常见曲霉菌的实时定量PCR诊断技术的研究

摘要

Objective The detected method of real-time quantitative PCR technology on common Aspergillus Species was developed. Methods The employing universal, fungus-specific primers and DNA probes, directed to the conservative internal transcribed spacer 2(ITS2) region of ribosomal DNA from A, terreus, A flavus, A. Fumigatus, A. Terreus, A. Nidulans and A niger were designed. Five kinds of common Aspergillus Species were detected by the quantitative real-time PCR and common PCR technology. Results DNA from each Aspergillus species for which DNA probes were designed produced am-plicons of approximately 350bp in size. Melting temperature of A, terreus, A. Flavus, A. Fumigatus,A terreus, A. Nidulajis and A. Niger was respectively 57℃ ± 0. 12℃ , 59℃ ± 0. 13℃ , 63℃ ± 0. 17℃ , 66°C ± 0. 15℃ , 66℃ ± 0. 14℃ and 68℃± 0. 12°C by melting peak analysis for five different Aspergillus spp. The minimum reaction concentration of DNA response template of A terreus, A flavus, A fumigatus, A nidulans and A niger was respectively 82. 4, 621. 3, 51. 2, 520. 8 and 19. 5 fg/μl by sensitivity analysis of common Aspergillus species. Conclusion The developed technology of real-time quantitative PCR is simple, rapid, and sensitive for the identification of medically important common Aspergillus species, which was a new option in invasive aspergillosis diagnosis.%目的 建立常见曲霉菌的实时定量PCR诊断技术.方法 针对黄曲霉菌、烟曲霉菌、构巢曲霉菌、黑曲霉菌和土曲霉菌内转录间隔区2(internal transcribed spacer 2,ITS2)保守基因设计引物和探针,通过普通PCR扩增其ITS2基因片段,作为实时定量PCR反应模板,通过实时定量扩增和分析其解链曲线来鉴定常见曲霉菌,同时分析该方法灵敏度.结果 真菌通用引物扩增五种常见曲霉菌ITS2基因DNA片段,大小为350 bp左右.实时定量PCR分析和探针解链曲线分析结果显示土曲霉解链温度(melting temperature,Tm)值为57℃±0.12℃,黄曲霉Tm值为59℃±0.13℃,烟曲霉Tm值为63℃±0.17℃,土曲霉Tm值为66℃±0.15℃,构巢曲霉Tm值为66℃±0.14℃,黑曲霉Tm值为68℃±0.12℃.方法 灵敏度分析显示各曲霉菌最低模板DNA反应浓度分别为烟曲霉51.2· fg/μl,黄曲霉621.3 fg/μl,黑曲霉19.5 fg/μl,土曲霉82.4 fg/μl和构巢曲霉520.8fg/μl.结论 创建一种可快速鉴定五种常见曲霉菌的实时定量PCR诊断技术,该方法特异度好、灵敏度高,符合临床需要,能为侵袭性真菌感染(invasive aspergillosis,IA)提供新的选择.