Experimental Investigation On The Rapid Diagnosis And Identification Of Pathogenic Molds

Part I Comparison and optimization of DNA extraction method for filamentous fungiObjective To find the best DNA extraction methods for filamentous fungi. Methods We compared the DNA quality of fungi cultured for 3d and 7-14d. As a sample, Aspergillus. niger DNA was extracted by four methods, including benzyl chloride method, CTAB method, Biospin kit and microwave method, and compared the purity and output of the products via agarose gel electrophoresis and PCR. The best method was used to extract 8 pathogenic mould. Results The extracted DNA from colony cultured for 3d was purer than that for 7-14d. Comparing the DNA agarose gel electrophoretogram made by the four methods, all the DNA was amplifiable using a standard PCR and the DNA extracted by CTAB showed the best result. Conclusions CTAB is suit to extract mould DNA and has the advantages of high extraction rate, good quality and simple operation.Part II Experimental investigation on the identification for eight invasive pathogenic molds by PCR-RFLPObjective To establish a diagnostic method for invasive mould infections using PCR-RFLP. Methods The ITS regions of Aspergillus. fumigatus, Aspergillus. flavus, Aspergillus. Terreus, Aspergillus. niger, Aspergillus. Versicolor, Aspergillus. Nidulans, Scedosporium apiospermum and Fusarium moniliforme were amplified with universal fungal primers, and RFLP analysis with Hhaâ… , Hinfâ… ,Haeâ…¢, Taqâ… and Mspâ… . Then 22 clinical strains and 2 environmental isolates were analyzed with PCR-RFLP. Results RFLP analysis of the PCR products with Hha I and Hinf I allowed discrimination of 8 invasive pathogenic molds. DNA extraction, PCR and restriction digestion could be carried out within one day.22 clinical strains and 2 environmental isolates were completely identical to those obtained by conventional morphological methods. Conclusions PCR-RFLP is a rapid method for the identification of deep mold infection.Partâ…¢Experimental investigation on the detection for pathogens of invasive aspergillosis by multiplex PCR Objective To develop a multiplex PCR method to identify simultaneously multiple aspergillus pathogens in a single reaction. Methods Four pairs of primers were selected in one multiplex PCR to identify the most frequent Aspergillus pathogens, A. fumigatus, A. flavus, A. terreus and A. niger. Mutiplex PCR with these 4 primer pairs was used to detect 1 template and 2 or 3 template mixtures. Sensitivity of multiplex PCR was tested with serial dilutions of PCR products. A total of 24 fungal isolates were tested with this method. Results The detection system demonstrated high specificity, that 4 aspergillus were identified through distinct amplicons of 250 bp,200 bp,450 bp and150 bp, respectively. Multiplex PCR could amplify the corresponding 1,2 or 3 DNA fragments, and the sensitivity of multiplex PCR was 100pg, which was less sensitive to single-primer-set PCR(10pg). Conclusions Multiplex PCR might be a rapid, sensitive and specific method for the diagnosis of invasive aspergillosis.Part IV Experimental investigation on the detection of mould by colony PCRObjective To detect the application of colony PCR in the field of identification of pathogenic mould. Methods 19 mould strains were examined using direct colony PCR to amplify the ITS region without DNA extraction. We compared parts of the strains by electrophoresis and PCR-RFLP using colony PCR and standard PCR. All of the strains were verified by analysis of fungal ITS sequences. Results 16 of the 19 strains(84.2%) yielded positive results on colony PCR. The cleavage maps of colony PCR-RFLP coincided with the standard PCR, and sequence analysis of the 16 strains were similar to those obtained by conventional morphological methods. Conclusions Colony PCR is a rapid method for the identification of mould, which not only reduces DNA template preparation time before PCR from mould colonies, but also reduces the cost of PCR.Part V Clinical application of colony PCR in fast fungi identificationObjective To study the application of colony PCR in the detection of clinical specimens. Methods Clinical specimens from a boy with subcutaneous fungal infections were collected, and examined using direct colony PCR to amplify the ITS region. Skin and nail samples were taken from patients with superficial mycosis and detected by colony multiplex PCR based on 3 pairs of primers, universal fungus-specific primer, dermatophyte-specific primer and yeast-specific primer. Results Pathogens identification of subcutaneous fungal infections using colony PCR was similar to those obtained by conventional morphological methods, and completed in 5d. Use of colony PCR reduced the time for identification of superficial mycosis to 3-5d, which was considerably faster than morphology identification (another additional 10d). Conclusions Colony PCR is a rapid and convenient method for the detection of clinical specimens, and may be routinely used in clinical laboratory.