{"title":"Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of <i>Staphylococcus aureus</i>.","authors":"Jin Xu, Dandan Jin, Zhengwei Wang","doi":"10.4014/jmb.2404.04012","DOIUrl":null,"url":null,"abstract":"<p><p>The accurate and rapid detection of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant <i>Staphylococcus aureus</i> (<i>S. aureus</i>) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of <i>S. aureus</i> in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence (\"3\" and \"4\" fragments). The \"3\" and \"4\" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the <i>S. aureus</i> concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.</p>","PeriodicalId":16481,"journal":{"name":"Journal of microbiology and biotechnology","volume":null,"pages":null},"PeriodicalIF":2.5000,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11239412/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of microbiology and biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.4014/jmb.2404.04012","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/4/19 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.
准确、快速地检测耐甲氧西林金黄色葡萄球菌(MRSA)具有重要的临床意义。本研究提出了一种检测临床样本中耐甲氧西林金黄色葡萄球菌(S. aureus)的新方法。该方法采用了一种基于适配体的比色测定法,将识别探针与放大信号的分裂 DNA 酶相结合,从而实现了高灵敏度和直接的耐甲氧西林分析。通过在临床样本中识别金黄色葡萄球菌膜上的 PBP2a 蛋白,可对识别探针进行异构,从而为近距离连接分裂 DNA 酶提供模板。分裂 DNA 酶的近接形成了一个完整的 DNA 酶,可识别 L 探针中的环状部分,并产生一个可释放环状序列("3 "和 "4 "片段)的切割位点。3 "和 "4 "片段形成一个完整的序列,诱导催化发夹组装,暴露出富含 G 的部分。释放出的 LR 探针富含 G 的序列会诱导形成 G-四联体-hemin DNA 酶,作为比色信号读数。在优化的实验参数下,在 5 个数量级的动态范围内,吸收强度与金黄色葡萄球菌浓度的对数呈强线性关系。计算得出的检测限为 23 CFU/ml,该方法对 MRSA 具有高选择性。
期刊介绍:
The Journal of Microbiology and Biotechnology (JMB) is a monthly international journal devoted to the advancement and dissemination of scientific knowledge pertaining to microbiology, biotechnology, and related academic disciplines. It covers various scientific and technological aspects of Molecular and Cellular Microbiology, Environmental Microbiology and Biotechnology, Food Biotechnology, and Biotechnology and Bioengineering (subcategories are listed below). Launched in March 1991, the JMB is published by the Korean Society for Microbiology and Biotechnology (KMB) and distributed worldwide.