羊口疮病毒和丝状支原体簇双重PCR检测方法的建立及应用

Establishment and application of a duplex PCR assay for detection of Orf virus and Mycoplasma mycoides cluster

为建立一种同时快速检测羊口疮病毒(ORFV)和丝状支原体簇(Mm cluster)的方法,本研究根据ORFV的B2L基因和Mm cluster的16S r RNA基因序列,设计合成了2对特异性引物,通过条件优化,建立了同时检测ORFV核酸和Mm cluster核酸的双重PCR方法。该方法可以同时扩增出ORFV和Mm cluster的特异性片段,而对其它常见病原的DNA模板均无扩增,对ORFV和Mm cluster的最低检出量分别为3.8×10~4拷贝/μL和1.3×10~3拷贝/μL。利用该方法对36份临床样品进行检测,结果显示双重PCR检测结果与单一PCR检测结果符合率为100%。本研究建立的双重PCR方法特异性强,敏感性高,可重复性好,为临床ORFV和Mm cluster的快速检测、诊断及流行病学调查提供了方法。

A rapid duplex PCR assay was established to detect Orf virus （ORFV） and Mycoplasma mycoides cluster simultaneously, two pairs of specific primers were designed based on the B2L gene of ORFV and 16S rRNA gene of M.mycoides cluster, the assay was established by optimizing the amplification conditions. The specific PCR products of 304 bp for ORFV and 1 232 bp for M.mycoides cluster were simultaneously amplified from the DNAs of ORFV and M.mycoides cluster by the duplex PCR, but no PCR products were amplified from genomic DNAs of other related pathogenic bacteria. The detection limits of the method were 3.8×104 copies/μL for ORFV and 1.3×103 copies/μL for M.mycoides cluster, respectively. Using the assay to detect 36 clinical samples from Fujian, the result showed that the coincidence between single PCR assays and the duplex PCR was 100%. The results indicated that the method was characterized by high sensitivity, stability and specificity, which could provide technical support for the pathogen identification, co-infection rapid detection and epidemiological investigation.