科研动态

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。

Development and Application of a Chip Digital PCR Assay for PPRV

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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