摘要
为建立一种高效准确检测猫传染性腹膜炎病毒(FIPV)的实时荧光定量PCR方法,以FIPV N基因为靶基因设计特异性探针和引物,构建质粒标准品。通过优化反应条件,建立real-time PCR方法,并进行临床检测。经方法优化,所建立方法的标准曲线为y=-3.137x+42.243,R^(2)值为0.999,扩增效率E为108.0%。该方法与猫瘟病毒(FPV)、猫疱疹病毒(FHV)和猫杯状病毒(FCV)无交叉反应,特异性良好;检测最低拷贝数为9.250 copies/μL,比常规PCR方法高出100倍;组内、组间变异系数均小于5.0%,重复性好。采用建立的方法和EvaGreen real-time PCR方法对39份临床样品进行检测,结果,检出阳性样品数分别为24和22,经Kappa检验,两者高度一致。上述结果表明,本研究成功建立了高效、敏感、特异的实时荧光定量PCR方法,可用于FIPV的临床病例检测。
In order to establish an efficient and accurate real-time PCR method for the detection of FIPV,specific probes and primers were designed with FIPV N gene as target gene,and plasmids were constructed.A real-time PCR assay was developed by optimizing the reaction system,and this method was used for the detection of clinical samples.After optimization,the standard curve of the real-time PCR was y=-3.137x+42.243 with an R^(2)value of 0.999,and the amplification efficiency was 108.0%.This method performed well with high specificity,sensitivity and repeatability.There was no cross-reac-tion with FPV,FHV and FCV.The minimum detection amounts of positive plasmids was 9.250 copies/μL,which was 100 times higher than that of the conventional PCR.The coefficient of variation between in-ter-groups and intra-groups were both less than 5.0%.The method and the Eva Green real-time PCR method were used to test 39 clinical samples,and the number of positive samples was 24 and 22,respectively,which were highly concordant using the kappa test.This study successfully established an efficient,sensitive and specific real-time PCR method that can be used to detect FIPV in clinical cases.
作者
刘妍
黄金龙
杜吉革
朱真
陈小云
王团结
罗玉峰
孙卫东
印春生
LIU Yan;HUANG Jin-long;DU Ji-ge;ZHU Zhen;CHEN Xiao-yun;WANG Tuan-jie;LUO Yu-feng;SUN Wei-dong;YIN Chun-sheng(Nanjing Agricultural University,Nanjing 210095,China;China Institute of Veterinary Drug Control,Beijing 102629,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第9期1095-1101,共7页
Chinese Veterinary Science
基金
国家重点研发计划项目(2022YFD1800603)。
作者简介
刘妍(1998-),女,山西临汾人,硕士生,研究方向为兽医临床诊断,E-mail:412870412@qq.com;通讯作者:孙卫东(1969-),男,江苏南京人,教授,博士,主要从事兽医临床诊断的研究,E-mail:swd100@njau.edu.cn;通讯作者:印春生(1977-),男,江苏响水人,研究员,博士,主要从事兽用生物制品检验、研发及相关标准物质的研制工作,E-mail:zjs62158844@sohu.com。
