应用荧光定量PCR-探针熔解曲线法快速鉴定分枝杆菌菌种方法的建立及初步评价

Development and preliminary evaluation on rapid identification of mycobacterium species by fluorescence quantitative PCR-probe melting curve method

目的应用荧光定量PCR-探针熔解曲线法建立一种快速、简便、高效的分枝杆菌菌种鉴定新方法,为临床医师正确诊断提供理论依据。方法以DNA直接测序法为对照,通过荧光定量PCR技术-探针熔解曲线法(包括反应A、B、C)分析22种分枝杆菌标准株、11种非分枝杆菌标准株和32株分枝杆菌临床分离株的菌种。结果应用荧光定量PCR-探针熔解曲线法分析22种分枝杆菌标准株和11种非分枝杆菌标准株,结果与标准株DNA测序一致,成功地建立鉴定方法。32株分枝杆菌临床分离株中,经DNA测序和荧光定量PCR-探针熔解曲线法分析显示,4株为结核分枝杆菌复合群(mycobacterium tuberculosis complex,MTC),7株为胞内分枝杆菌,6株为龟分枝杆菌脓肿亚种,4株为瘰疬分枝杆菌,4株为偶然分枝杆菌,3株为堪萨斯和胃分枝杆菌,2株为戈登分枝杆菌;另有2株分离株反应A的熔解曲线显示为分枝杆菌属的熔解温度(melting temperature,Tm)值,反应B和C的熔解曲线Tm值与22株分枝杆菌标准株的Tm值均不同,经DNA测序证实与戈登分枝杆菌标准株序列不同,与报道的戈登分枝杆菌分离株一致。结论用荧光定量PCR-探针熔解曲线法可简便、快速、灵敏、特异地将大多数分枝杆菌鉴定到种,提高分枝杆菌病的正确诊断率,指导临床合理用药。

Objective To develop a new rapid, simple and efficient method for the identification of mycobacterium species by fluorescence quantitative PCR-probe melting curve technique, to provide a basis for the correct diagnosis of mycobacterium diseases in clinic. Method DNA sequencing was used as the control, the target DNA fragments of 22 mycobacterium reference strains, 11 non-mycobacterium reference strains and 32 mycobacterium clinical isolates were analyzed by fluorescence quantitative PCR-probe melting curve method（including reaction A, B, C）. Result The results of 22 mycobacteria and 11 non-mycobacteria reference strains identified by fluorescence quantitative PCR-probe melting curve method were consistent with that by DNA sequencing, which suggests a new method for mycobacterium species identification was seccessfully developed. Of 32 mycobacterium clinical isolates identified by fluorescence quantitative PCR-probe melting curve method and DNA sequencing, 4 strains were identified as MTBC, 7 were M. intracellulare, 6 were M. abscessus, 4 were M. scrofulaceum, 4 were M. fortuitum, 3 were M. kansasii and M. gastri, 2 were M. gordonae, but the probe melting curve in reaction A of 2 strains display the Tm value of mycobacterium genus, and their probe melting curves in reaction B and C exhibited different Tm value with 22 strains of mycobacterium strains, DNA sequencing confirmed that the gene sequences of these two unknown mycobacterium strains were different from that of M. gordonae reference strains, but consistent with that of M. gordonae strain reported. Conclusion It might be a simple, rapid, sensitive and specific method for the identification of most mycobacterium species by fluorescence quantitative PCR-probe melting curve technique, and it can increase correct diagnosis rate of mycobacterium diseases, and direct the physicians rational chemotherapy.