Bekkaoui F, McNevin J P, Leung C H, Peterson G J, Patel A, Bhatt R S, Bryan R N
ID Biomedical Corporation, Burnaby, B.C., Canada.
Diagn Microbiol Infect Dis. 1999 Jun;34(2):83-90. doi: 10.1016/s0732-8893(99)00012-7.
A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme-immuno-assay (EIA) and uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5'-terminus and biotin at the 3'-terminus. The reaction occurs at a constant temperature that allows the target DNA to anneal to the probe. RNase H cuts the RNA portion, allowing the cut fragments to dissociate from the target, making it available for further cycling. CPT-EIA uses streptavidin-coated microplate wells to capture uncut probe followed by detection with horseradish-peroxidase conjugated anti-fluorescein antibody. The assay was compared to PCR and shown to accurately detect the presence or absence of the mecA gene in 159 staphylococcal clinical isolates. The CPT-EIA assay takes two hours starting from cultured cells compared with the 24-48 h required for detection of methicillin resistance by conventional susceptibility tests.
已开发出一种循环探针技术（CPT）检测方法，用于从耐甲氧西林葡萄球菌培养物中检测mecA基因。该检测方法基于比色酶免疫分析（EIA），并使用一种在5'-末端标记有荧光素、在3'-末端标记有生物素的mecA探针（DNA-RNA-DNA）。反应在恒定温度下进行，使靶DNA与探针退火。核糖核酸酶H切割RNA部分，使切割后的片段与靶标解离，从而可用于进一步循环。CPT-EIA使用包被有链霉亲和素的微孔板孔来捕获未切割的探针，随后用辣根过氧化物酶偶联的抗荧光素抗体进行检测。将该检测方法与聚合酶链反应（PCR）进行比较，结果表明其能准确检测159株葡萄球菌临床分离株中mecA基因的有无。与通过传统药敏试验检测耐甲氧西林情况所需的24 - 48小时相比，CPT-EIA检测方法从培养细胞开始只需两小时。
