多重PCR检测病死鸡中沙门氏菌方法的研究
Study on the detection of Salmonella from dead chicken by multiplex PCR
作者：杨帆(四川大学生命科学学院动物疫病防控与食品安全四川省重点实验室,生物资源与生态环境教育部重点实验室,“985工程”西南资源环境与灾害防治科技创新平台)；王红宁(四川大学生命科学学院动物疫病防控与食品安全四川省重点实验室,生物资源与生态环境教育部重点实验室,“985工程”西南资源环境与灾害防治科技创新平台)；张安云(四川大学生命科学学院动物疫病防控与食品安全四川省重点实验室,生物资源与生态环境教育部重点实验室,“985工程”西南资源环境与灾害防治科技创新平台)；雷昌伟(四川大学生命科学学院动物疫病防控与食品安全四川省重点实验室,生物资源与生态环境教育部重点实验室,“985工程”西南资源环境与灾害防治科技创新平台)；张志坤(四川大学生命科学学院动物疫病防控与食品安全四川省重点实验室,生物资源与生态环境教育部重点实验室,“985工程”西南资源环境与灾害防治科技创新平台)
Author：YANG Fan(Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Provience,Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, the “985 Project” Environment and Disaster Prevention and Control Technology Innovation Platform Southwest Resources, College of Life Sciences,Sichuan University)；WANG Hong-Ning(Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Provience,Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, the “985 Project” Environment and Disaster Prevention and Control Technology Innovation Platform Southwest Resources, College of Life Sciences,Sichuan University)；ZHANG An-Yun(Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Provience,Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, the “985 Project” Environment and Disaster Prevention and Control Technology Innovation Platform Southwest Resources, College of Life Sciences,Sichuan University)；LEI Chang-Wei(Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Provience,Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, the “985 Project” Environment and Disaster Prevention and Control Technology Innovation Platform Southwest Resources, College of Life Sciences,Sichuan University)；ZHANG Zhi-Kun(Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Provience,Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, the “985 Project” Environment and Disaster Prevention and Control Technology Innovation Platform Southwest Resources, College of Life Sciences,Sichuan University)
收稿日期：2013-05-09 年卷（期）页码：2015,52(1):163-169
期刊名称：四川大学学报: 自然科学版
Journal Name：Journal of Sichuan University (Natural Science Edition)
关键字：多重PCR；沙门氏菌；鸡白痢沙门氏菌；肠炎沙门氏菌
Key words：Multiplex PCR; Salmonella; Salmonella pullorum; Salmonella enteritidis
基金项目：国家基础研究计划973项目（2013CB127200）;国家公益性行业（农业）科研专项经费项目（201303044），（201403054）;国家“现代农业产业技术体系建设专项资金”项目（CARS-41-K09）;四川省公益性科研专项（2013NZ0025）;四川省蛋鸡产业链项目（2011NZ0073）;四川省肉鸡产业链项目（2012NZ0037）
中文摘要
为了建立能够同时检测病死鸡样品中的沙门氏菌、鸡白痢沙门氏菌和肠炎沙门氏菌的多重PCR方法. 采用煮沸法提取DNA做为模板, 选择分别针对沙门氏菌属、鸡白痢沙门氏菌和肠炎沙门氏菌的特异性基因(invA、fliC、sdfⅠ)设计引物, 优化反应体系和反应参数, 建立多重PCR检测方法. 应用该方法对国内17家鸡场369份病死鸡样品进行沙门氏菌的检测, 检测结果与传统细菌培养法的结果进行比较.结果表明, 优化过的多重PCR可同时对沙门氏菌种、属进行鉴定, 灵敏度为4.6×102 CFU/mL；多重PCR方法共检测出沙门氏菌34株, 其中鸡白痢沙门氏菌21株、肠炎沙门氏菌5株, 与传统细菌培养法检测结果的符合率分别为91.2%、90.5%和80.0%.
英文摘要
To establish the multiplex PCR which could detect Salmonella, Salmonella pullorum and Salmonella enteritidis, simultaneously.The genomic DNA of bacteria was extracted by the boiling method, and the specific genes (invA, fliC, sdfⅠ) of Salmonella, Salmonella pullorum and Salmonella enteritidis were selected to disign primers for establishing the multiplex PCR method , and then the specificity and sensitivity of multiplex PCR were tested; At the same time, 369 samples from 17 farms were detected by multiplex PCR, the results of the test were compared with the traditionaldetection methods. The results showed that the Salmonella species could be tested simultaneously by the optimized multiplex PCR, its sensitivity d was 4.6×102CFU/mL; The number of Salmonella, Salmonellapullorum and Salmonella enteritidis which detected by the multiplex PCR was 34, 21 and 5 , respectively, the coincidence rate with traditional bacteria culture method was 91.2%, 90.5% and 80.0% , respectively.
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