牛呼吸道合胞体病毒的恒温隔绝式荧光RT-PCR检测方法的建立及应用  

Establishment and application of an insulated isothermal RT-PCR for on-site detecting bovine respiratory syncytial virus

作　　者：常益铭 岳华[1,2] 汤承 CHANG Yi-ming;YUE Hua;TANG Cheng(College of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Chengdu 610041,China)

机构地区：[1]西南民族大学畜牧兽医学院,四川成都610041 [2]青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都610041

出　　处：《中国预防兽医学报》2023年第2期144-149,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十四五”国家重点研发计划课题(2021YFD1600203);国家农业产业技术体系四川肉牛创新团队专项(SCCXTD-2020-13);四川省高等学校重点实验室-动物医学实验室(2021PTJS34)。

摘　　要：为建立检测牛呼吸道合胞体病毒(BRSV)的恒温隔绝式RT-PCR(iiRT-PCR)方法,本研究根据BRSV N基因序列设计并合成一对引物和探针,通过反应体系和条件的优化,初步建立了检测BRSV的iiRT-PCR方法。利用该方法对BRSV、牛病毒性腹泻/粘膜病病毒(BVDV)、牛副流感病毒3型(BPIV-3)、牛传染性鼻气管炎病毒(IBRV)、牛冠状病毒(BCoV)、牛鼻病毒(BRV)、牛腺病毒3型(BAV-3)、多杀性巴氏杆菌(P.multocida)、溶血性曼氏杆菌(M.haemolytica)和牛支原体(M.Bovis)等临床可引起牛呼吸道疾病综合征的病原检测,结果显示,该方法仅特异性检测BRSV,对其它病原检测结果均为阴性,特异性强;将BRSV重组质粒标准品10倍倍比稀释(3.45×105拷贝/μL~3.45×10^(-1)拷贝/μL)后,利用该方法分别检测,结果显示,该方法对该重组质粒标准品的检测限为3.45拷贝/μL,敏感性高;重复性试验结果显示,批内重复性变异系数为1.92%~2.12%,批间重复性变异系数为1.67%~1.89%,重复性好。采用该iiRT-PCR方法和文献报道的3种方法同时对采自5个省146份肉牛呼吸道疾病患牛的鼻腔拭子和40份牦牛肺脏样品进行检测,结果显示,本实验建立的iiRT-PCR方法对146份鼻腔拭子中BRSV的平均检出率为36.30%,场阳性率为75.00%,5个省均有检出;牦牛肺脏样品中BRSV的检出率为45.00%,场阳性率为100%,该结果首次证实BRSV在牦牛中的存在和流行。另外本实验建立的方法对临床样品的检出率高于其他3种方法。本实验建立的iiRT-PCR方法配合使用PetNAD核酸萃取试剂盒和POCKITTM手持式核酸分析仪能够对BRSV现场检测,结果显示其与实验室检测结果一致。本研究为BRSV的快速检测提供了有利工具,丰富了国内BRSV的流行病学资料。Bovine respiratory syncytial virus(BRSV)is one of the important pathogens causing bovine respiratory disease complex(BRDC),and this study aimed to establish an insulated isothermal RT-PCR(iiRT-PCR)for detecting BRSV.Based on all N genes of BRSV in the GenBank database,a pair of primers and a fluorescent TaqMan probe were designed and synthesized.After optimizing the react system and conditions,an iiRT-PCR method for BRSV detection was preliminarily established.The assay only specifically amplifies the target fragment of BRSV and does not amplify bovine viral diarrhea virus,bovine parainfluenza virus type 3,infectious bovine rhinotracheitis virus,bovine coronavirus,bovine rhinitis virus,bovine adenovirus type 3,Pasteurellamultocida,Mannheimiahaemolytica,Mycoplasma bovis or other pathogens causing BRDC,indicating its strong specificity.After a 10-fold serial dilution(3.45×105 copies/μL-3.45×10^(-1) copies/μL)of the positive standard of BRSV recombinant plasmid,the method was used for detection.The detection limit of this assay was 3.45 copies/μL for the BRSV plasmid standard,showing a high sensitivity.Repeated experiments showed that intra-and inter-coefficients of variability were 1.92%-2.12%and 1.67%-1.89%,indicating its good repeatability.This method was conducted with PetNAD nucleic acid extraction kit and POCKITTM handheld nucleic acid analyzer,meeting the conditions for the field diagnosis of BRSV.A total of 146 nasal swabs from cattle with respiratory diseases were collected from 5 provinces during 2020-2021.Other 40 samples were collected from lung samples of Yaks by our iiRT-PCR and the other three assays reported in the literature.It was found that the detection rate of iiRT-PCR was higher than the other three assays reported in the literature.36.30%of samples tested as BRSV-positive using iiRT-PCR in 146 Nasal swabs,and the positive farm rate was 75%.The detection rate of BRSV in yak lung samples was 45.00%,and the positive farm rate was 100%.This study first confirmed the existence and prevalence o

关 键 词：牛呼吸道合胞体病毒 恒温隔绝式PCR 现场检测 

分 类 号：S852.65[农业科学—基础兽医学]