一种基于AllGlo^TM探针的侵袭性曲霉菌病诊断技术的建立与评价

The establishment and evaluation of diagnostic accuracy of AllGlo^TM probe-based techniques for invasive aspergillosis

目的建立并评价一种基于AllGlo^TM探针的侵袭性曲霉菌病诊断技术。方法对目标基因进行克隆化并制备成标准品，用自行设计的AllGlo^TM探针和引物对标准品进行核酸扩增，制定定量检测标准曲线，对方法的特异性、敏感性、重复性进行评价。结果黄曲霉和烟曲霉AllGlo^TM荧光核酸扩增的标准曲线分别为Y=-3．003X＋36．825和Y=-3．052X＋38．016，其批间变异系数分别为15．60％和12．94％，表示重复性良好，敏感性达10CFU／ml，相当于100-1000拷贝转录间隔区（ITS）2基因，与其他真菌、人类基因组及细菌无交叉阳性反应，特异性强。结论这种基于AllGlo^TM探针的侵袭性曲霉菌病诊断技术具有较好的重复性、特异性和较高的准确度。

Objective To establish and evaluate a diagnostic technique based on the AllGlo^TM probe for the invasive aspergillosis. Methods With the self-designed AllGlo^TM probes and primers and the standards, two standard curves of the real-time PCR based on AIlGlo^TM probes were established respectively for A. flavus and A. fumigatus, then its specificity, sensitivity and reproducibility were evaluated respectively. Results The findings indicated that the standard curce of A. flavus was Y = - 3. 003X ＋ 36. 825, and A. fumigatus＇ was Y = - 3. 052X ＋ 38.016, and their interassay coefficient of variation respectively were 15.60% and 12.94%, suggesting a good reproducibility. The lowest spore concentration they could be detected was 10 CFU/ml, which equated to 100-1000 copies of internal transcribed spacer （ITS）2 genes, suggesting a good sensibility. They didn＇t have cross-positive reaction with other fungus, human genome and bacteria, which indicated a good specificity. Conclusion The diagnostic technique based on the AllGloTM probe for the invasive aspergillosis possessed a good sensitivity, good specificity and deadly accuracy.