猫传染性腹膜炎病毒SYBR Green实时荧光定量PCR检测方法的建立与临床应用  

Establishment and Clinical Application of a SYBR Green Real-Time Fluorescence Quantitative PCR Detection Method for Feline Infectious Peritonitis Virus

作　　者：李星颖 谢梓民 原耀贤 孙铭澮 江文康 赵明明 何诗 何静 赖健仪 白银山 陈胜锋 陈志胜 马骏 王丙云 LI Xingying;XIE Zimin;YUAN Yaoxian;SUN Minghui;JIANG Wenkang;ZHAO Mingming;HE Shi;HE Jing;LAI Jianyi;BAI Yinshan;CHEN Shengfeng;CHEN Zhisheng;MA Jun;WANG Bingyun(College of Life Science and Engineering,Foshan University,Foshan 528225,China)

机构地区：[1]佛山大学生命科学与工程学院,广东佛山528225

出　　处：《中国兽医杂志》2025年第3期44-49,共6页Chinese Journal of Veterinary Medicine

基　　金：广东省普通高校动物干细胞工程技术研究中心项目(2021GCZX006)。

摘　　要：为了提高猫传染性腹膜炎(FIP)的临床检出率,本试验根据猫传染性腹膜炎病毒(FIPV)M基因设计特异性引物,将扩增的目的基因连接到p MD19-T载体上,获得重组质粒并作为标准阳性模板,建立针对FIPV的SYBR Green实时荧光定量PCR(q PCR)检测方法,并对其特异性、敏感性、重复性和临床检出率进行验证。结果显示,本方法梯度稀释的标准品与Ct值呈良好的线性关系,R^(2)=0.999 9,扩增效率为92.4%;与其他猫常见病毒未发生交叉反应;最低检测限为3.48×10^(2) copies/μL,敏感性是普通PCR检测方法的100倍;该方法重复性好,批内和批间变异系数均小于2.4%;应用该方法对临床阳性样本的检出率为100%,阴性样本检出率为0。结果表明,本试验建立的SYBR Green q PCR检测方法特异性强、敏感性高、重复性好、临床检测效果佳,可用于FIPV的临床诊断。In order to improve the clinical detection rate of feline infectious peritonitis(FIP),this study designed specific primers targeting the M gene of feline infectious peritonitis virus(FIPV),cloned the amplified target gene into the pMD19-T vector,and obtained recombinant plasmids to be used as standard positive templates.A SYBR Green real-time fluorescence quantitative PCR(qPCR)detection method targeting FIPV was then established,and its specificity,sensitivity,reproducibility,and clinical detection rate were validated.The results showed that the standard curve of serially diluted standards exhibited a good linear relationship with Ct values(R^(2)=0.9999),and the amplification efficiency was 92.4%.There was no cross-reaction with other common feline viruses.The lowest detection limit was 3.48×10^(2) copies/μL,which is at least 100 times more sensitive than conventional PCR methods.The method demonstrated excellent reproducibility,with intra-and inter-assay coefficient of variation both less than 2.4%.In clinical application,the detection rate for positive samples was 100%,while the detection rate for negative samples was 0%.The results indicate that the established SYBR Green qPCR method is specific,highly sensitive,reproducible,and effective for clinical FIPV diagnosis.

关 键 词：猫传染性腹膜炎病毒 M基因 SYBR Green 实时荧光定量PCR(qPCR) 

分 类 号：S858.65[农业科学—临床兽医学]