核酸外切酶-压电石英DNA传感器检测金黄色葡萄球菌的实验研究

Detection of Staphylococcus aureus by a lambda exonuclease based piezoelectric quartz crystal microbalance DNA sensor

目的构建一种用于检测金黄色葡萄球菌的核酸外切酶-压电石英DNA传感器新方法,同时对其重要影响因素进行探讨。方法采用λ核酸外切酶处理金黄色葡萄球菌PCR产物,EB染色、电泳后于紫外灯下进行观察,对杂交温度、杂交时间和杂交响应性能等因素进行实验研究,进一步将构建的核酸外切酶-压电石英DNA传感器法用于金黄色葡萄球菌检测,并对其灵敏度和特异性进行研究。结果λ核酸外切酶-压电石英DNA传感器法最适杂交温度为35℃,最适杂交时间为60min,响应性能显著大于普通压电DNA传感器(P<0.01),可以检测到1.0×104CFU/ml的金黄色葡萄球菌,特异性好。结论利用核酸外切酶-压电石英DNA传感器检测金黄色葡萄球菌,具有杂交效率高、技术难度低、频率响应性能高等优点,有较好的应用前景。

Objective To develop a new type of piezoelectric quartz crystal microbalance DNA sensor based on lambda exonuclease to detect Staphylococcus aureus （S. aureus） and optimize the main detection conditions. Methods After the DNA was extracted from the stain of S. aureus and the target fragment was amplified with self-designed universal primers, the PCR products were treated with Lambda exonuclease and visualized by agarose gel electrophoresis with ethidium bromide followed by a gel documentation system analysis. Then the products with or without exonuclease digestion were added into our piezoelectric quartz crystal microbalance DNA sensor for hybridization. The temperature and time of hybridization were optimized respectively. The specificity and sensitivity of our detection system were evaluated. Results The optimal temperature for hybridization was 35℃ and the optimal time was 60 min. The sensitivity of the lambda exonuclease based DNA sensor was better than that of common DNA sensor. The lowest detection limit of this new type quartz crystal microbalance system was 1.0×10^4 CFU/ml. Conclusion The lambda exonuclease based quartz crystal microbalance system we designed has the advantages of high efficiency in hybridization, easy to operate, and good response performance. Thus it can be applied to detect S. aureus infections.