nanjing Xiaozhuang University,Nanjing 211171;Zhangjiagang Technical Center of Entry-Exit Inspecton and Quarantine,Zhangjiagang 215600,Jiangsu)

机构地区：[1]上海海关动植物与食品检验检疫技术中心,上海200135 [2]南京农业大学动物医学院,南京210095 [3]南京晓庄学院,南京211171 [4]张家港海关综合技术中心,江苏张家港215600

出　　处：《中国食品学报》2020年第9期312-322,共11页Journal of Chinese Institute Of Food Science and Technology

基　　金：“十三五”国家重点研发计划项目(2018YFC1603603);上海市科委自然科学基金项目(19ZR1417500)。

摘　　要：本文自主研制出含内标的空肠弯曲菌实时荧光PCR快速检测试剂盒,并根据美国分析化学家协会(AOAC)微生物方法对试剂盒进行验证。结果表明,该试剂盒对115株目标菌和118株非目标菌的检测特异性为100%,灵敏度达6.4 CFU/mL。将试剂盒反复冻融30次,目的基因的Ct值无显著变化。稳健性测试结果显示,除了一株空肠弯曲菌对增菌液体积较为敏感外,增菌液体积、菌体裂解时间和DNA添加量均不影响其余10株空肠弯曲菌和5株同属的其它弯曲菌的检测结果。采用生产3 d、生产4个月和1年的3个批次试剂盒进行批间重复性测试和保质期测试,结果显示3批试剂盒对16株弯曲菌的检测结果一致,且11株空肠弯曲菌的Ct值无明显差异。对人工添加鸡肉、蔬菜和牛奶样品以及鸡肉实际样品检测结果显示,试剂盒方法与传统检测方法基本一致,未出现“假阴性”。试剂盒开放性好,可在不同品牌仪器上使用,含有的内部扩增对照可防止“假阴性”,可应用于食品中空肠弯曲菌的快速检测。The Campylobacter jejuni real-time PCR rapid detection kit was developed,and validated according to the AOAC microbiology method validation guidelines.The specificity of the kit was 100%for the 115 target strains and 118 non-target strains,and the sensitivity reached 6.4 CFU/mL.The Ct values for repeated freezing and thawing for 30 times didn't show significant differences.Robutstness results showed that the enrichment broth volume,heating lysis time and the DNA volume in PCR reaction didn't have any influence on the PCR result for the 11 Campylobacter jejuni and 5 non-target Campylobacter strains,except for one Campylobacter jejuni strain,for which high volume of enrichment broth can inhibit PCR reaction.The combined lot-to-lot and stability test showed the three lots(newly produced,4 months after production,1 year after production)gave consistent results for the 16 strains,and the Ct values for the 11 Campylobacter jejuni were similar.Consistent results were shown both for the kit method and conventional method in the innoculation of Campylobacter jejuni in chicken,vegetable and milk samples,as well as in practical chicken sample detection,and there was no“false negative”using the kit method.Our kit is open,can be used on various real-time PCR platforms,contained the internal amplification control which can avoid the“false negative result”,and can be applied in the rapid detection of Campylobacter jejuni from food.

关 键 词：空肠弯曲菌 实时荧光PCR AOAC方法验证 

分 类 号：TS207.4[轻工技术与工程—食品科学]