多重荧光定量PCR对三种主要致病性曲霉菌的检测研究

Development of multiplex fluorescent quantitative PCR for detection of common pathegenic aspergillus species

目的建立一种能同时鉴定烟曲霉、黄曲霉和黑曲霉菌的多重荧光定量PCR诊断方法，并将之应用于侵袭性曲霉菌病的诊断研究。方法构建多重荧光定量PCR方法，对该方法进行实验评价，并将之应用于侵袭性曲霉菌病患者的痰标本检测，初步评价该方法的临床诊断效能。结果建立的多重荧光定量PCR方法，制定标准曲线，R^2。在0．989～0．999之间，斜率分别为-3．117、-3．583、-3．674；只与烟曲、黄曲、黑曲的DNA发生特异性反应，无非特异性扩增；最低检测浓度为1×10^2孢子／ml；重复性检测试验，获得Ct值标准差为0．12～0．20，变异系数为0．37％～0．80％；对分离自26例侵袭性曲霉菌病患者的56份深部痰标本进行检测，敏感率为85．7％（48／56），特异性为92．0％（46／50），阳性预测值为92．3％（48／52），阴性预测值为85．2％（46／54）。结论研究建立的多重荧光定量PCR方法检测侵袭性曲霉菌病患者的深部痰标本有较高的敏感性、特异性和阴、阳性预测值，且快速、简便，将有助于侵袭性曲霉菌病的早期准确诊断。

Objective To establish a method for the early diagnosis of invasive aspergillosis （IA） using multiplex fluorescent quantitative polymerase chain reaction （FQ-PCR）, which can identify different species, including A. fumigatus, A. flavus and A. niger. Methods To establish a method of multiplex FQ-PCR and make a experimental evaluation. To deteete the patients＇ sputum samples who with IA using thetechnique that had been established, and the sensitivity, specificity, positive and negative predictive value of the technique were estimated with all the collected samples. Results The R2 of standard curve of each species for multiplex FQ-PCR was 0. 989 - 0. 999 , the slope were -3. 117,-3. 583,-3. 674, respectively. The present multiplex FQ-PCR system was exclusively positive for DNA extracted from the three species of aspergillosis, but not the other fungi, bacterium and human PBMC. The lowest limit of the system for the four aspergillosis was 1 × 10^2 spores/ml. The standard error of Ct value was 0.12-0.20 and coefficient of variation was 0. 37%-0. 80% , which demonstrated a high repeatability. Sensitivity, specificity, positive and negative predictive value of the the multiplex FQ-PCR were 85.7% （48/56）, 92.0%（46/50）, 92.3% （48/52） and 85.2% （46/54） respectively. Conclusions The multiplex FQ-PCR established for the detection of IA has good sensitivity, specificity and predictive value when using the sputum sample from IA-infected patients. This proved that it could greatly benefit the early diagnosis for IA infection in immunoeompromised patients and used as a routine diagnosis method in the future.