Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2
Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2
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DOI：
10.1128/jcm.01071-10
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发表时间：
2010-10-01
影响因子：
9.4
通讯作者：
Johnson, Elizabeth M.
中科院分区：
文献类型：
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作者：
Borman, Andrew M.;Linton, Christopher J.;Johnson, Elizabeth M.
从临床样品中快速鉴定酵母菌属分离株是特别重要的，因为它们具有固有的可变抗真菌药敏谱。在这里，我们已经评估了焦磷酸测序分析的一部分的内部转录间隔区2区（ITS 2）的病原性酵母菌的鉴定的效用。在严格盲法研究中，对包含43种不同真菌物种的总共477个临床分离株进行焦磷酸测序分析。通过焦磷酸测序产生的分子鉴定与使用常规生化试验（AUXACOLOR 2）和以下PCR扩增和核28 S大rRNA基因的D1-D2部分测序获得的分子鉴定进行了比较。超过98%（469/477）的菌株，包括40的43个真菌物种测试正确鉴定的焦磷酸测序只有35 bp的ITS 2。此外，用ITS 2焦磷酸测序特征序列对公共同步数据库进行BLAST搜索，结果显示在分析的ITS 2中仅存在最小的序列冗余。在所有情况下，焦磷酸测序特征序列对于所研究的酵母物种（或物种复合体）是独特的。最后，将焦磷酸测序技术与Whatman FTA纸技术相结合快速提取真菌基因组DNA，从纯培养物开始，可在6 h内完成分子鉴定。
Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures.