Detection of micrococcal nuclease for identifying Staphylococcus aureus based on DNA templated fluorescent copper nanoclusters
Detection of micrococcal nuclease for identifying Staphylococcus aureus based on DNA templated fluorescent copper nanoclusters
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基于 DNA 模板荧光铜纳米簇检测微球菌核酸酶鉴定金黄色葡萄球菌
DOI：
10.1007/s00604-019-3363-3
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发表时间：
2019-04-01
影响因子：
5.7
通讯作者：
Feng, Bo
中科院分区：
文献类型：
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作者：
Qing, Taiping;Long, Caicheng;Feng, Bo
摘要：微球菌核酸酶（MNase）是一种自然分泌的核酸降解酶，在细菌在感染宿主体内的传播中发挥着重要作用。 MNase的含量可用于估计金黄色葡萄球菌（S. aureus）的致病性。本文描述了一种荧光测定方法，用于测定 MNase 的活性并使用 DNA 模板荧光铜纳米簇 (CuNC) 鉴定金黄色葡萄球菌。具有富含 AT 区域和突出 3' 末端的双链 DNA (dsDNA) 被鉴定为 MNase 的高选择性底物和 CuNC 的模板。在没有 MNase 的情况下，富含 AT 的长双链 DNA 模板形成 CuNC，显示出明亮的黄色荧光，激发/发射峰位于 340/570 nm。然而，底物被酶消化成单核苷酸或短寡核苷酸片段，无法合成荧光 CuNC。该方法的工作范围为 1.0 × 10−3 - 5.0 × 10−2 U mL−1 MNase 活性范围，检测限为 1.0 mU mL−1，并且比其他核酸外切酶具有更高的选择性。该方法成功应用于金黄色葡萄球菌分泌的MNase的检测和金黄色葡萄球菌的鉴定。
图解摘要具有富含 AT 区域和 3' 突出末端的智能 dsDNA 被筛选为 MNase 的高选择性底物和形成铜纳米簇 (CuNC) 的模板。描述了一种通过智能 DNA 模板形成荧光 CuNC 来测定 MNase 活性和识别金黄色葡萄球菌的方法。
AbstractMicrococcal nuclease (MNase) is a naturally-secreted nucleic acid degrading enzyme with important role in the spread of the bacteria in an infected host. The content of MNase can be used to estimate the pathogenicity of Staphylococcus aureus (S. aureus). A fluorometric method is described here for determination of the activity of MNase and for identification of S. aureus using DNA templated fluorescent copper nanoclusters (CuNC). A double-stranded DNA (dsDNA) with AT-rich regions and protruding 3′-termini was identified as a high-selectivity substrate for MNase and as a template for CuNC. In the absence of MNase, the long AT-rich dsDNA templates the formation of CuNC that display bright yellow fluorescence, with excitation/emission peaks at 340/570 nm. However, the substrates are enzymatically digested to mononucleotides or short-oligonucleotide fragments, which fail to synthesize fluorescent CuNC. The method works in the 1.0 × 10−3 - 5.0 × 10−2 U mL−1 MNase activity range, has a 1.0 mU mL−1 detection limit, and is highly selective over other exonucleases. The assay was successfully applied to the detection of MNase secreted by S. aureus and to the identification of S. aureus.
Graphical abstractA smart dsDNA, with AT-rich regions and 3′-protruding termini, is screened as the high-selectivity substrate for MNase and template for the formation of copper nanoclusters (CuNC). A method is described for determination of the activity of MNase and for identification of S. aureus via smart DNA templated formation of fluorescent CuNCs.