超分支滚环扩增技术快速检测结核分枝杆菌感染的临床应用价值

Clinical Application on Rapid Detection of Mycobacterium tuberculosis Using Hyperbranched Rolling Circle Amplification

目的建立超分支滚环扩增技术（HRCA）对结核分枝杆菌的快速检测平台。方法对60例肺结核病人，38例非结核对照组和20例健康人的痰标本进行检测；同时对鸟分枝杆菌等非结核分枝杆菌检测以评价该平台的特异性。结果HRCA对H37RvIS6110基因的检测灵敏度达到740aM，检测H37Rv菌悬液的灵敏度为200cfu／ml。鸟分枝杆菌等非结核分枝杆菌的检测结果均为阴性。60例肺结核病人检测灵敏度（73．33％，44／60）与定量PCR相近（78．33％，47／60），显著高于涂片法（41．67％，25／60），差异具有统计学意义。结论超分支滚环扩增技术是一种简单实用，结果可靠和具有较强临床应用前景的结核菌检测技术。

Objective A highly sensitive, specific method, Hyperbranched Rolling circle amplification （HRCA）, was optimized and used to detect M. tuberculosis. Methods The sequences of the 5＇ terminal and 3＇ terminal of the padlock probe was designed according to the sequences of Insert Sequence 6110 （IS6110）, which was specific for the Mycobacterium tuberculosis complex.The padlock probe was used in HRCA to detect the sequence of IS6110 in MTB. Sputum from 60 TB patients, 38 lung cancer or pneumonia patients and 20 healthy persons were tested by HRCA. For testing the specificity of HRCA, eight kinds of None-tuberculosis Mycobacterium （NTM）, such as M.avium were also tested by HRCA. Results Through the optimization of HRCA, the sensitivity of HRCA in detection of purified DNA from H37Rv was 740 aM, and the sensitivity in detection of H37Rv culture suspension was 200 cfu/ml. The results of HRCA in eight kinds of NTM detection were negative. Conclusions The HRCA technique can be considered as a rapid and accurate method to detect H37Rv. The establishment of HRCA in detection of specific gene of H37Rv is useful for development of a cost-effective and reliable method in tuberculosis diagnosis.