热带假丝酵母菌实时荧光定量PCR检测方法的建立及临床应用初步评价

Study and clinical application of real-time PCR for Candida tropicalisinfection detection

目的建立、优化快速检测临床标本中热带假丝酵母菌的实时荧光定量PCR方法 ,并对其临床应用进行初步评价。方法用自行设计的高效引物、探针检测5种临床标本中的热带假丝酵母菌,对该方法的敏感性、特异性进行评价,并与真菌培养法进行比较。结果检测临床标本中热带假丝酵母菌的灵敏度可达10 copies/ml,与人类基因组、细菌及其他真菌无交叉阳性反应。与真菌培养法的检测一致性好(Kappa值为0.916,P<0.01)。结论实时荧光定量PCR检测热带假丝酵母菌灵敏度高、特异性强,可直接用于各种临床标本中热带假丝酵母菌的检测,而且可大大缩短报告时间,为临床诊断提供可靠依据。

Objective To develop a real-time PCR assay for rapid detection of Candida tropicalis in clinical specimens,and make a primary evaluation of the method.Methods Detecting the Candida tropicalis from five kinds of clinical specimens by using self-designed efficient primers and probe.The results were compared with those from fungal culture.The sensitivity,specificity and repeatability of real-time PCR assay were determined.Results Real-time PCR procedure was able to detect at least 10 copies of ITS2 gene and had no cross-reaction with human genomic DNA,other fungus and bacterium.Conclusion Real-time PCR assay can detect Candida tropicalis from different kinds of clinical specimens,and the time can be shortened by the technology.