Med Princ Pract. 2018;27(6):543-548. doi: 10.1159/000493426. Epub 2018 Sep 3.
Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates.
A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results.
Melting temperatures (Tm) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, Tm values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp.
The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.
白念珠菌和杜氏念珠菌是产芽管的致病性酵母种。准确鉴定这两种物种是必要的，因为白念珠菌是一种高度致病性的物种，而杜氏念珠菌对颊上皮细胞的黏附性增加，对唑类药物的敏感性降低，对氟胞嘧啶耐药。我们开发了一种用于快速检测和区分临床白念珠菌和杜氏念珠菌分离株的双重实时 PCR 检测方法。
通过使用两对种特异性引物和 SYBR Green 染料，通过实时 PCR 扩增产物的熔解曲线分析，开发了一种双重实时 PCR 检测方法，以区分白念珠菌和杜氏念珠菌分离株。还通过琼脂糖凝胶电泳分析扩增产物来确认实时 PCR 结果。
白念珠菌和杜氏念珠菌参考株的熔解温度（Tm）分别为 86.55°C 和 82.75°C。从 8 种其他常见念珠菌属参考株的 DNA 中未获得扩增产物。当将实时 PCR 应用于先前通过 Vitek 2 系统和/或 rDNA PCR 测序鉴定的 226 株临床分离株时，白念珠菌（n=113）和杜氏念珠菌（n=98）的 Tm 值分别为 86.68°C±0.529 和 82.616°C±0.535°C。结果通过琼脂糖凝胶电泳得到确认。从属于 9 种其他念珠菌属的 15 株分离物中未获得扩增产物。
本文所述的实时 PCR 检测方法不需要通过产芽管形成预先鉴定临床酵母分离物为白念珠菌/杜氏念珠菌，并且可以在 2 小时内准确报告结果。通过琼脂糖凝胶电泳检测扩增产物也适用于缺乏实时 PCR 设施的资源匮乏环境。
