Using PCR/Denaturing High-performance Liquid Chromatography For Detection And Identification Of Candida Species Of Clinical Suspected Samples

Objective To explore the clinical application of PCR/Denaturing high-performance liquid chromatography (PCR/DHPLC) in the detection and identification of candida species of clinical suspected samples.Method Sixty-three humoral clinical samples were included in this study. Polymerase Chain Reaction(PCR) was used to amplify the internal transcribed spacer(ITS) region of ribosomal DNA(r DNA) from the above-mentioned samples, using fungal universal primers. PCR products were verified by electrothoresis. If positive, it was then analyzed by DHPLC . The samples were detected by traditional culture as contrast at the same time. For verifying the veracity of this method, the amplified DNA was sequenced. The difference of positive rates of PCR/ DHPLC and culture methods was evaluated by fisher's exact test.Results The whole process of PCR/DHPLC took about 6.5 h, while which of culture methods took 2 days long. 15 out of 63 samples(23.81%) were positive by PCR/ DHPLC, among which 11 samples were Candida albicans, 2 samples were Candida tropicalis, 2 samples were Candida glabrata, Side peak was observed in 2 samples of Candida albicans. 13 out of 63 samples(20.63%) were positive by culture, among which 10 samples were Candida albicans, 2 samples were Candida tropicalis, 1 samples were Candida glabrata.. Acorrding to the analysis with SPSS13.0 software, there was no significant statistical difference in the positive rates of fungi between the PCR/DHPLC and culture methods (P=0.125) . The sensitivity of PCR amplification reaction by DNA dilutions was 2fg/μl. The results from DNA sequencing are in accordance with which from DHPLC.Conclusion To culture methods, the procedure of PCR/DHPLC is more rapid, convenient, sensitive and high-throughput for the detection and identification of the most frequently encountered candida species. It is of great value in the early diagnosis of candida species of clinical suspected samples, which can offer certain reference for making the Rx. PCR/DHPLC may become the beneficial supplyment of the traditional methods.