单管巢式实时PCR检测麻风杆菌方法的建立及初步应用

Establishment and preliminary application of single-tube nested realtime PCR in detecting Mycobacterium leprae

目的：明确单管巢式实时 PCR 检测麻风杆菌的特异性、灵敏性和重复性。方法：以麻风杆菌 hsp18基因为靶基因设计引物和探针，建立单管巢式实时 PCR 方法检测麻风杆菌并与抗酸染色及普通 PCR 进行特异性和灵敏性的比较。结果：单管巢式实时 PCR 检测下限为2.1 fg 质粒 DNA，普通PCR 为21 fg 质粒 DNA，敏感性高10倍；与8种非麻风杆菌无交叉反应；批内相对标准偏差（RSD）为0.24％~1.08％，批间 RSD 为0.09％~2.8％。对比检测54例麻风疑似标本中，抗酸染色、普通 PCR 和单管巢式实时 PCR 的敏感性分别为81.3％（26/32）、87.5％（28/32）和96.9％（31/32）。结论：单管巢式实时 PCR 特异性强、敏感性高且重复性好。

Objective： To detect the sensitivity of single-tube nested realtime PCR in detecting Mycobacte-rium leprae. Methods： The primers and probe were designed based on the hsp18 gene of Mycobacterium lep-rae. The single-tube nested realtime PCR was established and the sensitivity and specificity were compared with acid-fast staining and ordinary PCR. Results： The sensitivity of the single-tube nested realtime PCR for detecting Mycobacterium leprae was 2.1fg of plasmid DNA, which was 10 times more sensitive than that in the normal PCR. There was non-cross reactivity with other 8 Non-Mycobacterium leprae. Intra relative standard deviation （RSD） was 0.24%-1.08% and inter RSD was 0.09% -2.8%. The sensitivity of acid-fast staining, normal and single-tube nested PCR in detecting 54 suspected samples of leprosy was 81.3% （26/ 32）, 87.5%（28/ 32） and 96.9% （31/ 32）, respectively. Conclusion： The single-tube nested realtime PCR method in detecting Mycobacterium leprae is more sensitive and specific.