牛传染性鼻气管炎病毒和牛副流感病毒3型的双重荧光PCR方法的建立及应用  

Establishment and application of a dual real-time PCR for simultaneous detection of infectious bovine rhinotracheitis virus and bovine parainfluenza virus type 3

作　　者：龚宏毅 汤承[1,2] 岳华 GONG Hongyi;TANG Cheng;YUE Hua(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：《中国兽医科学》2024年第4期485-491,共7页Chinese Veterinary Science

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

摘　　要：为建立同时检测牛传染性鼻气管炎病毒(IBRV)和牛副流感病毒3型(BPIV3)的方法,通过对引物和探针筛选、反应体系和条件优化,成功建立了检测IBRV和BPIV3的双重荧光定量PCR方法。该方法只能特异性地检出IBRV和BPIV3,而对其他无关病原均不能检出;批内和批间变异系数均小于3%;对IBRV和BPIV3质粒标准品的检测下限分别为15.2 copies/μL和18.3 copies/μL。用建立的方法对采自四川、重庆、河北、安徽和吉林这5个地区的143份肉牛呼吸道疾病综合征(BRDC)样本以及四川省和青海省的59份牦牛BRDC样本进行检测,结果显示,肉牛样本中IBRV和BPIV3的阳性率分别为42.66%和39.86%,混感率为22.38%;牦牛样本中IBRV和BPIV3的阳性率分别为44.07%和30.51%,混感率为18.64%。上述结果表明,本研究建立的双重荧光定量PCR方法具有特异性和稳定性好、灵敏度高的特点,适用于临床上对IBRV和BPIV3的快速检测,临床样本的检测结果丰富了国内IBRV和BPIV3的流行病学资料。In order to establish a method for simultaneous detection of infectious bovine rhino-tracheitis virus(IBRV)and bovine parainfluenza virus type 3(BPIV3),a dual real-time PCR assay was successfully established by screening primers and probes,optimizing reaction system and conditions.This method could only detect IBRV and BPIV3 specifically,but other unrelated pathogens couldn't be detected;the coefficients of variation were less than 3%for both intra-group and inter-group;and detection limits of IBRV and BPIV3 plasmids were 15.2 copies/μL and 18.3 copies/μL,respectively.Furthermore,a total of 143 samples from beef cattles with bovine respiratory disease complex(BRDC)collected from Sichuan,Chongqing,Hebei,Anhui and Jilin Provinces and 59 samples of yaks with BRDC collected from Sichuan and Qinghai were detected by the dual real-time PCR assay,and the results showed that the positive rates of IBRV and BPIV3 in beef cattles were 42.66%and 39.86%,respectively,and co-infection rate was 22.38%;while the positive rates of IBRV and BPIV3 in yaks were 44.07%and 30.51%,respectively,and the co-infection rate was 18.64%.The results in this study indicated that the dual real-time PCR assay established has the characteristics of high specificity,stability and sensitivity,making it suitable for the rapid detection of IBRV and BPIV3 from clinical samples,and the detection results enrich the epidemiological data of IBRV and BPIV3 in China.

关 键 词：牛传染性鼻气管炎病毒 牛副流感病毒3型 双重荧光定量PCR 混合感染 

分 类 号：S852[农业科学—基础兽医学]