无酶点击化学连接链式反应结合试纸条用于金黄色葡萄球菌的检测

Detection of Staphylococcus aureus Based on Enzyme-free Click Chemical Ligation Chain Reaction Combined with Test Strip

为将无酶点击化学连接链式反应(CCLCR)结合试纸条用于金黄色葡萄球菌核酸的可视化检测。根据金黄色葡萄球菌16S rRNA的一段保守序列设计1对特异性核酸分子探针,一条探针5'端修饰生物素(Biotin),3'端修饰叠氮(N3);另一条探针5'端修饰环辛炔(DBCO),3'端修饰异硫氰酸荧光素(FITC),通过与目标基因进行特异性杂交,CCLCR和热循环作用下扩增产物进行试纸条分析。结果表明:CCLCR最佳探针浓度为0.05μmol/L,热循环扩增的最佳连接温度为25℃,该方法能够特异性检测出金黄色葡萄球菌核酸,而对肠炎沙门氏菌等其他5种食源性病原菌的检测结果呈阴性,同时对金黄色葡萄球菌核酸的检测灵敏度为1×10-8μmol/L。综上,将CCLCR与试纸条结合作为检测金黄色葡萄球菌的方法,具有操作简单、检测准确、检测成本低等优点,有望作为金黄色葡萄球菌的常规检测方法。

This study aims to develop a method for the visualization of Staphylococcus aureus nucleic acid by combining enzyme-free click chemical ligation chain reaction（ CCLCR） with test strips. A pair of specific nucleic acid probes was designed according to a conservative sequence of 16 sr DNA of Staphylococcus aureus. One probe modified biotin at the 5＇ end and N3 at the 3＇ end. The other probe modified DBCO and FITC,the product was hybridized with the target gene and the reaction product was detected by the test strip. The optimum temperature for CCLCR probe was 0. 05 μmol/L and the optimal temperature for thermocycling was 25 ℃. This method could specifically detect the nucleic acid of Staphylococcus aureus,and the detection result of other 5 foodborne pathogens such as Salmonella et. was negative,while the detection sensitivity of Staphylococcus aureus nucleic acid was 1×10^-8 μmol/L. In summary,the method of combining CCLCR with test strip for Staphylococcus aureus detection has the advantages of simple operation,accurate detection,low cost,etc.,and is expected to serve as a routine detection of Staphylococcus aureus.