摘要
为建立一种能够同时检测猪链球菌(SS)和副猪嗜血杆菌(HPS)的方法,本实验根据GenBank中登录的SS gdh和HPS 16S rRNA基因保守序列分别设计并合成引物和探针,经优化反应体系和条件,初步建立了一种可以同时鉴别检测SS和HPS的双重Taq Man荧光定量PCR方法。分别以SS、HPS、支气管败血波氏杆菌(Bb)、金黄色葡萄球菌(SA)、猪胸膜肺炎放线杆菌(APP)、无乳链球菌(GBS)基因为模板,利用该方法检测,结果显示,除SS、HPS外,与其他猪临床常见病原菌均无交叉反应,特异性强;利用建立的该双重Taq Man荧光定量PCR方法分别对10倍倍比稀释后的SS和HPS质粒标准品以及二者混合菌液检测,结果显示,该方法对SS和HPS质粒标准品的最低检测限分别为7.534×10^(1)拷贝/μL、6.350×10^(1)拷贝/μL,对细菌的最低检测限均为10~2cfu/μL,敏感性较高;组内和组间重复性试验的变异系数均小于3%,表明该方法重复性较好。利用建立的该双重Taq Man荧光定量PCR、常规PCR和国标法对在河南地区不同养猪场收集的54份疑似SS和/或HPS感染的病料样品进行检测,结果显示,双重Taq Man荧光定量PCR和常规PCR检测的阳性率分别为38.89%(21/54)和31.48%(17/54),二者的总符合率为92.59%;国标法与建立的方法检测结果一致。本研究建立的双重Taq Man荧光定量PCR方法为临床SS和HPS的鉴别检测和流行病学调查提供了有力的技术手段。
To establish a method for simultaneously detecting Streptococcus suis(SS)and Haemophilus parasuis(HPS)primers and probes were designed and synthesized based on the conservative sequence of SS gdh gene and HPS 16S rRNA gene registered in GenBank,and a duplex Taq Man real-time PCR method for simultaneous identification of SS and HPS was successfully established after optimizing the reaction system and conditions.The specificity test employed genome of SS,HPS Bordetella bronchiseptica(Bb),Staphylococcus aureus(SA),Actinobacillus pleuropneumoniae(APP)and Streptococcus agalactiae(GBS)as temples,and it showed that the method only identified SS and HPS and had no cross-reaction with other bacteria.The sensitivity test was performed to detect the continuous diluted SS and HPS recombinant plasmid standards and the bacterial cultures,it showed that the method was highly sensitive to SS and HPS with a minimum detection concentration of 7.534×10^(1)copies/μL,6.350×10^(1)copies/μL for each standard and of 10~2cfu/μL for each culture.The coefficients of variation in both intragroup and inter-group repeatability tests were less than 3%,indicating the good reproducibility of the method.Fifty-four samples of suspected SS and/or HPS infection from pig farms in Henan province were collected and tested to compare the established duplex Taq Man real-time PCR,conventional PCR and national standard method.The positive rates of the duplex Taq Man real-time PCR and conventional PCR were 38.89%(21/54)and 31.48%(17/54),respectively,and the total coincidence rate was 92.59%.The detection results of national standard method are consistent with those of established method.The established duplex Taq Man realtime PCR method provided a powerful technical means for clinical differential detection and epidemiological investigation of SS and HPS.
作者
靳曼玉
高树基
李金朋
张小玲
丁轲
董发明
汪洋
JIN Man-yu;GAO Shu-ji;LI Jin-peng;ZHANG Xiao-ling;DING Ke;DONG Fa-ming;WANG Yang(College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471000,China;Luoyang Key Laboratory of Animal Molecular Pathogens and Immunology,Luoyang 471003,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第10期1052-1058,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家十三五重点研发项目(2018YFD0500104)
国家自然科学基金项目(32172852、31902309)
河南省杰出青年科学基金项目(222300420005)。
