猪附红细胞体巢式PCR诊断方法的建立及初步应用

摘要

为建立一种更加准确、敏感的猪附红细胞体检测方法,本试验根据猪附红细胞体特异的DNA序列(AJ504999)设计2对巢式PCR引物,建立了巢式PCR检测方法.筛选该检测方法的最佳反应条件,并进行了特异性、敏感性及临床样本检测试验.结果表明,建立的巢式PCR对猪附红细胞体基因组DNA能扩增出特异性片段,而对弓形虫、链球菌、牛瑟氏泰勒虫、牛附红细胞体基因组DNA扩增反应结果均为阴性;DNA最低检测量为0.124 fg//μL;通过对55份临床样本的检测,该巢式PCR较常规PCR的阳性检出率高23.7%.本试验为猪附红细胞体病的诊断提供了一种更为特异、敏感的检测技术.%To establish a more exact and sensitive method to detect Eperythrozoon suis, two pairs of nested PCR primer were designed according to Eperythrozoon suis DNA sequence (AJ504999)and nested PCR assay was established.The optimum reaction conditions were screened, and sensitive, special and clinical samples tests were carried out.The results showed that Eperythrozoon suis genome DNA could be amplified by nested PCR,and Toxoplasma gondii, Streptococcus, Theileria sergenti and Eperythrozoon wenyonii genomic DNA could not.The minimum amount of DNA to detect was 0.124 fg/μL.After the detection of 55 clinical samples, results indicated that the positive detection rate of nested PCR was 23.7％ higher than conventional PCR.This research offers a more exact and sensitive detection method and it is good value for application.