常见致病性真菌核糖体RNA基因分子鉴定

Ribosomal RNA gene molecular identification of common pathogenic fungi

目的评价核糖体RNA基因（rDNA）分子鉴定用于常见致病性真菌鉴定的准确性。方法选取致病性酵母类真菌、丝状真菌和无绿藻共60株，其中包括13株标准菌株和47株临床／环境分离株，分别对其进行表型鉴定和rDNA分子鉴定，包括内转录间隔区1（ITSl）、内转录间隔区2（ITS2）及28S亚基rDNA5’端的D1／D2区（D1／D2）靶序列的测定与比对，比较表型鉴定及单个、多个rDNA靶序列分子鉴定的准确率。结果在23株酵母类真菌分离菌株中，D1／D2分子鉴定准确率最高（20／23，87．0％），而后依次为ITSl联合ITS2（19／23，82．6％）、单用ITS2（18／23，78．3％）、单用ITSl（14／23，60．9％）；在22株丝状真菌分离菌株中，ITSl联合ITS2分子鉴定准确率最高（16／22，72．7％），而后依次为ITsl（15／22，68．2％）、ITS2（14／22，63．6％）、D1／D2（13／22，59．1％）。而ITSl、ITS2、D1／D2三者线性联合鉴定准确率更高，可准确鉴定91．3％（21／23）的酵母类真菌分离菌株和72．7％（16／22）的丝状真菌分离菌株。结论rDNA分子鉴定应用于常见致病性真菌的鉴定准确率高，尤其是ITS联合D1／D2具有较好的应用前景。

Objective To evaluate the accuracy of ribosomal RNA gene （rDNA） molecular identification in identification of common pathogenic fungi. Methods Sixty pathogenic fungi including yeasts, filamentous fungi and Prototheca species were included in the study, among which 13 were reference strains and 47 were clinical/environmental isolates. Both phenotypic identification and rDNA molecular identification, including sequencing and alignment of internal transcribed spacer 1 （ITS1）, internal transcribed spacer 2 （ITS2） and D1/D2 region on 5＇ end of 28S subunit rDNA （D1/D2） target regions were performed for all the 60 strains. The accuracy of single region, multiple regions mo]）ecular and phenotypic identification methods was compared. Results The highest accuracy of species-level identification for yeast isolates was yielded by D1/D2 molecular identification（20/23, 87%）, followed by combining ITS1 with ITS2 （19/23, 82.6%）, ITS2 alone（18/23, 78. 3~）, and ITS1 alone （14/23, 60. 9%）. While for filamentous fungal isolates, the highest species-level identification accuracy was yielded by combination of ITS1 and ITS2 molecular identification （16/22, 72.7%）, followed by ITS1 alone（15/22, 68.2%）, ITS2 alone（14/22, 63.6%） and D1/D2 （13/22, 59.1%）. Combination bf ITS1, ITS2 and D1/D2 molecular methods showed the best performance, which yielded 91. 3% （21/23）species-level identification for yeast isolates, and 72. 70//oo （16/22）for filamentous isolates. Conclusions rDNA molecular identification has high accuracy for identification ofcommon pathogenic fungi, especially combination of ITS and D1/D2 regions, which might be a promising approach in the future.