小反刍兽疫病毒芯片式数字PCR检测方法的建立及应用
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S852.65

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国家重点研发计划项目(2017YFD0501800)


Development and Application of a Chip Digital PCR Assay for PPRV
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    摘要:

    为建立一种准确、快速、敏感性更高的小反刍兽疫病毒(peste des petits ruminants virus,PPRV)检测方法,建立了一种芯片式数字PCR(cdPCR)检测方法。根据GenBank中公布的PPRV Nigeria75 /1疫苗株N基因序列设计1对引物,PCR扩增大小为166 bp片段,构建pMDTM18-N标准质粒并优化反应条件,建立了PPRV N基因cdPCR检测方法,并与实时荧光定量 PCR(RT-qPCR)检测方法的灵敏性、重复性、特异性和临床样品检测做了比较分析。结果显示:当质粒标准品浓度在1.22×(105~102)copies/μL范围时,cdPCR检测方法比普通RT-PCR灵敏度高1 000倍,且稳定性好,特异性强;当标准品浓度在1.22×(105~10-3)copies/μL范围时,cdPCR与RT-qPCR相比具有相同的特异性,但灵敏性比RT-qPCR高100倍;与RT-qPCR 测定结果(13.29±6.74)copies/μL相比,cdPCR的最低检测限更低,约为(0.44±0.14)copies/μL;cdPCR对47份临床样本的PPRV核酸阳性检出率(25.5%)高于RT-qPCR(17.0%)。结果表明,建立的cdPCR方法特异性强、灵敏度高、重复性好,为预防PPR早期流行提供了一种快速有效的诊断和定量检测方法。

    Abstract:

    In order to establish an accurate、rapid and sensitive method to detect peste des petits ruminants virus(PPRV),a chip digital PCR(cdPCR)assay was developed. A pair of primers was designed based on the N gene sequence of PPRV Nigeria75/1 vaccine strain registered in GenBank. The fragment with the size of 166 bp was amplified by PCR to construct pMDTM18-N standard plasmid,followed by optimization of reaction conditions,then a cdPCR assay for PPRV N gene was established,its sensitivity,repeatability,specificity and clinical sample detection capacity were compared and analyzed with those of RT-qPCR. The results showed that cdPCR was with good stability and specificity,and its sensitivity was 1 000 times higher than that of RT-PCR when the concentration of plasmid standard ranged 1.22×(105~102)copies/μL;cdPCR was with the same specificity as RT-qPCR,but its sensitivity was 100 times higher than that of RT-qPCR when the concentration was 1.22×(105~10-3)copies/μL;the lowest detection limit of cdPCR(0.44±0.14)copies/μL was lower compared to that of RT-qPCR(13.29±6.74)copies/μL;and for 47 clinical samples,the detection rate of PPRV nucleic acid by cdPCR(25.5%)was higher than that by RT-qPCR(17.0%). In conclusion,the established cdPCR was with good specificity,sensitivity and repeatability,which could support to prevent any early prevalence of PPRV as a rapid and effective diagnostic and quantitative method.

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杨鸣发,马云云,于志亚,刘家森,康洪涛,姜骞,曲连东.小反刍兽疫病毒芯片式数字PCR检测方法的建立及应用[J].《中国动物检疫》编辑部,2021,38(12):91-98.

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