牛源乳房链球菌、无乳链球菌、停乳链球菌三重实时荧光定量PCR法的建立及初步应用  

Establishment and preliminary application of a triple real-time fluorescent quantitative PCR method for bovine-derived Streptococcus uberis, Streptococcus agalactiae, and Streptococcus dysgalactiae

作　　者：郑添予 于龙政[1,3] 徐晓琪 李长春 孙兴忠 金春梅[1,3] 王楠 ZHENG Tianyu;YU Longzheng;XU Xiaoqi;LI Changchun;SUN Xingzhong;JIN Chunmei;WANG Nan(College of Agriculture,Yanbian University,Yanji 133400,China;Jilin Provincial Academy of Animal Husbandry and Veterinary Medicine,Changchun 130062,China;Ministry of Education Engineering Research Center for North-East Cold Region Beef Cattle Science&Technology Innovation,Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学农学院,吉林延吉133400 [2]吉林省畜牧兽医科学研究院,长春130062 [3]延边大学东北寒区肉牛科技创新教育部工程研究中心,吉林延吉133002

出　　处：《黑龙江畜牧兽医》2022年第12期69-73,130,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：吉林省科技发展计划项目(20180623033TC);高等学校学科创新引智计划项目(D20034)。

摘　　要：为了建立快速检测牛乳腺炎主要病原菌乳房链球菌、无乳链球菌及停乳链球菌的方法,试验根据GenBank中3种细菌的非翻译区基因设计了1对特异性引物和3个探针,建立了针对乳腺炎链球菌属3种细菌的三重实时荧光定量PCR法;对温度梯度、探针引物配比浓度进行优化,并对该方法的灵敏性和特异性进行评价;应用建立的方法对吉林省9个地区18个养殖场720份奶牛奶样进行检测。结果表明:该方法最佳反应温度为60.2℃;引物和探针浓度最佳配比扩增体系为PCR Mix 10μL,上下游引物各1μL,Uberi-P 0.8μL,Dysg-P 0.7μL,Agala-P 0.8μL,DNA模板2μL,无核酸水3.7μL。该方法对无乳链球菌、乳房链球菌、停乳链球菌核酸浓度最低检出限分别为1.2×10^(-5)ng/μL、1.4×10^(-5)ng/μL和1.0×10^(-5)ng/μL,对大肠杆菌、金黄色葡萄球菌、单增李斯特菌、粪肠球菌、克雷伯菌、沙门氏菌、产气荚膜杆菌等多种病原均无交叉反应。应用该方法对720份奶样进行检测,其中无乳链球菌检出率为3.5%(25/720),乳房链球菌检出率为1.0%(7/720),停乳链球菌检出率为2.4%(17/720)。说明建立的三重实时荧光定量PCR法具有灵敏性高、特异性强等优点,可用于奶牛乳腺炎链球菌属细菌的病原学检测。In order to construct the method of quick detection of the main pathogens of bovine mastitisStreptococcus uberis(S.uberis),Streptococcus agalactiae(S.agalactiae)andStreptococcus dysgalactiae(S.dysgalactiae),a pair of specific primers and three probes were designed based on the untranslated region gene of the three bacteria according to GenBank,and a triple real-time fluorescent quantitative PCR method for the three bacteria of mastitis-relatedStreptococcusgenus was established.The temperature gradient and probe-primer concentration ratio were optimized,and the sensitivity and specificity of the method were evaluated.The method was used to detect 720 milk samples from 18 farms in 9 regions of Jilin Province.The results showed that the optimal reaction temperature of this method was 60.2℃,and the optimal ratio system of probe and primer concentration for this method was PCR Mix 10μL,forward primer and reverse primer 1μL,Uberi-probe 0.8μL,Dysg-probe 0.7μL,Agala-probe 0.8μL,DNA template 2μL,nucleic acid-free water 3.7μL.The minimum detection limits of the nucleic acid concentration of the method were 1.2×10^(-5)ng/μL forS.agalactiae,1.4×10^(-5)ng/μL forS.uberis,and 1.0×10^(-5)ng/μL for S.dysgalactiae.The method had no cross reactivity to many pathogens such asEscherichia coli,Staphylococcus aureus,Listeria monocytogenes,Enterococcus faecalis,Klebsiella,SalmonellaandClostridium perfringens.The method was used to detect 720 milk samples,in which the detection rate ofS.agalactiaewas 3.5%(25/720);the detection rate ofS.uberiswas 1.0%(7/720),and the detection rate ofS.dysgalactiae was 2.4%(17/720).The results suggested that the established triple real-time fluorescence quantitative PCR method had the advantages of high sensitivity and strong specificity,and could be used for etiological detection of cow mastitis-relatedStreptococcusgenus.

关 键 词：奶牛 乳腺炎 链球菌 实时荧光定量PCR 流行病学调查 

分 类 号：S852.61[农业科学—基础兽医学] Q78[农业科学—兽医学]