Das Sanchita, Brown Teresa M, Kellar Kathryn L, Holloway Brian P, Morrison Christine J
Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
FEMS Immunol Med Microbiol. 2006 Mar;46(2):244-50. doi: 10.1111/j.1574-695X.2005.00031.x.
Recently, a new flow cytometric technology to detect multiple DNA target sequences in a single microtiter well plate was developed [multianalyte profiling (MAP) System, Luminex Corp., Austin, TX]. DNA probes, directed to the internal transcribed spacer 2 region of ribosomal DNA, were therefore designed to detect and differentiate PCR amplicons from six medically important Candida species using this system. Each probe was covalently linked to one of 100 available microsphere (bead) sets. Biotinylated PCR amplicons were then hybridized to the complementary probe on each bead set. Bound amplicons were detected fluorometrically using a streptavidin-linked reporter dye, R-phycoerythrin. Specific hybridization was noted for all six Candida species probes (mean sample-to-background ratio+/-standard error: Candida albicans, 58.7+/-1.2; Candida tropicalis, 53.2+/-3.8; Candida glabrata, 46.9+/-2.1; Candida parapsilosis, 59.9+/-1.6; Candida krusei, 54.7+/-3.7 vs. 0.9+/-0.03 for all heterologous Candida species DNA targets and vs. 1.0+/-0.1 for samples containing water instead of DNA; P < 0.001). The limit of test sensitivity was 0.5 pg of DNA. A sample could be processed and analyzed within 1 h post-PCR amplification. Therefore, the multianalyte profiling system was rapid, sensitive and specific for the detection and differentiation of the most medically important species of Candida.
最近，一种用于在单个微量滴定板中检测多个DNA靶序列的新型流式细胞术技术被开发出来（多分析物谱分析（MAP）系统，Luminex公司，德克萨斯州奥斯汀）。因此，针对核糖体DNA的内转录间隔区2区域设计了DNA探针，以使用该系统检测和区分来自六种医学上重要的念珠菌属物种的PCR扩增子。每个探针与100种可用微球（珠子）组中的一组共价连接。然后将生物素化的PCR扩增子与每个珠子组上的互补探针杂交。使用链霉亲和素连接的报告染料R-藻红蛋白通过荧光法检测结合的扩增子。所有六种念珠菌属物种的探针均显示出特异性杂交（平均样品与背景比率±标准误差：白色念珠菌，58.7±1.2；热带念珠菌，53.2±3.8；光滑念珠菌，46.9±2.1；近平滑念珠菌，59.9±1.6；克柔念珠菌，54.7±3.7，而所有异源念珠菌属物种DNA靶标的比率为0.9±0.03，含有水而非DNA的样品的比率为1.0±0.1；P<0.001）。检测灵敏度极限为0.5 pg DNA。PCR扩增后1小时内即可对样品进行处理和分析。因此，多分析物谱分析系统对于检测和区分医学上最重要的念珠菌属物种具有快速、灵敏和特异的特点。
