Rapid Identification of Candida Species with Species-Specific DNA Probes

Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilliermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. norvegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans.

随着非白假丝酵母菌（Candida albicans）引发的感染病例逐年增多，其中包含天然对唑类抗真菌药物（azole antifungal drugs）耐药的假丝酵母菌属（Candida）菌种，快速鉴定假丝酵母菌物种的重要性日益凸显。本团队此前开发了一种酶联免疫吸附试验（enzyme immunoassay, EIA）格式的聚合酶链反应（Polymerase Chain Reaction, PCR）检测方法，通过使用通用真菌引物和针对核糖体RNA（ribosomal RNA, rRNA）基因内转录间隔区2（Internal Transcribed Spacer 2, ITS2）的物种特异性探针，实现对五种最常见假丝酵母菌物种扩增产物的检测。本团队额外设计了可检测API 20C糖同化鉴定试剂盒中包含的7种假丝酵母菌：C. guilliermondii、C. kefyr、C. lambica、C. lusitaniae、C. pelliculosa、C. rugosa及C. zeylanoides的探针；同时针对5种无法通过API 20C鉴定的假丝酵母菌——C. haemulonii、C. norvegica、C. norvegensis、C. utilis及C. viswanathii设计了特异性探针，并针对新报道物种杜氏假丝酵母菌（C. dubliniensis）设计了专属探针，最终构建了包含18种假丝酵母菌物种探针的检测体系。该PCR-EIA检测方法可准确鉴定受试各物种的多株菌株，其中包含5株经现有API 20C数据库鉴定为白假丝酵母菌，但通过基因型与非常规表型特征判定为杜氏假丝酵母菌的菌株。物种鉴定时长从传统鉴定方法的平均3.5天缩短至PCR-EIA方法的7小时。该方法操作简便、快速且具备临床实用性，适用于开展分子鉴定技术的临床实验室进行假丝酵母菌物种鉴定，同时为区分新报道物种杜氏假丝酵母菌与白假丝酵母菌提供了全新的技术手段。