双重实时荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌

目的 建立一种快速、灵敏、特异的鉴定克柔念珠菌和光滑念珠菌的双重实时荧光定量PCR方法.方法 以核糖体基因内转录间隔区Ⅱ(ITSⅡ)为靶目标,设计并合成分别针对克柔念珠菌、光滑念珠菌的种特异引物和探针.建立双重实时荧光定量PCR反应体系,并用该体系对呼吸道相关致病菌进行检测.鉴定结果与临床常规鉴定方法对照,评价其敏感度、特异度及重复性.结果 通过对100例样品的检测,结果显示该双重实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照,特异度为100%,敏感度为100%;最小能检测到10个拷贝数的重组质粒;批内重复实验和批间重复实验结果均与常规鉴定方法结果相符.结论 双重荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌,特异度和敏感度高,重复性好,且快速、简便,该方法将有助于念珠菌病的早期诊断和针对性治疗.

Identification of Candida krusei and Candida glabrata by duplex quantitative real-time PCR

Objective To set up a duplex quantitative real-time PCR (QPCR) assay with high sensitivity, specificity and rapidity to detect Candida krusei and Candida glabrata. Methods A pair of species-specific primers, combined with two probes which were selected from the internally transcribed spacers Ⅱ (ITS Ⅱ), were used to amplify the common pathogen associated with the respiratory system in order to evaluate its sensitivity, specificity and repeatability. Results The results obtained from the duplex QPCR assay and the general culture were the same by detecting the samples of 100 cases. Its specificity was 100%,and its sensitivity was 100%. The lower limit of detection of this duplex QPCR assay was 10 copys of recombinant plasmid DNA. The result within group and between groups were the same as the general culture.Conclusion The duplex quantitative real-time PCR assay can detect Candida krusei and Candida glabrata sensitively, specifically, rapidly, simply and stably, and is useful to early diagnosis and target treatment of the diseases caused by the Candida.