Great Basin Scientific, Longmont, CO 80503, USA.
Anal Biochem. 2010 Jan 15;396(2):284-9. doi: 10.1016/j.ab.2009.09.035. Epub 2009 Sep 25.
This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin-horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1 fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35 min using either a thin film biosensor chip or a microtiter plate-based assay.
本文描述了一种简单且廉价的信号放大方法，称为聚合酶检测（PED），可从阳性血培养瓶中快速、灵敏地检测到 tuf 基因中鉴定葡萄球菌属的保守序列、femB 基因中特异性检测金黄色葡萄球菌的保守序列以及 mecA 耐甲氧西林基因。微菌特异性捕获探针被固定在微量滴定板或硅芯片上。目标序列和生物素标记的、目标特异性探针与互补的捕获探针杂交，形成生物素标记的、表面固定的三部分复合物。在两步法中，通过将表面固定的生物素与链霉亲和素孵育来放大信号，然后加入与大约 80 个生物素结合的 500 kDa 葡聚糖聚合物。然后通过结合链霉亲和素-辣根过氧化物酶缀合物并孵育四甲基联苯胺来开发信号。PED 方法在模型 DNA 杂交测定中提高了检测下限 10-100 倍，检测下限低至 1 fmol/L 目标 DNA。这种灵敏度水平允许使用薄膜生物传感器芯片或基于微量滴定板的测定法，在 25 到 35 分钟内从耐甲氧西林金黄色葡萄球菌阳性血培养物中检测到基因组 DNA。
