焦磷酸测序技术快速检测鉴定临床尿液标本中真菌方法学的建立与应用

Rapid detection and identification of fungi in urine samples by pyrosequencing

目的建立一种快速检测鉴定临床尿液标本中常见真菌的方法学体系。方法收集临床尿液样本,提取DNA,利用PCR方法结合焦磷酸测序技术检测真菌的rDNA内转录间隔区,通过序列比对鉴定真菌种属。结果通过PCR-焦磷酸测序与常规培养相比较,1320例临床样本培养阳性180例,阳性率为13.6%,焦磷酸测序鉴定阳性192例,阳性率为14.5%。两种方法检测真菌阳性符合率为100.00%,阴性符合率为98.95%,总体符合率为99.09%,Kappa值为0.963,一致性较好。对13株标准菌株鉴定结果与实际结果相符。结论研究所建立的PCR-焦磷酸测序直接检测临床尿液标本真菌的方法,具有快速、准确性高、重复性好、高度自动化、低成本、高灵敏度、高通量(可一次性检测96个样品)等优点,无需常规培养,能在3h内一次性区分临床常见真菌,可作为一种快速检测鉴定临床标本中真菌的方法。

ObjectiveTo establish a rapid method for the clinical detection and identification of fungi in clinical urine samples.MethodsDNA was extracted from clinically collected urine sample, and the fungal ribosomal internal transcribed spacer was amplified by polymerase chain reaction (PCR) and followed by pyrosequencing. The fungal species were identified by sequence alignment.ResultsThe identification results were compared between PCR-pyrosequencing and conventional culture method. Among the 1320 urine samples, 180 were detected positive by conventional method with the positive rate of 13.6%, while 192 were positive by the pyrosequencing based method with the positive rate of 14.5%. The overall coincidence rate of the two methods was 99.09%, with the positive coincidence rate of 100% and the negative coincidence rate of 98.95%. The Kappa value was 0.963, suggesting a good consistency. The results of 13 standard strains were consistent with the actual results.ConclusionsA rapid culture-free method for the detection of fungi in urine sample has been successfully established. This method is based on PCR-pyrosequencing technology with highly accuracy, sensitivity and reproducibility. It is highly automated, cost effective and with high throughput (96 samples per run). The fungal pathogen in urine is identified by single step test within 3 hours without conventional culture. Thus, it is applicable in the clinical laboratory.