兔多杀性巴氏杆菌和兔出血症病毒双重PCR检测方法的建立及初步应用  

Establishment and preliminary application of a duplex PCR assay for simultaneous detection of Pasteurella multocida and rabbit hemorrhagic disease virus

作　　者：王豪杰 陈见兴 王梦杰 林欢[1] 夏长友 刘家森[1] 陈洪岩[1] 朱良全[2] 张贺 王玉娥[1] WANG Hao-jie;CHEN Jian-xing;WANG Meng-jie;LIN Huan;XIA Chang-you;LIU Jia-sen;CHEN Hong-yan;ZHU Liang-quan;ZHANG He;WANG Yu-e(Laboratory Animals and Comparative Medicine Innovation Team,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室实验动物与比较医学创新团队,黑龙江哈尔滨150069 [2]中国兽医药品监察所,北京100081

出　　处：《中国预防兽医学报》2023年第2期150-155,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：科技部重点研发计划青年科学家项目(2021YFF0703100)。

摘　　要：为建立兔多杀性巴氏杆菌(Pm)和兔出血症病毒(RHDV)的双重PCR检测方法,本研究根据Pm kmt1基因和RHDV VP60基因保守区域,设计2对特异性引物,经PCR分别扩增Pm kmt1基因和RHDV VP60基因保守区域,构建重组质粒pMD-Pm和pMD-RHDV,并经菌液PCR和测序鉴定后作为重组质粒标准品。经各反应条件优化后初步建立了检测Pm和RHDV的双重PCR方法。反应条件优化结果显示,该方法的最适退火温度为59℃,扩增kmt1和VP60基因的最优引物浓度均为0.5μL(10μmol/L)。对Pm和RHDV及其他病原包括兔源支气管败血波氏杆菌、产气荚膜梭菌等20种相关病原的特异性试验结果显示,除Pm和RHDV有特异性扩增条带外,其余相关病原均无扩增条带,特异性较强。将两种重组质粒标准品分别10倍倍比稀释并等体积混合后作为模板,经该双重PCR方法扩增。结果显示,该方法对pMD-Pm的检测限为17.9拷贝/μL,对pMD-RHDV的检测限为1260拷贝/μL,敏感性较高;对两种病原的DNA和cDNA混合物的批内和批间重复性试验结果均一致,重复性较好。对100份临床患病兔肺脏样品的检测结果显示,Pm和RHDV的阳性率分别为13%(13/100)和7%(7/100),二者混合感染率为37%(37/100),与已报道的Pm和RHDV单一PCR方法的检测结果均一致。表明该方法检测的准确性较高,可以用于临床样品的检测。本研究建立的同时快速检测Pm和RHDV的双重PCR方法为临床这两种病的诊断提供了技术支持。To establish a duplex PCR method for detection of Pasteurella multocida(Pm)and rabbit hemorrhagic disease virus(RHDV),two pairs of specific primers were designed according to the conserved regions of Pm kmt1 gene and RHDV VP60 gene.The conserved regions of Pm kmt1 gene and RHDV VP60 gene were amplified by PCR respectively,and the recombinant plasmids pMD-Pm and pMD-RHDV were constructed,which were identified by bacterial liquid PCR and sequencing as plasmid standards.After optimizing the reaction conditions,a duplex PCR method for monitoring Pm and RHDV was established.The result of optimization of reaction conditions showed that the optimum annealing temperature was 59℃,and the optimum primer concentration for amplification of kmt1 and VP60 genes was 10μmol/L(0.5μL).The results of specificity test showed that the specific bands were amplified only for Pm and RHDV,and no bands were amplified for other 20 related pathogens,including Bordetella bronchiseptica of rabbit origin,Clostridium perfringens,etc.,indicating strong specificity.The results of sensitivity test showed that the detection limit of this method was 17.9 copies/μL for pMD-Pm and 1260 copies/μL for pMD-RHDV,respectively,showing high sensitivity.The intra-batch and inter-batch repeatability test results of the mixture of two genomes consistent and the repeatability was good.The detection results of 100 lung samples of clinically ill rabbits showed that the positive rates of Pm and RHDV were 13%(13/100)and 7%(7/100),respectively,and the positive rate of mixed infection was 37%(37/100),which was consistent with the reported detection results of Pm and RHDV single PCR methods.It showed that the method had high accuracy and can be used for the detection of various clinical samples.The duplex PCR method established in this study for rapid detection of rabbit pasteurellosis multocida and rabbit haemorrhagia provides technical support for clinical diagnosis of these two diseases.

关 键 词：多杀性巴氏杆菌 兔出血症病毒 双重PCR 

分 类 号：S852.6[农业科学—基础兽医学]