Accuracy of a terminal-restriction fragment length polymorphism (T-RFLP) method to characterize oral fungi

Abstract

Terminal-restriction fragment length polymorphism (T-RFLP) method has been widely used for study profiling of microbial community, however, have some limitations and have never been studies in oral fungi. It is important to evaluate the accuracy of this method as a tool for investigating the oral fungal community. To evaluate accuracy of T-RFLP with quantitative PCR (qPCR) for characterize oral fungi. DNA was harvested from medically important fungi commonly found in the oral cavity: Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and Fusarium spp. cultures. Cryptococcus, Aspergillus and Fusarium DNA were mixed, and 10-fold dilutions of Candida specific DNA were added to the DNA mixtures to represent fungal community models with differential Candida abundance. Species-specific DNA and total fungal DNA was estimated by T-RFLP and SYBR qPCR. For T-RFLP, mixtures were amplified using pan-fungal fluorescent-labeled primers specific to ITS regions and analyzed after digestion with MspI or HaeIII. For qPCR, mixtures were amplified using species-specific and pan-fungal primers, and analyzed. Based on the weight of C.albicans genomic DNA, seven dilutions of Candida-specific targets in fungal community models, corresponding to 100 to 106 copies, were tested. Detection by qPCR was accurate when the abundance of Candida-specific targets was between 102 to 106 copies, whereas the range for T-RFLP detection was between 105 to 106 copies. Candida-specific T-RF proportion in DNA mixtures appeared to be underestimated by 10-fold. Aspergilllus-specific T-RF product was absent from the fungal community model suggesting that the T-RFLP method, or the pan-fungal primers, may have bias against Aspergillus detection. T-RFLP is advantageous for the detection of unknown fungal community, with accuracy when targets are highly abundant. However, it may have bias in the detection of some fungi. The species-specific qPCR assay is then required to validate the detection and target abundance.