CAJ | 학술논문

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.
목적 응용소식다중등위기인특이성확증(nMAS-PCR)검측체계,동보검측결핵분지간균rpoB 、katG 、inhA기인돌변,이쾌속사사결핵분지간균대리복평화이연정적내약성.방법 수집2012년7지8월엄주시흉과의원림상의사폐결핵환자165례,류취도양신담표본,제취결핵균DNA,용nMAS-PCR법동시진행rpoB기인516、526、531위점、katG기인315위점화inhA기인--15위점적돌변검측,초보판단대리복평여이연정적내약성,병장검측결과여선성탐침법비교,채용Kappa검험진행통계학분석.결과 165빈도양담표본중결핵분지간균148빈,기중리복평민감주121주,리복평내약주27주;이연정민감주115주,이연정내약주33주.nMAS-PCR법결과현시리복평급이연정민감주균미견돌변,특이도균위100％,이리복평내약주검측민감도위78％ (21/27),초저우선성탐침법(82％,22/27);이연정내약주검측민감도위82％ (27/33),여선성탐침법결과일치(82％,27/33);량충방법대이연정여리복평내약성검측결과비교,Kappa치분별위0.94、0.75.결론 nMAS-PCR법구유교고적민감도여특이도,간편경제,능구직접이용결핵도양담표본쾌속사사다약내약결핵.
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 rifampin(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 O.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.