Detection Of CAP10 Gene Of Cryptococcus Neoformans By Real-time Fluorescent Quantitative PCR And Its Preliminary Clinical Application

ObjectiveCopies of Cryptococcus neoformans CAP10 gene expression detection was developed using real-time Fluorescent Quantitative PCR technique with the objective of its preliminary application in the clinical laboratory diagnosis and therapeutic effect judgment.MethodsAccording to the constant sequence of five serological types(A,B,C,D,AD)of cryptococcus neoformans, specific primers and probes were designed. CAP10 gene was amplified with PCR and ligated into a pGEM-T Easy vector. A method for detection of CAP10 gene using FQ-PCR was established after determination of the sensitivity and specificity of the test as well as the reproducibility of the assay. And the results were analyzed by SPSS10.0 software.Results1. The sensitivity of this method was 101copy/μl and the CV of inter-assay and intra-assay were 0.31 % and 2.72 % respectively with excellent specificity and reproducibility.2. After therapy copies of Cryptococcus neoformans CAP10 gene decreased significantly in the group of improvement. The copies of Cryptococcus neoformans CAP10 gene had positive correlation with microbe amount and intracranial pressure. There was no significant difference in the declining of quantity between the group of amphotericin band fluconazole.ConclusionsA method of detection of cryptococcus neoformans CAP10 gene by real-time Fluorescent Quantitative PCR was established successfully and the results were stable and accurate. It might become a new method for evaluating the therapeutic effect and the prognosis of cryptococcus neoformans meningitis.