犬瘟热病毒和犬腺病毒2型双重TaqMan荧光定量PCR检测方法的建立  

Development of a duplex TaqMan real-time PCR assay for simultaneous detection and differentiation of CDV and CAdV-2

作　　者：扈富哥 宫英杰 王蕾 莫菲 毕振威[2,4] 钱晶 孙斌[1] 谭业平 HU Fuge;GONG Yingjie;WANG Lei;MO Fei;BI Zhenwei;QIAN Jing;SUN Bin;TAN Yeping(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Nantong Yishi Biotechnology Co.,Ltd.,Nantong 226010,China;Veterinary Biological Products(Taizhou)Guotai Technology Innovation Center,Taizhou 225300,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]江苏省农业科学院兽医研究所,江苏南京210014 [3]南通伊仕生物技术股份有限公司,江苏南通226010 [4]兽用生物制品(泰州)国泰技术创新中心,江苏泰州225300

出　　处：《畜牧与兽医》2024年第9期91-95,共5页Animal Husbandry & Veterinary Medicine

基　　金：江苏省农业自主创新资金项目[CX(22)3024]。

摘　　要：旨在建立犬瘟热病毒(CDV)和犬腺病毒2型(CAdV-2)双重TaqMan荧光定量PCR(RT-qPCR)检测方法。本试验针对CDV的N基因和CAdV-2的E3基因设计两对特异性引物与两条标记不同荧光基团的TaqMan-MGB探针,对引物、探针的浓度、反应体系和反应条件进行优化后检测该方法的灵敏度,特异性及重复性。结果:该方法在CDV和CAdV-2阳性参考质粒浓度1×10^(1)~1×10^(7) copies/μL的范围中,R2值为0.997和0.993,表明所建的标准曲线具有良好线性关系,能检测到最低浓度为5 copies/μL,并对其他病原无扩增现象,特异性良好;重复性试验中组内和组间变异系数小于1.54%。综上,建立的双重TaqMan RT-qPCR检测方法具有灵敏度高且特异性好等优点,可用于CDV和CAdV-2临床快速检测和鉴别诊断及流行病学调查等领域。This study was to establish a dual TaqMan RT-qPCR method for detection of CDV and CAdV-2.Two pairs of specific primers and two TaqMan-MGB probes labeled with different fluorescent groups were designed for the N gene of CDV and for the E3 gene of CAdV-2.The concentration of the primers and probes,the reaction system and the reaction conditions were optimized;and the sensitivity,specificity and repeatability of the method were detected.The results showed that the R2 values of the method were 0.997 and 0.993 in the range of 1×10^(1)-1×10^(7)copies/μL of the positive reference plasmids of CDV and CAdV-2,indicating that the established standard curve had a good linear relationship.The lowest concentration of the method was 5 copies/μL,and there was no amplification of the other pathogens,implying good specificity.The coefficient of variation within and between the groups in the repeatability test was lower than 1.54%.It was concluded here that the established dual TaqMan RT-qPCR method possessed the advantages of high sensitivity and good specificity,which would be useful and helpful for rapid detection,differential diagnosis and epidemiological investigation of CDV and CAdV-2.

关 键 词：犬瘟热病毒 犬腺病毒2型 荧光定量PCR 检测方法 

分 类 号：S852.4[农业科学—基础兽医学]