[Application of molecular biology techniques in the identification of pathogenic fungi and the diagnosis of fungal infection].
With the increasing incidence and mortality of fungal infection, the requirements for strict diagnostic approaches became a very urgent issue. Because of the traditional detective techniques, such as culture, gave poor diagnostic outcomes, the molecular biological techniques are expected to develop the potential diagnostic approaches. During the past decades, we have carried out serial studies on the molecular properties of pathogenic fungi, and we would like to review as following. Firstly, we applied several molecular tools in classification and identification of pathogenic fungi. We performed random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and other techniques in studying the typing, to classify and identify the properties of Dermatophytes, Candida spp., Cryptococcus neoformans, Dematiaceous fungi, and Aspergillus spp. Interestingly, we found the same T. rubrum strain might infect different sites of the host, while a site-specificity displayed in T. mentagrophytes. This finding indicated the genetic discrepancies among the fungi. Beside, we also found that the E. dermatitis strains with different virulences possessed some discrepancies at gene level. We then developed a PCR-based molecular procedure to identify the novel species in Exophiala spp. As the applicable strategy, we also investigated the rDNA sequence properties in several fungi. And as a result, we submitted for the first time to GenBank the complete sequence of Aspergillus fumigatus rDNA/ITSI/ITSII, which provided the basis for designing the species-specific probes and for its further clinical applications. Secondly, we have tried to develop the molecular diagnostic approaches based on our DNA sequence data which were used for identification studies previously. By analyzing the DNA sequence of Aspergillus fumigatus rDNA/ITSI/ITSII, we developed a nested PCR method to detect Aspergillus fumigatus genes. Our preliminary results indicated that this PCR-based molecular approach has great importance in the diagnosis of invasive aspergillosis. We also designed the species-specific probes and then established several in situ hybridization procedures. We found these hybridization methods could get the positive rate up to 81% (13/16), which suggests that these methods have potential diagnostic value for invasive candidiasis and aspergillosis. Based on our experiences, we would conclude that the molecular biological techniques possess great value to investigate the biological properties of pathogenic fungi, and we are looking forward to see more and more molecular tools will be used in the pathogenic mechanisms of fungal infections and antifungal activity studies.
Li
Ruo-yu
RY
Department of Dermatology, Peking University First Hospital, Peking University Research Center for Medical Mycology, Beijing 100034, China. lrywdh@public.bta.net.cn
Li
Dong-ming
DM
Yu
Jin
J
Liu
Wei
W
Ji
Zhao-hui
ZH
Wang
Duan-li
DL
D001228:Aspergillosis / Q000175:diagnosis / Q000382:microbiology; D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification; D002175:Candida / Q000235:genetics / Q000302:isolation & purification; D002177:Candidiasis / Q000175:diagnosis / Q000382:microbiology; D003881:Dermatomycoses / Q000175:diagnosis* / Q000382:microbiology; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016533:Mycological Typing Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length