建立实时定量PCR检测临床样品中的麻风杆菌DNA

Establishment of a real-time quantitative PCR for detection of Mycobacterium leprae DNA in clinical samples

目的探讨建立实时定量PCR检测麻风病的方法,评价该方法对于麻风病的诊断价值。方法用17种可培养的分枝杆菌和4种常见球菌和杆菌检测RT-PCR的特异性,并系列稀释已知量的菌液至10-9以检测RT-PCR的敏感性。临床标本40例,包括TT 2,BT 17,BL 17,LL 1,I 3例。其中取皮损组织26份,常规组织液27份及血液样品34份。结果 RT-PCR最低检测限约为4.9个麻风菌/反应,17种其他分枝杆菌和4种细菌常见球菌和杆菌DNA均未扩增出RLEP片段。检测皮损、组织液和血液三种标本,发现皮损标本中麻风菌量最多;BI值与皮损中麻风菌DNA的Ct值之间存在负性相关,提示RT PCR可作为参考值用于定量患者麻风菌载量。结论用RLEP RT-PCR定量麻风菌细菌负荷,该方法敏感性高、特异性强,具有临床参考价值。

Objective To establish a real-time quantitative polymerase chain reaction （RT-PCR） and evaluate the diagnostic value of the method for leprosy. Methods The cultivable specificity of RT-PCR was tested by using 17 species of mycobacteria, 4 sepieies of cocci and other bacilli, and the sensitivity of PCR was determined by using serially diluted known bacteria solution at concentration of 10^-9 . Forty cases were collected including 2 TT eases, 17 BT cases, 1 LL cases, 3 I cases by taking 26 skin lesion samples, 27 fluids and 34 blood samples. Results The minimum detection limit was about 4.9 leprosy bacillus/response by RT-PCR, there were not amplificated M.leprae DNA fragment from 17 speicies of other myeobaeteria branches and 4 kinds of bacterium. BI value of leprosy in the skin was a negative correlation with Ct value, RT-PCR can be used for detection of the bacteria load. Conclusion RLEP quantitative RT-PCR is sensitivie, specific for clinical quantificaiton of M.laprae load.