巢式多重等位基因特异性PCR直接检测痰标本结核分枝杆菌耐药性临床应用研究

Application of nested multiplex allele-specific polymerase chain reaction for detecting multidrug- resistant Mycobacterium tuberculosis in sputum

目的应用巢式多重等位基因特异性扩增（nMAS—PCR）检测体系，同步检测结核分枝杆菌rpoB、katG、inhA基因突变，以快速筛查结核分枝杆菌对利福平和异烟肼的耐药性。方法收集2012年7至8月广州市胸科医院临床疑似肺结核患者165例，留取涂阳晨痰标本，提取结核菌DNA，用nMAS—PCR法同时进行rpoB基因516、526、531位点、katG基因315位点和inhA基因--15位点的突变检测，初步判断对利福平与异烟肼的耐药性，并将检测结果与线性探针法比较，采用Kappa检验进行统计学分析。结果165份涂阳痰标本中结核分枝杆菌148份，其中利福平敏感株121株，利福平耐药株27株；异烟肼敏感株115株，异烟肼耐药株33株。nMAS—PCR法结果显示利福平及异烟肼敏感株均未见突变，特异度均为100％，而利福平耐药株检测敏感度为78％（21／27），稍低于线性探针法（82％，22／27）；异烟肼耐药株检测敏感度为82％（27／33），与线性探针法结果一致（82％，27／33）；两种方法对异烟肼与利福平耐药性检测结果比较，Kappa值分别为0．94、0．75。结论nMAS—PCR法具有较高的敏感度与特异度，简便经济，能够直接利用结核涂阳痰标本快速筛查多药耐药结核。

Objective To evaluate a nested multiplex allele-specific polymerase chain reaction （nMAS-PCR） simoutanously detecting mutations in rpoB, katG and inhA gene mutations to screen multidrug-resistant Mycobacterium tuberculosis （MDR-TB） in sputum directly. Methods Totally 165 cases of smear-positive morning specimens were collected from clinical suspected tuberculosis in Guangzhou Chest Hospital from Jul to Aug 2012. The tuberculosis （TB） mutations of 5 codons （codon 351 of the katG gene, the 15th nucleotide preceding the mabA-inhA operon and codon 516,526,531 of the rpoB gene） by nMAS- PCR to speculate its fifampin（RIF） and isoniazid（INH） resistance. The results were compared with PCR- LiPA, and the Kappa test were used. Results Of the 165 acid-fast bacilli smear-positive specimens, 148 were comfirmed as Mycobacterium tuberculosis. Among the 148 Mycobacterium tuberculosis specimens, no mutation was detected in the sensitive specimens and the specificity was 100%. The sensitivity was 78% （21/27） for RIF resistance by nMAS-PCR while 82% （22/27） by PCR-LiPA, and 82% （27/33） for INH resistance by nMAS-PCR while 82% （27/33） by PCR-LiPA. The Kappa value of the two methods to detect INH and RIF resistance was 0. 94 and 0. 75 respectively. Conclusion The performance of nMAS-PCR suggests that it be a relatively inexpensive and technically feasible method with high sensitivity and specificity for rapid detection of MDR-TB directly in respiratory specimens.