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

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.

本研究报道了一种反向杂交线探针检测法（reverse-hybridization line probe assay, LiPA），该方法结合聚合酶链式反应（polymerase chain reaction, PCR）扩增技术，可检测并鉴定具有临床意义的真菌病原体，涵盖念珠菌属（Candida）、曲霉菌属（Aspergillus）及隐球菌属（Cryptococcus）的相关物种。研究人员从白色念珠菌（Candida albicans）、副近平滑念珠菌（Candida parapsilosis）、光滑念珠菌（Candida glabrata）、热带念珠菌（Candida tropicalis）、克柔念珠菌（Candida krusei）、都柏林念珠菌（Candida dubliniensis）、新生隐球菌（Cryptococcus neoformans）、烟曲霉（Aspergillus fumigatus）、杂色曲霉（Aspergillus versicolor）、构巢曲霉（Aspergillus nidulans）以及黄曲霉（Aspergillus flavus）的内部转录间隔区（internal transcribed-spacer, ITS）设计了特异性DNA探针，并将其整合至LiPA体系中，用于检测生物素标记的ITS区域PCR扩增产物，同时对探针的特异性进行了评估。本研究确定了针对白色念珠菌、烟曲霉及新生隐球菌的基因组DNA，在ITS1区域以及完整ITS区域扩增子的LiPA检测限。后续我们对临床分离的真菌菌株开展了LiPA的进一步性能评估：本次共纳入127株菌株，涵盖双相型酵母菌、皮肤癣菌及丝状真菌，经LiPA检测后，该方法可正确鉴定77株双相型酵母菌与23株丝状真菌分离株；剩余27株菌株所属的真菌物种未在本LiPA体系中设计对应探针。研究团队将该真菌-PCR-LiPA技术应用于接种念珠菌细胞的血液样本：样本先经微珠研磨进行机械破壁，随后采用已报道的硫氰酸胍-硅胶法或商业化QIAamp组织试剂盒完成DNA提取。对提取的DNA进行PCR扩增，并在LiPA体系中完成DNA探针杂交后，该方法的检测限可达2~10个细胞/毫升。同时在PCR扩增体系中加入内标对照，用于监测PCR反应的抑制情况。该真菌PCR-LiPA检测法具备良好的稳定性与灵敏度，可轻松整合至临床检验实验室中，有望实现真菌感染的当日诊断。