Identification of Clinically Important Dermatophytes Using Non-Culture Based Nested Polymerase Chain Reaction
| Autor: | Chia-Yi Yang, 楊佳懿 |
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| Rok vydání: | 2005 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 The routine procedures for species identification of dermatophytes rely on the examination of colony and microscopic morphology. Important characteristics include the growth rate, colony pigmentation, size and shape of macroconidia or microconidia. Also important are several physiological properties. Dermatophyte strains frequently vary in the expression of these phenotype characteristics; the identification of dermatophytes is therefore often difficult. In addition, the media and other growth conditions can affect the macroscopic and microscopic morphology of dermatophytes. In the development of specific nucleotide information, species- or strain-specific DNA variabilities can be detected by performing the nested polymerase chain reaction (nested PCR) with arbitrary primers. We introduce a direct DNA sequence-based approach from clinical specimens for the accurate and timely identification of medically important fungi by nested polymerase chain reaction assay with a rapid automated DNA extraction system. Two pairs of universal fungal primers, ie, ITS-F5&ITS-F4 and ITS-F1&ITS-F2 were used to amplify ribosomal DNA directly from clinical specimens even if they could not be identified by conventional culture. A sensitivity test was determined by serial dilution in nested PCR. The least detectable amount of fungi DNA by nested PCR would be 6 copies. An evaluation using 196 clinical specimens revealed the total culture yield rate was 32% and genotyping rate was 69%. It showed that the skin scrapings with scanty amount of dermatophyte hyphae could be detected without compromising sensitivity and was less labor-intensive. Even if they cannot be identified by accepted phenotypic features, the pathognomic dermatophytes can be identified better by non-cultured based nested PCR method than traditional procedures. Because dermatophyte species could be now identified without countering the problems of culture due to non-viable hyphye, non-sporulation, atypical spore and time consuming. Identification methods based on the genotype of an isolate of dermatophytes are considered more stable. We conclude that this approach is a rapid and might be a more accurate diagnosis than most phenotypic methods for identification of many medically important dermatophytes infection frequently encountered in a routine diagnostic microbiology laboratory. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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