摘要
目的采用TaqMan-MGB探针作为检测信号,建立快速检测光滑念珠菌的实时荧光定量PCR方法。方法以光滑念珠菌核糖体RNA编码基因(rDNA)中的内转录间隔区2(ITS2)作为靶基因,设计特异性引物和TaqManMGB探针。在梯度光滑念珠菌纯菌液标本中评价所建方法灵敏度和重复性;在102株光滑念珠菌临床分离株、非光滑念珠菌的其他4种念珠菌临床分离株、7种细菌临床分离株和人基因组的DNA中评价所建方法特异度;并在模拟光滑念珠菌血症的血标本中初步探讨其临床应用价值。结果所建方法对光滑念珠菌纯菌液的检测下限为101 CFU/ml,相应的批内、批间变异系数(CV)分别为1.68%和2.84%。对102株光滑念珠菌临床分离株检出率为100%;对非光滑念珠菌无交叉反应,显示该方法特异性为100%。对模拟光滑念珠菌血症血标本的最低检测下限为101 CFU/ml。结论所建方法具有特异性和灵敏度高、重复性好的特性,适于临床上光滑念珠菌血症的快速检测。
Objective To develop a TaqMan-MGB probe-based quantitative real-time PCR assay for rapid identification of Candida giabrata （C. glabrata）. Methods The internal transcribed spacer 2 （ITS2） on ribosomal DNA （rD- NA） of C. glabrata was used as a target for designing specific primers and TaqMan-MGB probe. The sensitivity and reproducibility of the present method were evaluated with serial diluted samples of C. glabrata. The specificity of the method was validated in DNA samples from clinical isolates of 102 strains of C. glabrata,4 other species of non-glabrata Candida, 7 species of bacteria and human blood specimen, respectively. We also evaluated the clinical application values of the assay using simulated C. glabrata eandidemia in blood samples. Results The lowest counts of C. glabrata that the assay can measure accurately was 101CFU/ml,and its coefficients of variability （CV） of intra-assay and interassay were 1.68 % and 2.84% ,respectively. The detectable rate was 100% for 102 clinical isolates of C. glabrata. No eross reaction by the assay were observed indieating the specifieity was 100%. For simulated infection blood samples, the detection limit of C. glabrata was 101 CFU/ml by the assay. Conclusion The newly developed method was a highly sensitive and specific assay,which could be applied to rapid diagnosis of bloodstream infections induced by C. glabrata.
出处
《中国实验诊断学》
2016年第2期178-181,共4页
Chinese Journal of Laboratory Diagnosis
基金
北京大学航天临床医学院科研基金(YN201322)
