Study On The Rapid Diagnostic Methods Of Deep-seated Mycosis

Objective To establish rapid methods to detect and identify deep-seated fungi in clinical samples by using two-step PCR.Methods According to the sequence of the fungal large subunit rRNA gene, we designed an universal primer for the fungal kingdom and species-specific primers for Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus respectively. Using two-step PCR and common fungi culture methods, we detected and identified fungal strains, simulated clinical samples and 95 clinical samples from 90 patients who suffered from skeptical deep-seated mycosis. Two methods were compared with each other.Results All strains of standard and clinical isolated fungi strains showed a 550 bp to 650 bp DNA fragment with the universal primer pair. Using 3 specific primer pairs, we obtained specific fragment of 156bp,183bp and 192bp from strains of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus respectively. Common bacteria, virus and human genomic DNA had no specific amplification. The detection limit of Candida albicans in simulated blood sample, Cryptococcus neoformans in simulated cerebrospinal fluid sample and Aspergillus fumigatus in simulated bronchoalveolar lavage (BAL) sample with universal primer PCR was lOCFU/ml, lOCFU/ml and 15CFU/ml respectively. 29 samples were universal PCR positive and 18 of these samples were also culture positive. The universal PCR positive rate was significantly higher than the culture positive rate. The three pathogen fungal species identified in fungi cultures were the same as that revealed by two-step PCR.Cone I ua i on Two-step PCR is a rapid, sensitive and specific method for clinical laboratory to detect and identify fungal pathogens in clinical samples.