CAJ | 학술논문

目的探讨PCR-反向斑点杂交技术用于检测结核分枝杆菌及其耐药基因突变的效果,了解绍兴地区结核分枝杆菌耐药基因的突变情况.方法根据结核分枝杆菌基因特征,运用反向斑点杂交技术构建特异性的PCR反应体系,并用10株非结核分枝杆菌、3株其他菌株(金黄色葡萄球菌、大肠埃希菌和鲍曼不动杆菌)和1株H37Rv结核分枝杆菌对该反应体系进行验证,同时用该体系对来源于绍兴市立医院的125份抗酸杆菌涂片阳性痰液样本及150株结核分枝杆菌进行5种耐药突变基因(rpoB、katG、inhA、embB、rpsL)的检测.结果构建的PCR-反向斑点杂交体系用于10株非结核分枝杆菌、3株其他菌株的检测结果均为阴性,H37Rv标准株可检出,但未检出耐药突变位点.275份结核分枝杆菌样本中对4种抗结核药物均敏感的菌株有201份(73.1％),单药耐药20份(7.3％),耐多药54份(19.6％);共检出133个耐药突变位点,其中对利福平、异烟肼、链霉素和乙胺丁醇的耐药突变位点数分别为41个、51个、30个、11个;前5位突变位点为315M、43M、S531L、15M和H526Y.结论绍兴地区耐药结核分枝杆菌以耐多药为主.构建的PCR-反向斑点杂交体系用于检测结核分枝杆菌特异性强、灵敏度高,可快速检测结核分枝杆菌的耐药基因.
목적 탐토PCR-반향반점잡교기술용우검측결핵분지간균급기내약기인돌변적효과,료해소흥지구결핵분지간균내약기인적돌변정황.방법 근거결핵분지간균기인특정,운용반향반점잡교기술구건특이성적PCR반응체계,병용10주비결핵분지간균、3주기타균주(금황색포도구균、대장애희균화포만불동간균)화1주H37Rv결핵분지간균대해반응체계진행험증,동시용해체계대래원우소흥시립의원적125빈항산간균도편양성담액양본급150주결핵분지간균진행5충내약돌변기인(rpoB、katG、inhA、embB、rpsL)적검측.결과 구건적PCR-반향반점잡교체계용우10주비결핵분지간균、3주기타균주적검측결과균위음성,H37Rv표준주가검출,단미검출내약돌변위점.275빈결핵분지간균양본중대4충항결핵약물균민감적균주유201빈(73.1％),단약내약20빈(7.3％),내다약54빈(19.6％);공검출133개내약돌변위점,기중대리복평、이연정、련매소화을알정순적내약돌변위점수분별위41개、51개、30개、11개;전5위돌변위점위315M、43M、S531L、15M화H526Y.결론 소흥지구내약결핵분지간균이내다약위주.구건적PCR-반향반점잡교체계용우검측결핵분지간균특이성강、령민도고,가쾌속검측결핵분지간균적내약기인.
Objective To discuss the effect of PCR-reverse dot blot hybridization technique for detection of Mycobacterium tuberculosis and drug-resistant gene mutation,and understand Mycobacterium tuberculosis drug resistance gene mutation in Shaoxing area and its relationship with the drug resistance.Methods According to the characteristics of Mycobacterium tuberculosis gene,we used reverse dot blot hybridization technique construct specific PCR reaction system,and 10 strains of non Mycobacterium tuberculosis,3 strains of other strains (Staphylococcus aureus,Escherichia coli and A cinetobacter.baumannii sp.)and 1 strain of Mycobacterium tuberculosis (H37Rv) to validate the reaction system.The system was also used to detect five kinds of resistance mutation detection of the gene (rpoB,katG,InhA,embB and rpsL)of 125 acid fast bacilli smear positive sputum samples and 150 strains of Mycobacterium tuberculosis sourcing from the Shaoxing Municipal Hospital.Results By construction of PCR reverse dot blot hybridization system,the detection result of 10 strains of non Mycobacterium tuberculosis and 3 strains of other strains were negative.H37Rv standard strains could be detected,but they were not detected in the resistant mutations.Among 275 cases of tuberculosis patients,201 cases (73.1％)were sensitive to four kinds of anti-TB drugs;20 patients (7.3％)were single drug;54 cases were multi-drug resistance (19.6％).133 resistance mutations loci were detected in 275 samples,of which there were 41,51,30 and 11 resistant mutation loci of rifampicin,isoniazid,streptomycin and ethambutol respectively.The top 5 gene mutation detection loci Mycobacterium tuberculosis of were 315M,43M,S531 L,15M and H526Y.Conclusions The drug resistance of Mycobacterium tuberculosis in Shaoxing area is mainly based on the multi drug resistance.Construction of PCR-reverse dot blot hybridization system for detecting Mycobacterium tuberculosis is of specificity and high sensitivity,which can quickly detect the resistance gene of Mycobacterium tuberculosis.