检测猪繁殖与呼吸综合征病毒及鉴别类NADC30毒株的双重荧光定量PCR方法  

Discrimination PRRSV and NADC30 strain with a duplex TaqMan real-time PCR

作　　者：周茜 于孔森 林青青 李玮卓 李文霞 余琦 王秋霞[3] 陈蕾 王永强 ZHOU Xi;YU Kong-sen;LIN Qing-qing;LI Wei-zhuo;LI Wen-xia;YU Qi;WANG Qiu-xia;CHEN Lei;WANG Yong-qiang(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;College of Animal Science and Technology,Henan University of Science and Technology,Xinxiang 453003,China;Taizhou LeilingBio Technology Co.,Ltd.,Taizhou 225300,China;Beijing Center for Disease Prevention and Control,Beijing 100013,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]扬州大学动物科学与技术学院,江苏扬州225009 [3]河南科技学院动物科技学院,河南新乡453003 [4]泰州蕾灵百奥生物科技有限公司,江苏泰州225300 [5]北京市动物疫病预防控制中心,北京100013

出　　处：《中国兽医科学》2023年第4期462-467,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32102680);江苏省科学技术厅基础研究计划项目(BK20200931)。

摘　　要：为建立可同时检测猪繁殖与呼吸综合征病毒(PRRSV)保守区和类NADC30毒株的双重TaqMan探针荧光定量PCR方法,分别针对保守的N蛋白基因和Nsp2基因设计特异性引物和探针,通过优化反应体系及反应条件对该方法的灵敏度、特异性、重复性进行评估。结果显示,双重标准曲线的相关系数(R^(2))均大于0.990,具有良好的线性关系;最低检测限分别为5.27 copies/μL和3.00 copies/μL,具有较高的灵敏度;不与其他常见猪病病原发生非特异反应,且重复性良好,批内批间变异系数均小于3%。对168份临床样本的检测结果显示,PRRSV总阳性检出率为68.45%(115/168),其中类NADC30检出率为32.14%,混合感染率为20.24%,与临床诊断结果相一致。结果表明,本方法可用于检测PRRSV和鉴别类NADC30毒株以及猪繁殖与呼吸系统综合征的流行病学调查。In order to develop a duplex TaqMan real-time PCR assay which could simultaneously detect and discriminate porcine reproductive and respiratory syndrome virus(PRRSV)and NADC30-like strain,the specific primers and probes were designed based on conserved N and Nsp2 gene,respectively.The sensitivity,specificity and reproducibility were evaluated by the optimized reaction condition.The results showed that the correlation coefficient(R^(2))of the duplex standard curves were both higher than 0.990,indicating a good linear relationship.The lowest detection limits were 5.27 and 3.00 copies,respectively.There was no non-specific reaction with common infectious diseases in pigs.Intra-and inter-batch coefficients of variation were both less than 3%.A total of 168 clinical samples detection showed that positive rate of PRRSV was 68.45%(115/168),of which NADC30-like strain was 32.14%positive,and mixed infection rate was 20.24%,which was consistent with clinical diagnosis,indicating that this method can be used to early detection of PRRSV,NADC30-like strain discrimination,and epidemiological investigation of this disease.

关 键 词：猪繁殖与呼吸综合征病毒 类NADC30毒株 TAQMAN探针 荧光定量PCR 鉴别检测 

分 类 号：S852.659.6[农业科学—基础兽医学]