三种非洲猪瘟病毒荧光PCR检测试剂盒和两种核酸提取方法的比对
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S855.3

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吉林省动物疫病监测流调和防治项目(2130108)


Comparison of Three Fluorescent PCR Kits for African Swine Fever Virus and Two Nucleic Acid Extraction Methods
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    摘要:

    为评价不同荧光PCR检测试剂盒对非洲猪瘟病毒(African swine fever virus,ASFV)的检测效果,以及不同核酸提取方法对ASFV的提取差异,选取ASFV B646L基因标准物质作为模板参照,通过荧光PCR检测对标准曲线、最低检出限(LOD)、特异性、检测时间等进行比较,分析3个厂家商品化ASFV荧光PCR检测试剂盒(A、B、C)的综合性能;选取磁珠法和柱式法两种核酸提取方式,对标准物质核酸提取后进行ASFV荧光PCR扩增,以Ct值大小评价试剂的提取效果。结果显示:3种ASFV荧光PCR试剂盒决定系数(R2)分别为0.985、0.996、0.985,且特异性较好;3种ASFV荧光PCR试剂盒LOD介于10-1~10-2 copies/μL,A试剂盒反应循环数最多且反应时间最长(105 min),LOD为10-2 copies/μL。全自动核酸提取工作站与手工核酸提取Ct值无统计学差异(P>0.1),批内变异系数(CV)在1.5%以内。结果表明:3种荧光PCR检测试剂盒的综合性能较为理想,A试剂盒更适用于临床含毒量低的样品检测;两种核酸提取方法均可有效提取ASFV B646L基因质粒标准物质。本试验为科学、合理选择ASFV检测和提取试剂提供了数据参考。

    Abstract:

    In order to evaluate different fluorescent PCR kits for African swine fever virus(ASFV)and extraction difference by different nucleic acid extraction methods,taking ASFV B646L gene reference materials as the template,the comprehensive performance of commercial ASFV fluorescent PCR kits(A,B and C)provided by three manufacturers were analyzed and compared by fluorescent PCR in the aspects of standard curve,limit of detection(LOD),specificity and detection time;reference material nucleic acids were extracted by immunomagnetic beads and column method,then amplified by fluorescent PCR to evaluate the extraction efficiency of the kits by Ct value. The results showed that the determination coefficients(R2)of the three kits were 0.985,0.996 and 0.985,respectively,with good specificity;and their LOD ranged from 10-1 to 10-2 copies/μL,for kit A,its reaction cycles was maximum with longest reaction time(105 min),and its LOD was 10-2 copies/μL. No significant difference(P>0.1)was observed in Ct value between automatic nucleic acid extraction system and manual nucleic acid extraction,and the intra-assay coefficient of variation(CV)was within 1.5%. In conclusion,the comprehensive performance of the three kits was ideal,especially kit A was more suitable for detecting samples with low viral load;ASFV B646L gene plasmid could be effectively extracted by the two extraction methods. A reference was provided for scientific and reasonable selection of detecting or extracting kits for ASFV.

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曹东阳,朱奕霏,马晓媛,白翠,杨金鑫,胡泽宇,吴丹.三种非洲猪瘟病毒荧光PCR检测试剂盒和两种核酸提取方法的比对[J].《中国动物检疫》编辑部,2022,39(12):112-117.

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