应用PCR-反向点杂交技术快速检测结核分枝杆菌耐药突变基因

Rapid detection of Mycobacterium tuberculosis resistant genes using PCR reverse dot blotting hybridization

目的 评价PCR-反向点杂交技术在检测结核分枝杆菌耐药突变基因中的应用价值。方法选取2014年8月至2015年12月江西省胸科医院住院的部分肺结核患者作为研究对象，共1071例。研究对象均送检了痰液标本，共1071份。所有患者均经病原学或病理学证实，或符合菌阴肺结核诊断标准。采用PCR-反向点杂交技术对研究对象痰标本进行异烟肼（H）、利福平（R）、链霉素（s）、乙胺丁醇（E）耐药基因检测，并以痰培养阳性标本的药物敏感性试验（简称“药敏试验”）检测结果作为金标准，来验证检测结果的可靠性及评价其检测效能。结果PCR-反向点杂交检测的1071例患者中，596例结核分枝杆菌阳性，阳性率为55．65％。其中，218例（36．58％）检出H、R、S、E耐药基因突变，分布于所测13个位点。H、R、S、E最常见突变位点分别位于KatG的315M（82．53％，137／166）、rpoB的$531L（69．50％，98／141）、rpsl的43M（78．65％，70／89）、embB的306M2（55．13％，43／78）和306M1（39．74％，31／78）。433例患者标本同时行结核分枝杆菌培养，培养阳性且同时PCR反向点杂交检测阳性157例。以培养及药敏试验结果为金标准，PCR-反向点杂交法对H、R、S、E的耐药检测经Kappa检验具有较好的一致性（Kappa值分别为0．77、0．73、0．66、0．49）；敏感度分别为88．89％（40／45）、79．66％（47／59）、67．57％（25／37）、63．16％（12／19）；特异度分别为91．07％（102／112）、91．84％（90／98）、95．00％（114／120）、91．30％（126／138）。结论PCR-反向点杂交技术用于结核分枝杆菌耐药基因检测较为可靠，对早期临床用药具有较好的指导作用。

Objective To explore the value of PCR reverse dot blotting hybridization in detecting drugresistant gene mutation of Mycobacterium tuberculosis. Methods A total of 1071 inpatients were enrolled from Jiangxi Chest Hospital in interval between August 2014 and December 2015. All patients were with positive pathology or positive pathogeny, or identified by the criteria of pulmonary tuberculosis with negative smear or culture. PCR reverse dot blot hybridization which was used to detect Mycobacterium tuberculosis drug-resistant genes, which were included （isoniazid （H）, rifampin （R）, streptomycin （S） and ethambutol （E） in 1071 sputa samples）. The results were compared with result of drug susceptibility test, which usually considered as golden standard, and the efficiency was evaluated. Results Of the 1071 cases were detected by using PCR reverse dot blot hybridization, 596 （55. 650/oo） were TB positive, and H, R, S or E mutant genes distributing in 13 loci were found in 218 cases （36.58%）. The most common mutant-gene of H, R, S and E were KatG 315M （82.53%, 137/166）, rpoB S531L （69.50%, 98/141）, rpsl-43M （78.65%, 70/89）, embB-306M2 （55.13%0, 43/78） and embB 306M1 （39.74%ff0, 31/78）, respectively. Additionally, 433 clinical samples were tested with TB culture, and 157 cases had obtained positive results in both of TB culture and dot blot hybridization. By using culture and drug sensitivity results as gold standard, PCR reverse dot blot hybridization had excellent consistency in detecting H, R, S and E compared by adopting Katapa test （Kappa index were 0.77, 0.73, 0.66 and 0.49, respectively）. The sensitities were 88.89% （40/45）, 79.66% （47/59）, 67.57% （25/37） and 63.16% （12/19）, and the specificities were 91. 07% （102/112）, 91. 840/00 （90/98）, 95.00% （114/120） and 91. 30% （126/138）, respectively. Conclusion PCR reverse dot blot hybridization was reliable to detect the drugresistant gene mutation of Mycobacterium tuberculosis, and it was helpful in drug usage guidance.