...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Clinical Microbiology >Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens
【24h】

Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens

机译：基于PCR的线型探针鉴定真菌病原菌的开发

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida,Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans,Aspergillus fumigatus, Aspergillus versicolor,Aspergillus nidulans and Aspergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans, A. fumigatus, and C. neoformans. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.
机译：我们报告了反向杂交谱线探针测定法（LiPA），与PCR扩增结合使用时，它可以检测并鉴定具有临床意义的真菌病原体，包括 Candida ，曲霉菌和隐球菌物种。 DNA探针是由白色念珠菌，寄生念珠菌，光滑念珠菌，念珠菌的内部转录间隔区（ITS）设计的Tropicalis ，克鲁迪假丝酵母，杜比里尼假丝酵母，新型隐球菌，烟曲霉，曲霉云芝，构巢曲霉和黄曲霉。将探针掺入LiPA中以检测生物素化的ITS PCR产物，并评估探针的特异性。我们为ITS 1和来自 C的基因组DNA的完整ITS扩增子建立了LiPA检测极限。白色的， A。烟熏和 C。新甲虫。 LiPA的进一步评估是在临床真菌分离物中进行的。用LiPA检测了由二形酵母，皮肤真菌和丝状真菌组成的127个分离株，正确鉴定出77个双形酵母和23个丝状分离株。其余的27种分离物代表的真菌种类，其探针未包含在LiPA中。将真菌PCR-LiPA技术应用于接种了 Candida 细胞的血样，该血样通过微珠打浆进行预处理，以机械方式破坏细胞，通过先前描述的硫氰酸胍-二氧化硅法或DNA提取DNA。市售的QIAmp组织套件。 LiPA分析中提取的DNA的PCR扩增和随后的DNA探针杂交产生2至10个细胞/ ml的检测限。 PCR扩增中包括内标对照，以监测PCR抑制情况。这种真菌PCR-LiPA分析方法功能强大且灵敏，可以轻松整合到临床测试实验室中，有可能在当日诊断出真菌感染。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号