基于非洲猪瘟病毒标准物质的荧光PCR检测试剂盒初步评价

为探究非洲猪瘟病毒(African swine fever virus,ASFV)标准物质作为试剂盒评价体系的可行性,比较了市场上5种主流品牌ASFV荧光PCR检测试剂盒的检测性能。使用ASFV P72基因核酸标准物质作为模板,根据5种试剂盒说明书分别进行相应的荧光定量PCR检测,结合扩增曲线、Ct值,分析不同试剂盒的敏感性、可重复性以及所需反应时间。结果显示:5种试剂盒的阴性、阳性对照均成立,最低检测限均为5.9×10^-1拷贝/μL;4个厂家的试剂盒线性关系R2>0.98,其中最优的R²=0.994,离散度最小;各试剂盒的实际反应耗时与理论反应耗时均有一定差异(0.20~0.96 h)。结果表明,各生产厂家使用P72基因作为靶基因研制的ASFV荧光PCR检测试剂盒都可以使用ASFV标准物质作为评价体系,来评判试剂盒的检测性能。本试验为各实验室不同样品检测的ASFV荧光PCR检测试剂盒选择提供了一种可用的评价方法。

Evaluation on Fluorescent PCR Kit Based on ASFV Reference Materials

In order to study the feasibility of African swine fever virus(ASFV)reference materials as a kit evaluation system,the performance of five major brands of fluorescent PCR kits in market was compared.Fluorescent PCR assay was carried out in accordance with corresponding instructions of the five kits by taking ASFV P72 gene nucleic acid reference materials as a template,and the sensitivity,repeatability and reaction time of the five kits were analyzed in combination with amplification curve and Ct value.The results showed that the negative and positive controls of the five kits were all valid,and the minimum limit was 5.9×10^-1 copies/μL.The linear relation of four kits was R²>0.98,and the best was R2=0.994 with minimum dispersion,the actual reaction time was different from theoretical ones to some extent(0.20 to 0.96 h).In conclusion,ASFV reference materials could be used as an evaluation system for all ASFV fluorescent PCR kits developed by use of P72 as a target gene,which could evaluate the performance of kits.In view of the above,an accessible evaluation method was provided to select an appropriate kit to test different samples in all laboratories.