胞内劳森氏菌PCR检测方法的建立与应用

Development and application of PCR assay for the detection of Lawsonia intracellularis

为建立一种适用于临床检测胞内劳森氏菌(Lawsonia intracellularis,LI)的检测方法,本研究通过比较提取粪便样品基因组DNA的不同方法,结合PCR方法特异性扩增aspA基因检测临床样品中的LI。敏感性试验表明,阳性样本最低检出量为1.7×102copies/μL;特异性试验表明,该方法对大肠杆菌、沙门氏菌、流行性腹泻病毒和伪狂犬病病毒扩增结果均为阴性;对102份临床样品阳性检出率为12.75%,与ELISA试剂盒检测结果符合率达88.04%;不同方法提取粪便样品基因组DNA对PCR检测敏感性的影响结果表明,固相吸附法最低检出量为2.9×102copies/μL,显著优于Chelex-100+Triton X-100法(3.32×103copies/μL)和蛋白酶K-CTAB法(2.45×106copies/μL)。因此,固相吸附法结合PCR方法进行检测具有快速、特异、敏感、重复性好等优点,适用于猪增生性肠炎的临床发病诊断及流行病学监测。

To detect Lawsonia intracellularis （LI） which cause Porcine proliferative enteritis （PPE）, a PCR detection method was developed with a pair of specific primers for amplification of a 294 bp DNA product from LI DNA extracted by guanidine thiocyanate-diatomaceous earth （GuSCN-DE）. The test results indicated that limit detection for LI DNA was 1.7×102 copies and no amplification for E. coli, Salmonella, coronavirus, and pseudorabies virus. Tested on 102 clinic fecal and mucosal samples, the positive rate was 12.75% by PCR detection and the coincidence rate was 88.04%, comparing with the commercial ELISA kit for LI detection. This assay could be used for LI rapid detection and PPE diagnosis.