实时定量PCR检测白色念珠菌方法学的建立

目的 采用Taq Man MGB探针技术, 建立白色念珠菌实时荧光定量PCR (RQ-PCR) 检测方法, 为临床白色念珠菌的检测提供快捷的方法.方法 以真菌D1/D2区基因的一段高度保守序列为扩增靶序列, 合成白色念珠菌特异性引物和探针, 提取白色念珠菌标准菌株的DNA, 构建含有靶基因片段的重建质粒, 扩增后DNA作为标准品, 建立白色念珠菌RQ-PCR的标准曲线, 并对其重复性、敏感性及特异性等方面进行方法学评价.结果 对构建好的克隆质粒测序, 插入片段与预期片段序列一致, 相似性达100%.白色念珠菌标准曲线为Y=-3.824x+41.36, R2=0.990, 该扩增曲线形态良好.结论 成功建立了实时荧光定量PCR检测白色念珠菌的方法, 其敏感性和特异性较好.

Establishment of Detection Methodology of C.albicans by Real-time PCR

Objective To use TaqMan MGB probe technology to establish a detection methodology of Candida albicans (C.albicans) by real-time fluorescence quantitative PCR (RQ-PCR) and to provide a quick method for the detection of candida infection in clinical settings. Methods D1/D2 region of candida species which is a highly conserved sequence gene was selected as amplification target sequence and specific primers and probes for C.albicans were synthesized. The DNA of standard strains of C.albicans was extracted and rebuilt plasmid containing the target gene fragment was constructed. The DNA was amplified as a standard to establish the standard curve of C.albicans and methodological evaluation was conducted to identify the linear range, repeatability, sensitivity, specificity and so on. Results After sequencing the recombinant cloning vector, the sequence of the fragment inserted was consistent with the expected sequence, with the similarity of 100%. C.albicans standard curve Y =-3.824 x +41.36, R2= 0.990, which was in good shape and illustrated the detection method of C.albicans by real-time PCR had successfully established. Conclusion The detection method of C.albicans by real-time PCR has better sensitivity and specificity.