Identification of fungal pathogens by visible microarray system in combination with isothermal gene amplification.
Identification of fungal pathogens by visible microarray system in combination with isothermal gene amplification.
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DOI：
10.1007/s11046-014-9756-2
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发表时间：
2014-08
期刊：
影响因子：
5.5
通讯作者：
Gonoi, Tohru
中科院分区：
文献类型：
--
作者：
Sakai, Kanae;Trabasso, Plinio;Moretti, Maria Luiza;Mikami, Yuzuru;Kamei, Katsuhiko;Gonoi, Tohru
真菌引起的传染性疾病在免疫功能低下患者中的发病率不断上升，促使研究人员开发快速准确的诊断方法。病原真菌种类的鉴定是决定适当治疗的关键，但由于真菌培养和临床样品形态学鉴定的困难，不易获得满意的结果。在本研究中，我们建立了一个微阵列系统，通过在检测过程中使用化学显色反应，可以识别来自24属临床重要真菌病原体的42种。该阵列利用rRNA基因的内部转录间隔区在物种水平上鉴定真菌DNA。该阵列对355种目标和非目标真菌进行了特异性测试。全血103 CFU/ml、血清50 fg /ml直接检测成功，表明所建立的阵列系统对临床样品中感染真菌的鉴定具有敏感性。此外，我们用等温扩增代替PCR扩增和标记。pcr扩增和等温扩增目标基因的成功鉴定表明，我们的微阵列系统是一种有效和稳健的方法，可用于鉴定样品中的多种真菌物种。本文的在线版本（doi:10.1007/s11046-014-9756-2）包含补充材料，仅供授权用户使用。
The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 103 CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample. The online version of this article (doi:10.1007/s11046-014-9756-2) contains supplementary material, which is available to authorized users.
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