為建立一種準(zhǔn)確,、快速,、敏感性更高的小反芻獸疫病毒（peste des petits ruminants virus，PPRV）檢測(cè)方法,，建立了一種芯片式數(shù)字PCR（cdPCR）檢測(cè)方法,。根據(jù)GenBank中公布的PPRV Nigeria75 /1疫苗株N基因序列設(shè)計(jì)1對(duì)引物，PCR擴(kuò)增大小為166 bp片段,，構(gòu)建pMDTM18-N標(biāo)準(zhǔn)質(zhì)粒并優(yōu)化反應(yīng)條件,，建立了PPRV N基因cdPCR檢測(cè)方法，并與實(shí)時(shí)熒光定量 PCR（RT-qPCR）檢測(cè)方法的靈敏性,、重復(fù)性,、特異性和臨床樣品檢測(cè)做了比較分析。結(jié)果顯示：當(dāng)質(zhì)粒標(biāo)準(zhǔn)品濃度在1.22×（105~102）copies/μL范圍時(shí),，cdPCR檢測(cè)方法比普通RT-PCR靈敏度高1 000倍,，且穩(wěn)定性好，特異性強(qiáng),；當(dāng)標(biāo)準(zhǔn)品濃度在1.22×（105~10-3）copies/μL范圍時(shí),，cdPCR與RT-qPCR相比具有相同的特異性,，但靈敏性比RT-qPCR高100倍；與RT-qPCR 測(cè)定結(jié)果（13.29±6.74）copies/μL相比,，cdPCR的最低檢測(cè)限更低,，約為（0.44±0.14）copies/μL；cdPCR對(duì)47份臨床樣本的PPRV核酸陽(yáng)性檢出率（25.5%）高于RT-qPCR（17.0%）,。結(jié)果表明,，建立的cdPCR方法特異性強(qiáng)、靈敏度高,、重復(fù)性好,，為預(yù)防PPR早期流行提供了一種快速有效的診斷和定量檢測(cè)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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