Molecular Identification Of Common Pathogenic Fungi And Comparative Study Of In Vitro Drug Susceptibility Assay Of Penicillium Marneffei By FUNGUS3

Objective:Evaluating the most useful universal primers for identificat ion common pathogenic fungi,and establishing stable PCR(Polymerase Cain Reaction) molecular diagnosis platform for Mycosis.Methods:1. Amplification Testing the most useful universal primersf or PCR. First of all, we collected the clinical pathogenic strains, and ext-racted the DNA from the fungal sample used the Kit. The second, PCR amplify the internal transcribed spacer(ITS) area of the DNA samples usi-ng the selected primers. At the end, these products were be examined by delete gel electrophores. It was successful if there was a single strip ap-peared. Doing a comparison between these primers on the basis of the re-sults.2.DNA sequence align Delete sequence the PCR products and blast on Pubmed Blast tool, got the name of the pathogen and compared with the traditional culture diagnostic result, and compared it with the tradition-al diagnostic methods.Result:There were no amplification in all the negative control. Am-ong the4pairs of primers,ITS4and ITS-86were the most stable primers which could amplified all the samples.Amplification and sequence the ITS region of the fungi could identify most of the common fungal species.Su-ccessfully, some species could been identified by the molecular diagnostic method, which had been misdiagnosed by the traditional culture diagnose.Conclusion:The ITS universal primers PCR combined gene sequencin Objective:Evaluation of ATB FUNGUS3with the Clinical and Lab-oratory Standards Institute (CLSI)M27-A3document for testing in vitro P-enicillium marneffei antifungal susceptibility.Methods:The agreement between ATB FUNGUS3and CLSI M27-A-3was assessed with the in vitro susceptibilities of21Penicillium marnef-fei strains to the common antifungal agents, including amphtericinB(AMB), flucytosine(5-Fc), fluconazole(FLC), itraconnazole(ITC) and coriconazole (VRC). No discrepancies of MIC endpoints between the two methods w-ere considered to be essential agreements.Results:?MIC readings for the quality control strain was within the limits;?The two method shown highly intra-laboratory reproducibility;?When the final inocula concentrations in the drug dilutions of ATB FUNGUS3were0.5?2.5×104CFU/mL, the essential agreements of th e five antifungal agents were85.7%for AMB,95.2%for5-Fc,95.2%fo r FLC,100%for ITC and VRC. ITC and VRC presented the highest con c-ordan,and AMB was the lowest one.The overall essential agreements wit h the two method were95.2%.But there were2minor errors and3maj or errors.?In16of all the Penicillium marneffei strians, we tried inocul a concentrations of3x105CFU/mL in the drug dilutions of ATB FUNG US3.Then compared the results with CLSI M27-A3.We found that the ess entialagreements of the five antifungal agents have been improved. They were87.5%for AMB,93.75%for FLC,100%for5-Fc, ITC and VRC. The overall essential agreements with the two method were96.25%.But there were1minor errors and2major errors.?All of the antifungal ag ents' MICs range in ATB FUNGUS3were too high to get the exact num erical value excepting FLC.Conclusion:The ATB FUNGUS3method is a simple, rapid, accurate, and reproducible method for the determination of MICs for Penicillium marneffei which can be considered a valuable alternative to the CLSI me-thod.