微量液体培养法快速鉴别分枝杆菌  

Fast identification of mycobacteria in microtiter liquid culture

作　　者：崔振玲[1] 王洁[1] 黄晓辰[1] 陆俊梅[1] 胡忠义[1] 

机构地区：[1]同济大学附属上海市肺科医院结核病诊疗中心上海市结核病(肺)重点实验室,200433

出　　处：《中华预防医学杂志》2011年第1期17-20,共4页Chinese Journal of Preventive Medicine

基　　金：基金项目：国家科技重大专项（2008ZX10003-003-02）;上海市科委科研课题（10QA1405800）;高等学校博士学科点专项科研基金资助项目（20090072120049）

摘　　要：目的 建立微量液体培养法快速鉴别MTB和非结核分枝杆菌（NTM）方法,探讨其临床应用价值.方法 将不同浓度的对硝基苯甲酸（PNB）及噻吩-2-羧酸肼（TCH）和液体培养基加入96孔板中,种入待检菌株,37 ℃培养7～10 d观察结果.根据15种分枝杆菌标准株和30株已知MTB临床分离株不同PNB和TCH浓度时生长试验结果,确定PNB和TCH最佳浓度.用建立的微量液体培养法对424株分枝杆菌临床分离株进行菌种鉴定,并与PCR鉴定及测序鉴定结果进行比较.结果 微量液体培养法中PNB浓度200μg/ml时可有效区分MTB和NTM标准株;TCH浓度0.5 μg/ml时可有效区分结核分枝杆菌与牛结核分枝杆菌标准株.本法鉴定424株分枝杆菌临床分离株,以PCR法鉴定结果为标准,可鉴定出313株结核分枝杆菌复合群菌株中306株和全部NTM菌株,鉴定结核分枝杆菌复合群灵敏度为97.8%（306/313）,特异度为100.0%（107/107）;鉴定NTM的灵敏度为100.0%（107/107）,特异度为96.5%（306/317）;可鉴定出全部的MTB菌株,鉴定MTB的灵敏度可以达到100.0%（305/305）,特异度尚待研究.结论 微量液体培养法可以在7～10 d内对分枝杆菌菌种进行快速鉴定,结果准确度高,操作简便,成本低廉,符合临床快速诊断的需要,适合在我国各级医疗机构推广使用.Objective This research was to establish a method for fast identification of mycobacteria in microtiter liquid culture and to evaluate its clinical value. Methods 2-thiophenecarboxylic acid hydrazide （TCH） and paranitrobenzoic acid（PNB） at different concentrations were added into liquid culture in 96-well plate. Different mycobacterium standard strains were incubated in liquid culture with PNB and TCH for 7 to 10 days. According to the growth assay for 15 mycobacterium strains and 30 mycobacterium tuberculosis strains, the best PNB and TCH concentration were determined. A total of 424 clinical mycobacterium isolates were identified by microtiter liquid culture at the best PNB and TCH concentration. The results of microtiter liquid culture were compared with those of PCR and DNA sequencing. Results The best concentration of PNB was 200 μg/ml in microtiter liquid culture. Compared with the results of PCR, the sensitivity and specificity for identification of mycobacterium tuberculosis complex in microtiter liquid culture were 97. 8%（306/313） and 100. 0% （107/107） respectively and those for non-tuberculosis mycobacteria in microtiter liquid culture were 100. 0% （107/107） and 96. 5% （306/317） respectively. The best concentration of TCH was 0. 5 μg/ml. Compared with the results of PCR,the sensitivity of mycobacterium tuberculosis in microtiter liquid culture was 100.0% （305/305）. The specificity remained under and more studies were needed. Conclusion In microtiter liquid culture with PNB and TCH, mycobacteria can be identified in 7 to 10 days. The results were accurate and the process was simple without expensive equipments. This method meets clinical needs and can be used in all level hospitals in China.

关 键 词：分枝杆菌属 生物学鉴定法 培养技术 评价研究 

分 类 号：R446.5[医药卫生—诊断学]