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High specific MNase assay for rapid identification of Staphylococcus aureus using AT-rich dsDNA substrate.
Talanta ( IF 5.6 ) Pub Date : 2019-06-13 , DOI: 10.1016/j.talanta.2019.06.029 Xuan Wang 1 , Caicheng Long 1 , Xiaoyan Xu 1 , Taiping Qing 1 , Peng Zhang 1 , Bo Feng 1
Talanta ( IF 5.6 ) Pub Date : 2019-06-13 , DOI: 10.1016/j.talanta.2019.06.029 Xuan Wang 1 , Caicheng Long 1 , Xiaoyan Xu 1 , Taiping Qing 1 , Peng Zhang 1 , Bo Feng 1
Affiliation
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Micrococcal nuclease (MNase) is a nonspecific endo-exonuclease that digests single-stranded/double-stranded DNA and RNA. The existence of MNase can serves as an important diagnostic biomarker of Staphylococcus aureus (S. aureus) infection. However, most of the substrates in MNase-based sensors are single-stranded DNA, which could also be digested by exonuclease I or S1 nuclease and interfere the MNase detection. In this work, we developed a highly selective fluorescent method for MNase detection using a specific dsDNA and nucleic acid dye SYBR Green I (SGI) as the indicator. After rational design, an AT-rich dsDNA with 3' protruding termini was screened as the high-specific substrate of MNase assay and efficient enhancer of SGI. The AT-rich dsDNA substrate can resist the digestion of other exonuclease and greatly enhance the fluorescence of SGI. This high-specific substrate-based probe can detect MNase in buffer as well as biological sample with highly selectivity. Moreover, this method was also applied to monitor the MNase secreted by S. aureus. Thus, the proposed MNase-based assay has a strong potential to identify S. aureus in food safety and microbial infection due to its excellent analytical sensitivity and high selectivity.
中文翻译：
使用富含AT的dsDNA底物快速鉴定金黄色葡萄球菌的高特异性MNase检测。
微球菌核酸酶（MNase）是一种非特异性内切核酸外切酶，可消化单链/双链DNA和RNA。MNase的存在可以作为金黄色葡萄球菌（金黄色葡萄球菌）感染的重要诊断生物标志物。但是，基于MNase的传感器中的大多数底物是单链DNA，也可以被核酸外切酶I或S1核酸酶消化，并干扰MNase的检测。在这项工作中，我们开发了一种高度选择性的荧光方法，使用特定的dsDNA和核酸染料SYBR Green I（SGI）作为指示剂进行MNase检测。经过合理的设计，筛选出具有3'突出末端的富含AT的dsDNA作为MNase检测的高特异性底物和SGI的有效增强子。富含AT的dsDNA底物可以抵抗其他核酸外切酶的消化，并大大增强SGI的荧光。这种基于底物的高特异性探针可以高选择性地检测缓冲液和生物样品中的MNase。此外，该方法还用于监测金黄色葡萄球菌分泌的MNase。因此，所提出的基于MNase的测定法由于其优异的分析灵敏度和高选择性而具有在食品安全和微生物感染中鉴定金黄色葡萄球菌的强大潜力。
更新日期：2019-06-13
中文翻译：
使用富含AT的dsDNA底物快速鉴定金黄色葡萄球菌的高特异性MNase检测。
微球菌核酸酶（MNase）是一种非特异性内切核酸外切酶，可消化单链/双链DNA和RNA。MNase的存在可以作为金黄色葡萄球菌（金黄色葡萄球菌）感染的重要诊断生物标志物。但是，基于MNase的传感器中的大多数底物是单链DNA，也可以被核酸外切酶I或S1核酸酶消化，并干扰MNase的检测。在这项工作中，我们开发了一种高度选择性的荧光方法，使用特定的dsDNA和核酸染料SYBR Green I（SGI）作为指示剂进行MNase检测。经过合理的设计，筛选出具有3'突出末端的富含AT的dsDNA作为MNase检测的高特异性底物和SGI的有效增强子。富含AT的dsDNA底物可以抵抗其他核酸外切酶的消化，并大大增强SGI的荧光。这种基于底物的高特异性探针可以高选择性地检测缓冲液和生物样品中的MNase。此外，该方法还用于监测金黄色葡萄球菌分泌的MNase。因此，所提出的基于MNase的测定法由于其优异的分析灵敏度和高选择性而具有在食品安全和微生物感染中鉴定金黄色葡萄球菌的强大潜力。
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