Establishment And Clinical Application Of Real-time PCR For Detecting Pathogenic Fungus

Objective: To set up a real-time method using SYBR Greenâ… technology to detect disease fungus and evaluate its clinical application value.Method: We extracted disease fungus DNA from 5 positively incubated samples by using phenol-guanidine thiocyanate method. A pair of universal primers complementing to the 18S rDNA of fungus ribosomal were chose to set up a experimental method by using SYBR Greenâ… real-time PCR after optimized conditions. 37 clinical samples were tested by using this method and compared to conventional method, the purpose is to evaluate test effectiveness of this method.Result: DNA extracted with guanidine thiocyanate and phenol method have good purity and quality for PCR experiment, and whole process of DNA extraction can be completed in one hour. The real-time PCR has a good amplification curve without non-specific amplification. The correlation coefficient of standard curves was 1.0 and its sensitivity was 20pg DNA. All the 5 positive incubated disease fungus samples were detected, the Tm value of Monilia was different from mold fungus which was the criteria for discrimination. 9 of the 37 clinical samples were positive by microscopic examination that came from cornea samples. Of the 12 positive cases confirmed by fungus culture, 9 cases were from cornea samples while the other 3 ones from samples of deep mycosis patients. The results of Real-time PCR were consistent with that by fungus culture.Conclusion: Guanidine thiocyanate and phenol method to extract disease fungus DNA is convenient, quick, low cost and good quality which is easy for routine work in clinical laboratory. This real-time PCR reaction system is highly specific, reliable, high sensitivity for detecting pathogenic fungus which could be useful for the diagnosis of clinical fungus infection.