犬冠状病毒和犬细小病毒双重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 canine coronavirus and canine parvovirus

作　　者：宫英杰 扈富哥 王蕾 毕振威[2,4] 钱晶 王建发[1] 谭业平 GONG Yingjie;HU Fuge;WANG Lei;BI Zhenwei;QIAN Jing;WANG Jianfa;TAN Yeping(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Nantong Engens Biotechnology Co.,Ltd.,Nantong 226010,China;Guotai(Taizhou)Center for Technology Innovation for Veterinary Biologicals,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》2025年第1期74-78,共5页Animal Husbandry & Veterinary Medicine

摘　　要：旨在建立犬冠状病毒(CCoV)和犬细小病毒(CPV)双重TaqMan荧光定量PCR检测方法。针对CCoV的3′UTR与CPV的VP2设计引物与探针,通过对反应体系与条件进行优化,建立了CCoV与CPV的双重荧光定量PCR检测方法,并对该方法进行敏感性、特异性、重复性验证与临床样品检验。结果:CCoV与CPV阳性参考质粒浓度在10^(2)~10^(7) copies/μL,CCoV与CPV的标准曲线R^(2)分别为0.999与0.996,线性关系良好,CCoV与CPV的最低检测限均为5 copies/μL,与其他病原无交叉反应,组内与组间的变异系数均小于1.38%,说明该方法灵敏度高、特异性强、重复性良好;对113份临床样本进行检测,与普通PCR比较,该方法对CCoV和CPV的检出率较高,分别为37.2%和40.7%。研究表明,本试验建立的CCoV和CPV双重荧光定量PCR方法检测效果良好,为相关疾病的临床诊断、流行病调查等提供了有力工具。A dual TaqMan fluorescence quantitative PCR assay for detection of canine coronavirus and canine parvovirus was established in this study.Primers and probes were designed for 3′UTR of CCoV and VP2 of CPV.After the reaction system and conditions were optimized,the sensitivity,specificity and repeatability of the method were verified and clinical samples were tested.The results showed that the standard curves R^(2)of CCoV and CPV positive reference plasmids ranged from 1×10^(7)copies/μL to 1×10^(2)copies/μL,and the linear relationship was good.The minimum detection limits of CCoV and CPV were both 5 copies/μL.There was no cross-reaction with other pathogens,and the co-efficient of variation within and between groups was less than 138%.The results indicated that this method had high sensitivity,strong spe-cificity and good repeatability.Compared with ordinary PCR,the detection rate of CCoV and CPV using this method was 372%higher and 407%higher.Therefore,the PCR method established in this study had a good effect and could provide help for clinical diagnosis and epide-miological investigation.

关 键 词：犬冠状病毒 犬细小病毒 双重实时荧光定量PCR TAQMAN探针 

分 类 号：S854[农业科学—临床兽医学]