基于非洲猪瘟病毒B646 L和I177L基因双重实时荧光定量PCR鉴别ASFV-G-ΔI177 L疫苗株的检测方法  

Detection Method Based on Dual Real-time Fluorescence Quantitative PCR of African Swine Fever Virus B 646 L and I 177 L Genes to Identify ASFV-G-ΔI177L Vaccine Strain

作　　者：郭瑞 韩小改 娄亚坤 杨楠 任宝红 GUO Rui;HAN Xiaogai;LOU Yakun;YANG Nan;REN Baohong(State Key Laboratory of Market Regulation(Food Safety Rapid Testing and Smart Supervision Technology),Zhengzhou 450001,China;Zhengzhou Zhongdao Biotechnology Co.,Ltd.,Zhengzhou 450001,China)

机构地区：[1]国家市场监管重点实验室(食品安全快速检测与智慧监管技术),河南郑州450001 [2]郑州中道生物技术有限公司,河南郑州450001

出　　处：《中国兽医杂志》2024年第12期52-57,共6页Chinese Journal of Veterinary Medicine

基　　金：国家市场监管重点实验室(食品安全快速检测与智慧监管技术)科研项目(ZDSYS202305)。

摘　　要：为建立一种可鉴别非洲猪瘟病毒(ASFV)野毒株与ASFV-G-ΔI177L(I177L基因缺失株)疫苗株的实时荧光定量PCR检测方法,本试验针对非洲猪瘟病毒B646L与I177L两种基因保守性高的区域,分别设计了引物和探针,通过引物、探针最佳工作浓度筛选和退火温度优化,建立了一种双重实时荧光定量PCR核酸检测方法,并验证该方法的敏感性、特异性、重复性。结果显示,该检测方法建立的标准曲线线性关系良好,R2均>0.99;对pUC57-B646L和pUC57-I177L质粒标准品最低检出限均为10 copies/μL;与其他常见猪病病原无交叉反应,特异性强;批内变异系数为0.11%~1.39%,批间变异系数为0.21%~0.96%,均小于1.4%,重复性和稳定性良好。结果表明,本试验成功建立了一种性能良好的非洲猪瘟病毒B646L与I177L基因双重实时荧光定量PCR核酸检测方法,可为临床上非洲猪瘟病毒野毒株和ASFV-G-ΔI177L疫苗株的检测和鉴别提供技术支撑。To develop a real-time fluorescence quantitative PCR method that differentiates between wild-type African swine fever virus(ASFV)and the ASFV-G-ΔI177L(I177L gene deletion strain)vaccine strain,primers and probes were designed targeting highly conserved regions of the ASFV B 646 L and I 177 L genes.This dual-target assay was optimized by screening for the optimal working concentrations of primers and probes and adjusting annealing temperatures.Sensitivity,specificity,and reproducibility of this dual real-time fluorescence quantitative PCR nucleic acid detection method were validated.The results showed a strong linear relationship in the standard curves,with R 2 values over 0.99.The detection limit was as low as 10 copies/μL for both pUC57-B646L and pUC57-I177L plasmid standards,with high specificity and no cross-reaction with other common swine pathogens.Intra-assay and inter-assay coefficients of variation were 0.11%-1.39%and 0.21%-0.96%,respectively,all below 1.4%,indicating excellent reproducibility and stability.These findings demonstrate the successful establishment of a reliable dual-target real-time fluorescence quantitative PCR method for detecting and distinguishing between wild-type ASFV and the ASFV-G-ΔI177L vaccine strain,providing valuable technical support for clinical ASFV strain differentiation.

关 键 词：非洲猪瘟病毒(ASFV) B646L基因 I177L基因 双重实时荧光定量PCR 

分 类 号：S852.651[农业科学—基础兽医学]