鸡毒支原体套式PCR检测方法的建立及应用

Establishment and application of a nested PCR for the detection of Mycoplasma gallisepticum by mgc2 gene sequence

为了建立鸡毒支原体(Mycoplasma gallisepticum,MG)套式PCR检测方法并初步将其应用于临床检测,对目前常用的5对引物(gapA-F/gapA-R、16SrRNA-F/16SrRNA-R、mgc2-F1/mgc2-R1、mgc2-F2/mgc2-R2、LP-F/LP-R)检测MG的灵敏度进行了比较,从中选出灵敏度较强的引物建立套式PCR检测方法,同时进行了特异性、敏感性及临床检测试验。结果表明,以mgc2基因设计的引物灵敏度最高,能检测出的MG的最低质量浓度为57.6pg/μL,用此引物建立的套式PCR能够扩增的基因片段的大小为300bp,与GenBank中收录的相关序列的同源性为98%,而与大肠杆菌、沙门菌、鸡滑液囊支原体均无交叉反应,能够检测出DNA的最小质量浓度为0.18fg/μL。通过对山东地区疑似MG的50只病死鸡进行检测,阳性检出率为80%。本研究建立的鸡毒支原体套式PCR具有敏感性高、特异性强等优点,可用于MG的临床诊断和分子流行病学调查。

To develop a nested PCR for detection of Mycoplasma gallisepticum and apply it to clinical testing,a comparison of the detection sensitivity of five primer pairs（gapA-F/gapA-R, 16S rRNA-F/16S rRNA-R, mgc2-F1/mgc2-R1, mgc2-F2/mgc2-R2, LP-F/LP-R） for M. gallisepticurn was carried out, the stronger sensitivity primers were chosen to establish a set of nested PCR, and the specificity, sensitivity and clinical tests were carried out. The results of verification experiments showed that mgc2 primers had the highest sensitivity and the lowest detected concentration of M. galliselticum was 57.6 pg/μL. The targeted gene fragment was 300 bp in length and homology of the nested PCR product was 98% with that available in GenBank,and this detection method had no cross reaction with Escherichia coli, Salmonella gallina- rum ,and Mycoplasma synoviae ,and the sensitivity of the assay was reached to 0.18 fg of DNA. The posi- tive rate was 80% in 50 clinical samples suspected M. gallisel）ticum in Shandong by the nested PCR. In conclusion, this method, with good specificity and sensitivity, could be used as one for the diagnosis and de- tection of clinical cases,and molecular epidemiological investigation of M. gallisepticum.