不同靶点环介导等温扩增技术对结核分枝杆菌检测的比较研究

Detection of Mycobacterium tuberculosis by loop-mediated isothermal amplification assay targeting different genes

目的探讨针对hspX、gyrB、IS6110靶基因设计引物，采用环介导等温扩增技术（LAMP）检测对结核病的诊断价值。方法肺结核组为我院收治的68例肺结核患者，对照组为我院收治的45例肺癌患者和20名健康体检者，收集痰标本后采用LAMP、定量PCR及涂片法进行分析，评价LAMP技术的临床应用价值。采用LAMP及定量PCR分别对结核分枝杆菌标准株H37Rv的DNA样品进行检测，评价LAMP技术的检测限；同时对鸟分枝杆菌、胞内分枝杆菌、瘰疬分枝杆菌、偶发分枝杆菌、耻垢分枝杆菌、海分枝杆菌、龟分枝杆菌和堪萨斯分枝杆菌等8种非结核分枝杆菌菌株及4株普通菌标准株包括大肠埃希菌（ATCC25922）、阴沟肠杆菌（ATCC700323）、铜绿假单胞菌（ATcc27853）和金黄色葡萄球菌标准菌株（ATcc29213）进行鉴定，评价LAMP的敏感度和特异度。结果本研究采用hspX、IS6110和gyrB靶基因进行LAMP检测，对H37Rv的DNA样品检测限分别为320amol／L，320amol／L和32amol／L。LAMP对8种非结核分枝杆菌和4种普通菌检测结果为阴性。对照组中健康志愿者定量PCR，涂片法及LAMP方法的结果均为阴性，对照组中肺癌患者中hspX，gyrB，IS6110LAMP方法和定量PCR阳性结果2例，3种靶基因位点的LAMP和定量PCR检测的特异度均为96．9％（63／65）。针对hspX、gyrB、IS6110靶基因进行LAMP检测，对68例肺结核患者痰标本检测的敏感度分别为75．0％（51／68），77．9％（53／68），73．5％（50／68）；定量PCR检测的敏感度为79．4％（54／68）。3种不同基因位点LAMP技术与定量PCR检测的敏感度比较差异无统计学意义（x^2=0．82，P〉0．05），但均高于涂片法的38．2％（26／68），LAMP技术与涂片法之间差异均有统计学意义（x^2=18．71，x^2=22．02，x^2=17．18，P值均〈0．01）。结论hspX、gyrB、IS6110LAMP技术检测的敏感度和特异度均较高，尤其是针对gyrB靶基因位点进行LAMP检测的敏感度与定量PCR接近，并且高于涂片法；如果结合不同基因位点LAMP技术的检测结果进行评判可以提高该技术的敏感度。

Objective To evaluate a loop-mediated isothermal amplification （LAMP） assay targeting the hspX, gyrB, and IS6110 genes for the diagnosis of tuberculosis （TB）. Methods Mycobacterium tuberculosis pre- sent in sputum samples from 68 pulmonary tuberculosis patients and control groups consisting of 45 lung cancer pa- tients and 20 healthy controls were tested by LAMP, quantitative real time PCR and smear microscopy to evaluate the value of the LAMP assay. The detection limit of the assay was evaluated by detecting Mycobacterium tuberculosis H37Rv reference strains with PCR and LAMP targeting the three genes. Specificity was evaluated using the follo- wing non-tuberculous mycobacteria （NTM） ： M. avium, M. intracellulare, M. srofulaceum, M. fortuitum, M. smeg- matis, M. marinum, M. chelonae and M. kansasii, and four other species of bacteria： E. colt （ATCC25922）, Enter-obacter cloacae （ATCC700323）, Pseudomonas aeruginosa （ATCC 27853） and Staphylococcus aureus （ATCC29213）. Results The H37Rv reference strain was successfully detected by this method and no false-positive results were ob- tained for NTM or common bacteria. The detection limit of PCR for purified DNA from H37Rv was 32 amol/L and that for hspX LAMP, gyrB LAMP, and IS6110 LAMP were 320 amol/L, 32 amol/L and 320 amol/L, respective- ly. The detection sensitivity of the three LAMP methods for sputum from 68 pulmonary tuberculosis patients was 75.0% （51/68）, 77.9% （53/68）, and 73.5% （50/68）, all of which were higher than that of smear microscopy, 38. 2% （26/68）, （x^2 =18.71, x^2 =22.02, x^2 =17.18, P〈0. 01）. There was no significant difference between the sensitivity of LAMP and that of quantitative reabtime PCR （x^2 =0.82 ,P〉0.05）. The specificity of detection using hspX LAMP, gyrB LAMP, and IS6110 LAMP and PCR was 96.9% （63/65） in each case. Conclusion The sen sitivity and specificity of the TB LAMP methods targeting the hspX, gyrB and 1S6110 genes were higher than that of smear microscopy and close to that of PCR. Sensitivity could be improved by using a combination of TB LAMP methods targeting different genes.