多重PCR检测病死鸡中沙门氏菌方法的研究

Study on the detection of Salmonellafrom dead chicken by multiplex PCR

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%.

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 Salmo-nella 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,3 6 9 samples from 1 7 farms were detected by multiplex PCR,the results of the test were compared with the traditionaldetec-tion methods.The results showed that the Salmonella species could be tested simultaneously by the op-timized multiplex PCR,its sensitivity d was 4.6×10^2 CFU/mL;The number of Salmonella,Salmonel-lapullorum and Salmonellaenteritidis which detected by the multiplex PCR was 34,21 and 5 ,respec-tively,the coincidence rate with traditional bacteria culture method was 91.2%,90.5% and 80.0% , respectively.