摘要
基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)和荧光定量PCR技术建立一种猪伪狂犬病病毒(pseudorabies virus,PRV)LAMP-Taqman快速检测体系。针对PRV保守序列设计LAMP引物组,并将环引物修饰荧光基团和淬灭基团作为Taqman探针,以实际阳性样本和重组质粒为模板,测试体系组分添加量、特异性、灵敏度和重复性;同时,使用市售恒温检测试剂盒平行测试38份实际样本,验证LAMP-Taqman检测体系的实际检测效果。结果显示,各组分最佳终浓度为:PRV-FIP/BIP 0.8μmol/L,Bst DNA聚合酶0.7 U/μL,TaqDNA聚合酶0.24 U/μL,dNTPs 1.6 mmol/L,MgSO_(4)7.2 mmol/L;体系特异性好,不与其他病毒样本产生交叉反应,梯度样本的线性相关系数(R^(2))为0.995,重复性测试的变异系数小于3.000%,最低检出限可达2.81×10^(2)拷贝/μL;对实际样本的测试结果与市售恒温荧光法检测试剂完全一致。结果表明,基于LAMP-Taqman方法建立的PRV检测体系特异灵敏、稳定准确,是一种适用于猪群PRV精准检测的可靠技术方法。
A LAMP-Taqman rapid detection system for porcine pseudorabies virus(PRV)was developed based on LAMP and quantitative PCR.LAMP primers were designed for PRV conserved sequences,and the loop primer modified by the fluorescent quenching group was used as the Taqman probe.The composition optimization,specificity,sensitivity and repeatability of the LAMP-Taqman system were tested using positive samples and recombinant plasmid as templates.Thirty-eight samples of pork swabs were tested with the commercial LAMP detection kit in parallel to verify the actual detection effect of the LAMP-Taqman detection system.The results showed that the optimal final concentration of each component was as follows:PRV-FIP/BIP 0.8μmol/L,Bst DNA polymerase 0.7 U/μL,Taq DNA polymerase 0.24 U/μL,dNTPs 1.6 mmol/L,MgSO_(4)7.2 mmol/L.This system had good specificity and did not cross-react with other virus samples.The linear correlation coefficient of gradient samples was 0.995,the coefficient of variation of repeatable tests was less than 3.000%,and the minimum detection limit could reach 2.81×10^(2) copies/μL.The test results of the actual swab samples were consistent with the commercial isothermal fluorescence detection reagents.In conclusion,the PRV detection system established by LAMP-Taqman method in this study is specific,sensitive,stable and accurate,and is a reliable technical method suitable for the accurate detection of porcine PRV.
作者
李宇
石磊
时国强
张莹露
董振国
LI Yu;SHI Lei;SHI Guoqiang;ZHANG Yinglu;DONG Zhenguo(Hebei Sanshibio-tech Co.,Ltd.,Shijiazhuang 050035,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2024年第10期2110-2115,共6页
Chinese Journal of Veterinary Science
基金
石家庄市高层次科技创新创业人才基金资助项目(05202001)。
