鸡源细菌性腹泻病原多重PCR检测方法的建立和应用  

Establishment and application of a multiplex PCR method for thedetection of diarrheic chicken derived bacteria

作　　者：王旭涛 刘康平 崔亚男 姜朋欣 崔佳美 侯紫娟 陈宁 李茜[2] 李妍[1] WANG Xu-tao;LIU Kang-ping;CUI Ya-nan;JIANG Peng-xin;CUI Jia-mei;HOU Zi-juan;CHEN Ning;LI Qian;LI Yan(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China;Hebei Animal Husbandry and Veterinary Institute,Baoding 071000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071000 [2]河北省畜牧兽医研究所,河北保定071000

出　　处：《中国预防兽医学报》2025年第2期159-165,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省重点研发计划项目(22326615D)。

摘　　要：为建立一种可同时检测大肠杆菌O1、O2、O78和肠炎、鼠伤寒及鸡白痢沙门菌的多重PCR方法,本研究分别针对大肠杆菌O1 wxz、O2 wxz、O78 wxz基因、肠炎沙门菌prot6E基因、鼠伤寒沙门菌STM4495基因、鸡白痢沙门菌ipaJ基因设计引物,以上述6种菌DNA的等体积混合物作为模板,分别对6对引物的退火温度、浓度进行优化,初步建立了检测上述细菌的多重PCR检测方法。优化结果显示,多重PCR方法中大肠杆菌O2和O78血清型及肠炎沙门菌的最适引物浓度均为0.1μmol/L;鸡白痢沙门菌、大肠杆菌O1血清型最适引物浓度均为0.2μmol/L;鼠伤寒沙门菌最适引物浓度为0.4μmol/L,最适退火温度为58℃。提取肠炎、鼠伤寒及鸡白痢沙门菌标准株及大肠杆菌O1、O2、O78型标准菌株,链球菌、多杀性巴氏杆菌的DNA作为模板,采用建立的多重PCR检测,评估该方法的特异性。将上述6种血清型标准菌株的DNA等体积混合,10倍倍比稀释后分别作为模板,采用本研究建立的多重PCR方法扩增,以评估该多重PCR方法的敏感性。结果显示,该方法除两种菌各3种血清型标准菌株为阳性外,其余菌株均为阴性;且该方法对上述两种菌各血清型标准菌株DNA等体积混合物的检测限为1.9×10~2拷贝/μL。利用该方法和玻片凝集法分别检测河北(共40份)及四川地区(共7份)鸡源腹泻性大肠杆菌和沙门菌的流行现状。结果显示,河北地区大肠杆菌O2及O78血清型的检出率分别为25%(10/40)与20%(8/40);大肠杆菌O2、O78血清型混合感染的检出率为5%(2/40),未检测到大肠杆菌O1血清型和沙门菌;四川地区大肠杆菌O2及O78血清型的检出率均为57.1%(4/7);肠炎沙门菌的检出率为14.3%(1/7);大肠杆菌O2、O78血清型混合感染的检出率为14.3%(1/7),未检测到O1血清型大肠杆菌、鼠伤寒和鸡白痢沙门菌。玻片凝集试验结果显示,河北地区大肠杆菌O2及O78血清型的检出率分别为22.5%(9/40To establish a multiplex PCR method for simultaneous detection six serotypes of Escherichia coli(E.coli)O1,O2,O78 and enteritidis(S.enteritidis),typhimurium(S.typhimurium),and Salmonella pullorum(S.pullorum),primers were designed for E.coli O1 wxz,O2 wxz,and O78 wxz genes,the prot6E gene of S.enteritidis,the STM4495 gene of S.typhimurium,and the ipaJ gene of S.pullorum.The equal volume mixture of the above six bacteria DNA was used as the template.The annealing temperature and concentration of 6 pairs of primers were optimized,and a multiplex PCR method was established for the detection of the above bacteria.The optimization results showed that the optimal concentration of the primers was 0.1μmol/L for S.enteritidis,E.coli O2 and O78 serotypes,0.2μmol/L for S.pullorum,and E.coli O1 serotype,0.4μmol/L for S.typhimurium,and the optimal annealing temperature of the multiplex PCR primers was 58℃.The DNA of standard strains of S.enteritidis,S.typhimurium and S.pullorum,E.coli O1,O2 and O78 standard strains,Streptococcus and Pasteurella multocida were extracted as templates and the specificity of the method was evaluated by the established multiplex PCR.The DNA of each of the above six serotype standard strains was mixed in equal volume,diluted 10-fold as template and amplified using the multiplex PCR method established in this study to assess the sensitivity of the multiplex PCR method.The results showed that the method was negative except for 6 serological standard strains.The detection limit of the method for the DNA aliquot mixture of the 6 serotype standard strains was 1.9×102 copies/μL.The method and slide agglutination were used to detecting the epidemic situation of diarrheic chicken derived E.coli and Salmonella in regions of Hebei(40 samples)and Sichuan(7 samples).The results showed that the detection rates of E.coli O2 and O78 serotypes in Hebei were 25%(10/40)and 20%(8/40),respectively;the detection rate of mixed infections of E.coli O2 and O78 serotypes was 5%(2/40),and E.coli O1 serotype and Salmon

关 键 词：大肠杆菌 肠炎沙门菌 鼠伤寒沙门菌 鸡白痢沙门菌 多重PCR 

分 类 号：S852.61[农业科学—基础兽医学]