交叉引物恒温扩增技术快速检测金黄色葡萄球菌

The establishment of rapid detection of Staphylococcus aureus by cross primer amplification technology

目的建立一种快速、特异、灵敏的交叉引物恒温扩增(crossing priming amplification,CPA)技术检测金黄色葡萄球菌(Staphylococcus aureus,SA)耐热核酸酶基因(nuc)。方法从GenBank上下载多条金黄色葡萄球菌nuc基因序列,在其保守区域设计CPA引物及探针,建立CPA恒温扩增快速检测技术,并对反应条件进行优化,同时验证方法的特异性和敏感性。结果本方法对金黄色葡萄球菌检测具有高度特异性,对构建的阳性质粒DNA灵敏度达到1.0×10^1 copy/μl,对12份临床标本的检测阳性率与荧光PCR法相同,同时CPA法实验过程操作简便、反应迅速,且无需使用昂贵仪器,最快可在40 min内出检测结果。结论本研究建立的CPA法快速、简便、灵敏、特异,适用于金黄色葡萄球菌核酸快速检测。

Objective To develop cross priming amplification(CPA) technology for specific, sensitive, and simple detection of the thermostable nuclease gene(nuc) of Staphylococcus aureus. Methods The gene sequences of nuc of Staphylococcus aureus downloaded from the Genbank were aligned using the biological software and the specific primers and probes were designed, then isothermal nucleic acid amplification reactions were established to improve the sensitivity and specificity of the assay. Results The method was highly specific for the detection of the Staphylococcus aureus. The detection limit of the positive plasmid was up to 1.0×10~1 copy/μl. The positive rate of CPA method in 12 clinical specimens was the same as the fluorescent PCR method. And the CPA method operation was simpler, rapider, without expensive instruments, the fastest can be out of the test results within one hour. Conclusion The CPA assay is a rapid, simple, sensitive and specific tool for diagnosis of Staphylococcus aureus.