TB AccuProbe、INNO-LIPA^(TM)RIF.TB探针和rpoβDNA测序三种方法鉴定结核分枝杆菌的研究

Study on identification of mycobatertium tuberculosis with TB AccuProbe and INNO-LIPA^(TM) RIF.TB and rpo β sequencing

目的用 3种先进的技术 -TBAccuProbeDNA杂交法、INNO LIPATMRIF TB(LiPA)探针试验和rpoβDNA基因序列测定的前瞻性研究 ,以鉴定结核分枝杆菌复合物 (MTBC)和非结核分枝杆菌(NTM)及其成本效益。方法在BactecMGIT 96 0培养系统中将获得的 10 5例阳性培养物筛选出 70例MTBC和 35例NTM ,用 3种技术鉴定并检测其rpoβ基因序列突变位点。 结果TBAccuProbe杂交阳性率是 95 7% (6 7/70 ) ,LiPADNA探针阳性率是 98 6 % (6 9/70 ) ,rpoβDNA序列测定阳性率是 97 1% (6 8/70 )。 3种方法两两比较差异在统计学上均无显著意义 (P >0 0 5 )。每份阳性培养物花费时间 :TBAc cuProbe杂交法和LiPADNA探针法在 2 4小时内完成 ,全部完成需 6～ 37天 ,平均为 15天 ;rpoβ基因序列测定需 4～ 5天完成 ,全部完成需 8～ 4 0天 ,平均 17天。比传统的 4～ 8周鉴定时间减少了很多。每份标本所需费用 3种方法各为 12 0、194和 96元。在 35例NTM中 ,3种方法均为MTBC阴性 ,同时rpoβDNA序列测定鉴定了NTM的种类。结论TBAccuProbe杂交和LiPADNA探针操作简便 ,所需时间短 ,只能鉴定MTBC和NTM。但LiPADNA探针还能鉴定rpoβ基因位点突变 ;rpoβ基因序列测定所需时间稍长 ,要有特定仪器和专门人员 ,但所需费用较低 ,除能鉴定MTBC外 。

Objective A prospective study was organized to determine the accuracy and costeffectiveness of the TB AccuProbe DNA hybridization test,the PCR bases LiPA assay,and a verifying automated DNA sequencing of the rpoβ gene,for the routine rapid and direct identification of the Mycobacterium tuberculosis complex (MTBC) and Non Tuberculosis Mycobacterium(NTM) and rifampin(RMP) susceptibility.Methods Three methods were used to identify the MYBC and rpoβ gene mutation or NTM in the Bactec MGIT 960 system.Results The study for 70 cases MTBC and 35 cases NTM revealed that the TB AccuProbe test detected 67 of the 70 (95.7%),the LiPA probe assay identified 69 of the 70 (98.6%) and rpoβ DNA sequencing identified 68 of 70 (97.1%) M.tuberculosis isolates.TB AccuProbe test and LiPA probe assay can readily detect the MTBC with 24 hours of positive growth in MGIT 960 vials and need 6～37 days for identification(average turnaround time was 15 days) and rpoβ DNA sequencing required 4～5 days of positive growth and need 8～40 days for identification (average turnaround time was 17 days) after receiving the specimen.Besides the identification of the MTBC and its susceptibility to RMP and rpo β DNA sequencing was also capable to identify the NTM.In addition,the molecular methods were able to rapidly identify RMP susceptible and resistant MTBC in mixed mycobacerial cultures weeks earlier than the conventional methods.The average reagent and labor costs for each specimen associated with single TB AccuProbe and LiPA and rpoβ DNA sequencing tests were 120 yuan,194 yuan,96 yuan,respectively.Conclusion All three molecular tests have high sensitivity and specificity and also can be reliable used in combination with the MGIT 960 system.The application of these methods should determine by the level of service provided by a particular laboratory.