聚合酶链反应-反向线点杂交技术鉴定分枝杆菌的多中心评价

Evaluation PCR-reverse Line Hybridization Assay for Detection and Identification of Mycobacterium Species:a Multi-center Study

目的评价以16S-23S rDNA间隔序列作为靶基因的聚合酶链反应-反向线点杂交(PCR-RLB)技术用于鉴定分枝杆菌。方法在5个中心同时进行试验。采用PCR-RLB技术检测60株属于50个种的分枝杆菌标准株;10株非分枝杆菌标准株。以胶体金法结合测序法或是气相色谱(gas chromatography,GC)法作为对比,检测鉴定383株分枝杆菌临床分离株。结果所有的分枝杆菌标准株和临床分离株PCR扩增产物均可与分枝杆菌属探针杂交,而种特异性探针仅与相应的种杂交,非分枝杆菌PCR扩增产物均不与分枝杆菌属及种探针杂交。与胶体金法或是GC法结合测序法相比,PCR-RLB法准确率100%,成功地将380株分枝杆菌鉴定到种(另外3株仅鉴定到分枝杆菌层未鉴定到种),包括11株其他方法不能准确鉴定的混合感染。结论以16S-23S rDNA间隔序列作为靶基因的PCR-RLB技术,鉴定分枝杆菌敏感性和特异性高,快速、实用,具有很好的临床应用前景。

Objective To evaluate the PCR-reverse line dot hybridization assay（RLB） based on 16S-23S rDNA internal transcribed spacer（ITS） sequence for detection and identification of mycobacterium species.Methods This study was performed in five different centers simultaneously,and the performance of PCR-RLB was estimated and verified by detection of 60 reference strains belonging to 50 mycobacterium species,10 nonmycobacterial speices and 383 clinical isolates identified by immunochromatographic assay（ICA） combined sequencing and gas chromatography（GC）.Results The genus-specific probe hybridizied with the amplification of PCR of all mycobacteria and species-specific probes hybridized only with corresponding species,however,neither of them hybridized with the amplification of PCR of nonmycobacterial speices.Compared with ICA combined sequencing and GC,accuracy of PCR-RLB was nearly 100%, and PCR-RLB identified 380 of 383 in species-level successfully,including 11 mixed clinical isolates which could not be identified with other assays correctly.Conclusion The mycobacterial PCR-RLB based on 16S-23S rDNA ITS is rapid,convenient,sensitive and specific,and practical,and it is promising to be applied in clinical diagnosis.