科研動態(tài)

為建立一種準確、快速、敏感性更高的小反芻獸疫病毒（peste des petits ruminants virus，PPRV）檢測方法，建立了一種芯片式數(shù)字PCR（cdPCR）檢測方法。根據(jù)GenBank中公布的PPRV Nigeria75 /1疫苗株N基因序列設(shè)計1對引物，PCR擴增大小為166 bp片段，構(gòu)建pMDTM18-N標準質(zhì)粒并優(yōu)化反應條件，建立了PPRV N基因cdPCR檢測方法，并與實時熒光定量 PCR（RT-qPCR）檢測方法的靈敏性、重復性、特異性和臨床樣品檢測做了比較分析。結(jié)果顯示：當質(zhì)粒標準品濃度在1.22×（105~102）copies/μL范圍時，cdPCR檢測方法比普通RT-PCR靈敏度高1 000倍，且穩(wěn)定性好，特異性強；當標準品濃度在1.22×（105~10-3）copies/μL范圍時，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 測定結(jié)果（13.29±6.74）copies/μL相比，cdPCR的最低檢測限更低，約為（0.44±0.14）copies/μL；cdPCR對47份臨床樣本的PPRV核酸陽性檢出率（25.5%）高于RT-qPCR（17.0%）。結(jié)果表明，建立的cdPCR方法特異性強、靈敏度高、重復性好，為預防PPR早期流行提供了一種快速有效的診斷和定量檢測方法。

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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