真菌通用引物结合高分辨熔解曲线分析检测鉴定常见曲霉菌

Detection and identification of aspergillus with panfungal primers and high-resolution melting curve analysis

目的探索一种能够快速检测鉴定临床常见侵袭性曲霉菌病原的新型分子生物学方法。方法以真菌核糖体RNA基因的内转录间隔区2为靶基因,在5.8S、28S rRNA基因上设计泛真菌通用引物,扩增临床常见曲霉菌。同时用新生隐球菌基因组DNA、人基因组DNA及临床常见细菌基因组DNA进行引物特异性检测。将通用真菌引物扩增产物进行高分辨熔解曲线分析。以新鲜提取的烟曲霉基因组DNA为模板按照1∶10的比例梯度稀释,进行敏感性检测。检测7个浓度梯度的烟曲霉基因组DNA。结果设计的真菌通用引物可以扩增临床常见的4种曲霉菌及新生隐球菌,但与人基因组DNA和临床常见细菌基因组DNA无扩增反应,检测限可低至1.5 pg/μl,且4种曲霉菌及新生隐球菌扩增产物的熔解曲线不同。该方法敏感性和特异性均较好。结论利用真菌通用引物的扩增产物结合高分辨熔解曲线分析可以达到检测鉴定临床常见4种曲霉菌的目的,此方法对侵袭性曲霉菌的快速检测鉴定具有重要意义。

Objective To develop a new method to detect and identify invasive Aspergillus species.Methods Panfungal primers for the 5.8 S and 28 S rDNA were designed and tested for the specificity for the clinically important Aspergillus species and Cryptococcus neoformans.Then perform high resolution melt curve analysis on the panfungal amplicon.The DNA template extracted from Aspergillus fumigatus was 10-fold serially diluted and amplified to get its sensitivity.Results The panfungal primers were able to amplify four clinically important Asergillus species and Cryptococcus neoformans.There was no nonspecific amplification of human genome DNA and bacteria genome DNA.The detection limit was 1.5 pg / μl.The high resolution melting curve for the four Aspergillus species and Cryptococcus neoformans differs with good sensitivity and specificity.Conclusion The four clinically important Aspergillus species could be detected and identified with the designed panfungal primers and analyzed by the amplicon-high resolution melting curves.It provides a rapid method for the detection and identification of invasive Aspergillus pathogens.