Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology
Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology
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DOI：
10.1128/jcm.38.7.2525-2529.2000
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发表时间：
2000-07-01
影响因子：
9.4
通讯作者：
Bekkaoui, F
中科院分区：
文献类型：
--
作者：
Fong, WK;Modrusan, Z;Bekkaoui, F
建立了一种检测耐甲氧西林金黄色葡萄球菌(MRSA)mecA基因的循环探针技术(CPT)。该方法使用mecA探针(DNA-RNA-DNA)，在5‘端标记荧光素，在3’端标记生物素。CPT反应在恒定的温度下进行，这使得探针可以与目标DNA退火。RNaseH切割探针的RNA部分，允许被切割的片段与目标DNA解离，使目标可用于进一步的循环。试条检测步骤使用表面浸渍有链霉亲和素和免疫球蛋白G抗体的硝酸纤维素膜。在没有mecA基因的情况下，未切割的探针与抗荧光素-金偶联物结合，然后被链霉亲和素捕获，形成测试线。在mecA基因存在的情况下，探针被切割，试条上不形成测试线。用纸片法检测324株S金黄色葡萄球菌mecA基因的敏感性为99.4%，特异性为100%，与一组mecA阴性的非S金黄色葡萄球菌无交叉反应。CPT-试纸分析方法简单，不需要复杂的设备。此外，从原代培养到检测到金黄色葡萄球菌S的mecA基因，需要1.5h。
A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe Is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S, aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene, Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S, aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S, aureus isolates.