以巢聚式聚合酶鏈結反應法直接鑑定臨床檢體表皮皮癬菌的研究

傳統上，在鑑定皮癬菌的菌種方法完全依賴菌落特徵及顯微鏡下形態學的檢查，其中比較重要的特徵包括皮癬菌的生長速率、菌落的顏色、大小、大孢子及小孢子的形態等；另外，皮癬菌的某些生理特性也很重要。但是，同一皮癬菌的菌種在形態上的表現差異性常常很大，因此，皮癬菌的鑑定就顯得相對困難。此外，皮癬菌的培養基及其他生長條件也會影響大孢子及小孢子的形態表現，而影響鑑定。因此，本實驗想要利用分子生物學的方法，找出可鑑定的基因序列，以輔助鑑別臨床上具有重要性的菌種。本實驗利用自動系統，萃取微量的genomic DNA分別來自skin scrapping及cultured fungi。在保守區的一段特異性DNA sequence，設計出ITS-5F & ITS-4F primer及ITS-1F & ITS-2F進行巢聚式聚合酶鏈結反應法(nested PCR)。將這一段在各黴菌菌種間差異很大的DNA片段放大，然後對菌株的ITS1-5.8S-ITS2 定序，再與傳統形態學做種名對照再確認，ㄧ方面針對已知菌名的菌株，隨機抽樣做ITS1-5.8S-ITS2 DNA序列比對，另ㄧ方面比較與黴菌室以傳統方法鑑定的表皮皮癬菌菌名符合程度。利用連續稀釋法進行實驗敏感度的測試，在本實驗方法可測得的最少量DNA檢體為63個複製。分析130個病人的196個表皮皮癬菌菌株，表現型(phenotype)的鑑定率在32%，基因型(genotype)的鑑定率在69%。目前證實一些無法以表現型鑑定的表皮皮癬菌，可以不需經由菌種的培養，以基因型較正確地鑑定出種名及屬名，許多因培養過程產生的鑑定問題，像是細菌污染、孢子變形、甚至完全不產生孢子、黴菌培養曠日費時、菌絲死亡(non-viable hyphae)等，因而不再成為表皮皮癬菌種鑑定及確認的干擾因素。以基因型鑑定法鑑定皮癬菌較傳統黴菌實驗室方法穩定、可靠，而且可以節省許多人力及時間。

關鍵字

基因型；表現型；黴菌培養；巢聚式聚合酶鏈結反應； ITS ；基因序列

並列摘要

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.

並列關鍵字

genotyping ； phenotyping ； fungal culture ； nested PCR ； ITS ； sequencing

參考文獻

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以巢聚式聚合酶鏈結反應法直接鑑定臨床檢體表皮皮癬菌的研究

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