传染性脾肾坏死病毒TaqMan荧光定量PCR检测方法的建立及应用  

Establishment and Application of a TaqMan Real-time PCR for Infectious Spleen and Kidney Necrosis Virus

作　　者：李望东 李勇 张淑瓶 盘润洪 蓝间媛 曾俊霞 侯月娥 Li Wangdong;Li Yong;Zhang Shuping;Pan Runhong;Lan Jianyuan;Zeng Junxia;Hou Yuee(Zhuhai Modern Agriculture Development Center,Zhuhai,Guangdong 519070,China;Zhuhai Kerric Testing Technolongy Co.Ltd.,Zhuhai,Guangdong 519070,China)

机构地区：[1]珠海市现代农业发展中心,广东珠海519070 [2]珠海科艺普检测科技有限公司,广东珠海519070

出　　处：《中国动物检疫》2023年第9期98-103,共6页China Animal Health Inspection

基　　金：珠海市现代农业产业园项目(DHH22-ZH1CFGW-241)。

摘　　要：为建立传染性脾肾坏死病毒(infection spleen and kidney necrosis virus,ISKNV)的定量快速检测方法,根据GenBank中的ISKNV衣壳蛋白(MCP)编码基因保守序列设计一对特异性引物和探针,建立了ISKNV TaqMan荧光定量PCR检测方法,并对其特异性、敏感性和重复性进行了检测。结果显示:以重组质粒标准品构建的标准曲线相关系数为0.997,具有良好的线性关系;检测下限为10 copies/μL,敏感性是常规PCR的10倍;对传染性造血器官坏死病毒、病毒性神经坏死病毒、黄颡鱼杯状病毒、大口黑鲈虹彩病毒、鳜鱼弹状病毒的检测结果均为阴性,仅ISKNV检测结果为阳性;批次内和批次间的变异系数均小于2%。应用建立的方法对已知结果的233份临床样品进行检测,检出76份阳性、157份阴性,与已知结果一致。结果表明,建立的TaqMan荧光定量PCR检测方法具有敏感、快速、特异以及可定量、重复性好等优点,可用于ISKNV感染的快速鉴别诊断、定量检测以及分子流行病学调查。In order to establish a quantitative and rapid detection method for infectious spleen and kidney necrosis virus(ISKNV),a pair of specific primers and probes were designed based on the conserved sequence of the gene encoding ISKNV capsid protein(MCP)registered in GenBank,and an TaqMan fluorescence quantitative PCR for detection of ISKNV was established,then its specificity,sensitivity,and repeatability were tested.The results showed that the correlation coefficient of the standard curve constructed with the recombinant plasmid was 0.997,with a good linear relationship;the detection limit of the established method was 10 copies/μL,and its sensitivity was 10 times that of normal PCR;it could not reacted with infectious hypodermal and haematopoietic necrosis virus(IHNV),viral nerve necrosis virus(VNNV),yellow catfish calicivirus(YCCV),largemouth bass ranavirus(LMBV)and siniperca chuatsi rhabdovirus(SCRV),and react with ISKNV only;the inter-assay and intra-assay variable coefficients were both less than 2%.233 clinical samples with known results were detected by the established method,obtaining 76 positive and 157 negative samples,which was consistent with the previous results.In conclusion,the established method,with the advantages of sensitivity,rapidity,specificity,quantification and good reproducibility,could be used for rapid differential diagnosis,quantitative detection and molecular epidemiological investigation of ISKNV infection.

关 键 词：传染性脾肾坏死病毒 TaqMan荧光定量PCR 定量检测 

分 类 号：S851.34[农业科学—预防兽医学]