采用聚合酶链反应-酶免疫分析及手持式电化学传感器检测金黄色葡萄球菌A 型和B型肠毒素基因
Detection of Staphylococcus aureus enterotoxin A and B genes with PCR-EIA and a hand-held electrochemical sensor.
作者信息
Aitichou Mohamed, Henkens Robert, Sultana Afroz M, Ulrich Robert G, Sofi Ibrahim M
机构信息
Clinical Research Management, 1425 Porter Street, Frederick, MD 21702, USA.
出版信息
Mol Cell Probes. 2004 Dec;18(6):373-7. doi: 10.1016/j.mcp.2004.06.002.
Two electrochemical assays for detecting Staphylococcus aureus enterotoxin A and B genes were developed. The assays are based on PCR amplification with biotinylated primers, hybridization to a fluorescein-labeled probe, and detection with horseradish peroxidase-conjugated anti-fluorescein antibody using a hand-held electrochemical detector. The limit of detection (LOD) for both assays was approximately 16 copies of the sea and seb genes. The assays were evaluated in blinded studies, each with 81 samples that included genomic and cloned S. aureus DNA, and genomic DNA from Alcaligens, Bacillus, Bacteroides, Bordetella, Borkholderia, Clostridium, Comanonas, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Listeria, Moraxella, Neisseria, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Streptococcus, Vibrio and Yersinia species. Both assays showed 100% sensitivity. The specificity was 96% for the SEA assay and 98% for the SEB assay. These results demonstrate the feasibility of performing probe-based detection of PCR products with a low-cost, hand-held, electrochemical detection device as a viable alternative to colorimetric enzyme-linked assays of PCR products.
开发了两种用于检测金黄色葡萄球菌肠毒素A和B基因的电化学检测方法。这些检测方法基于用生物素化引物进行PCR扩增、与荧光素标记探针杂交以及使用手持式电化学检测器用辣根过氧化物酶偶联的抗荧光素抗体进行检测。两种检测方法的检测限（LOD）约为sea和seb基因的16个拷贝。这些检测方法在盲法研究中进行了评估，每项研究有81个样本，包括基因组和克隆的金黄色葡萄球菌DNA，以及来自产碱杆菌属、芽孢杆菌属、拟杆菌属、博德特氏菌属、伯克霍尔德菌属、梭菌属、丛毛单胞菌属、肠杆菌属、肠球菌属、大肠杆菌属、弗朗西斯菌属、嗜血杆菌属、克雷伯菌属、李斯特菌属、莫拉克斯氏菌属、奈瑟菌属、变形杆菌属、假单胞菌属、沙门氏菌属、沙雷氏菌属、志贺氏菌属、链球菌属、弧菌属和耶尔森氏菌属的基因组DNA。两种检测方法均显示出100%的灵敏度。SEA检测方法的特异性为96%，SEB检测方法的特异性为98%。这些结果证明了使用低成本的手持式电化学检测设备对PCR产物进行基于探针的检测作为PCR产物比色酶联检测的可行替代方法的可行性。
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