光滑假丝酵母菌感染实时荧光定量PCR检测方法的建立

Establishment of real-time polymerase chain reaction for detection of Candida glabrata infection

目的:建立、优化快速检测临床标本中光滑假丝酵母菌的实时荧光定量PCR方法,并评价其临床应用价值。方法:以光滑假丝酵母菌ITSⅡ基因的一段高度保守序列为扩增靶序列,合成引物和探针,并与pMD19-T构建重组质粒作为标准品,建立检测光滑假丝酵母菌的实时定量荧光PCR方法。从线性范围、重复性、敏感性、特异性等4个方面对反应体系进行方法学评价。用建立的实时荧光定量PCR方法对1 042例临床标本(包括血液、尿液、痰液、胸水、支气管肺泡灌洗液)进行检测,并与真菌培养法进行比较。结果:建立的光滑假丝酵母菌实时荧光定量PCR方法的灵敏度可达10 CFU/ml;与人类基因组、细菌及其他真菌无交叉阳性反应。重复检测样本,其循环阈值的试验内及试验间变异系数均小于10%。用于临床标本检测时共检出96份阳性标本,与真菌培养法(检出93份阳性标本)的一致性好(Kappa值为0.925)。结论:实时荧光定量PCR方法检测光滑假丝酵母菌灵敏度高、特异性及重复性好;可直接用于各种临床标本中光滑假丝酵母菌的检测,能大大缩短报告时间,为临床诊断提供可靠依据。

Objective：To develop a real-time polymerase chain reaction（PCR） assay for rapid detection of Candida glabrata in clinical specimens, and to evaluate it in clinical application. Methods： Primers and probe were de signed against the ITS Ⅱ sequences of the Candida glabrata, and a recombined plasmid with pMD-19T was contructed. Then the real-time PCR for diagnosing infection of Candida glabrata was established. The linear range, sensitivity, specificity and repeatability of the method were assessed. Candida glabrata from 1 042 clinical specimens, including blood, urine, sputum, pleural effusion, bronchoalvelor lavage fluid, was detected by self-designed efficient primers and probe, and the results were compared with that of fungal culture. Results： Real-time PCR procedure was able to detect at least 10 CFU/ml of ITS Ⅱ gene without cross-reaction with human genomic DNA, other fungi, or bacteria. The intra- and inter-assay coefficients of variation of CT values were less than 10% with repetition. Ninety-six positive results were found with the new method, and the result was identical with that of fungal culture method for 93 specimens （kappa value was 0. 925）. Conclusions： Real-time PCR assay has good sensitivity, specificity and repeatability. It can detect Candida glabrata from many kinds of clinical specimens, and the detection time can be shortened.