基于绵羊无浆体Groel基因的PCR方法的建立

Establishment of a PCR assay based on Groel gene of Anaplasma ovis

利用绵羊无浆体(Anaplasma ovis)高度保守的Groel基因,建立了快速、特异、敏感的绵羊无浆体检测方法。根据GenBank上登录的绵羊无浆体Groel基因序列(FJ460438),设计1对特异性引物,建立了绵羊无浆体PCR检测方法,并对其进行特异性、敏感性、重复性试验及临床样品检测。结果显示,该方法能特异性地检测绵羊无浆体,与山羊无浆体、嗜吞噬细胞无浆体、牛无浆体、边缘无浆体、吕氏泰勒虫、绵羊泰勒虫、羊附红细胞体、弓形虫基因组均无交叉反应;该方法检测下限可达到7.7×10-15 g·μL-1,比文献报道的PCR敏感10倍(77×10-15 g·μL-1);具有良好的重复性;对81份羊临床血液样品检测结果表明,绵羊无浆体感染率为32.1%(26/81),高于文献报道的PCR(MSP4基因)检测结果(18.5%,15/81)。

A fast,specific,and sensitive PCR assay was established to amplify the highly conserved region of Groel gene from the genomic DNA of Anaplasma ovis.According to the published A.ovis Groel gene sequence in GenBank(FJ460438),a pair of primers were designed and a PCR assay were established to detect the A.ovis.At the same time,the specificity,sensitivity,reproducibility test and detection of clinical samples were performed by the PCR.The results showed that the method can be used to specifically detect A.ovis,with no cross reaction with A.capra,A.phagocytophilum,A.bovis,A.maginale,Theileria luwenshuni,T.ovis,Mycoplasma ovis and Toxoplasma gondii;The sensitivity reached 7.7×10-15gμL-1,which is 10 times as sensitive as the PCR reported in the literature(77×10-15gμL-1);This method had good repeatability;The 32.1%positive rate in 81 clinical samples by the PCR assay was higher than that of the PCR(MSP4 gene)test results(18.5%,15/81)reported in the literature.