{"title":"Rapid and simple detection of <i>Candida albicans</i> using closed dumbbell-mediated isothermal amplification.","authors":"Yanli Zhang, Xuhan Chen, Yeling Zhong, Fei Guo, Guifang Ouyang, Rui Mao","doi":"10.3389/fcimb.2025.1484089","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong><i>Candida albicans</i>, a human fungal pathogen, multiplies to invade body cells and causes fungal diseases in the condition of insufficient body's immune function. Early detection of <i>C. albicans</i> is required to guide appropriate prevention and treatment.</p><p><strong>Methods: </strong>The purpose of this study was to establish a <i>C. albicans</i> assay based on newly developed closed dumbbell-mediated isothermal amplification (CDA) to achieve rapid and simple point of care diagnostic. The CDA technique was carried out by specific primers targeting at the conserved <i>C. albicans</i> ITS2 gene. All primers were selected and evaluated by real-time fluorescence monitoring and endpoint visual judgement indicated by hydroxy naphthol blue (HNB). Optimal primers and accelerate primers (out primers and loop primers) were designed and selected after confirmation of the fundamental CDA primers to achieve more efficient CDA reaction for <i>C. albicans</i> detection (CA-OL-CDA).</p><p><strong>Results: </strong>After establishment of the assay, 9 <i>non-Candida albicans</i> strains, including 3 <i>Candida</i> species were tested to negative by adopting the established CA-OL-CDA assay, indicated high specificity. The limit of detection of <i>Candida albicans</i> DNA by CA-OL-CDA assay was 6.2×10<sup>-6</sup> ng/μL of DNA (10 copies/μL), 10-fold more sensitive than real-time quantitative PCR (qPCR).</p><p><strong>Discussion: </strong>The CA-OL-CDA assay exhibited advantages of high specificity, sensitivity, simpler and more efficient operation. In addition, the CA-OL-CDA method holds potential in on-site detection for <i>C. albicans</i> using color shift by adopting the reaction mixture based on HNB.</p>","PeriodicalId":12458,"journal":{"name":"Frontiers in Cellular and Infection Microbiology","volume":"15 ","pages":"1484089"},"PeriodicalIF":4.6000,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11830661/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Cellular and Infection Microbiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3389/fcimb.2025.1484089","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Introduction: Candida albicans, a human fungal pathogen, multiplies to invade body cells and causes fungal diseases in the condition of insufficient body's immune function. Early detection of C. albicans is required to guide appropriate prevention and treatment.
Methods: The purpose of this study was to establish a C. albicans assay based on newly developed closed dumbbell-mediated isothermal amplification (CDA) to achieve rapid and simple point of care diagnostic. The CDA technique was carried out by specific primers targeting at the conserved C. albicans ITS2 gene. All primers were selected and evaluated by real-time fluorescence monitoring and endpoint visual judgement indicated by hydroxy naphthol blue (HNB). Optimal primers and accelerate primers (out primers and loop primers) were designed and selected after confirmation of the fundamental CDA primers to achieve more efficient CDA reaction for C. albicans detection (CA-OL-CDA).
Results: After establishment of the assay, 9 non-Candida albicans strains, including 3 Candida species were tested to negative by adopting the established CA-OL-CDA assay, indicated high specificity. The limit of detection of Candida albicans DNA by CA-OL-CDA assay was 6.2×10-6 ng/μL of DNA (10 copies/μL), 10-fold more sensitive than real-time quantitative PCR (qPCR).
Discussion: The CA-OL-CDA assay exhibited advantages of high specificity, sensitivity, simpler and more efficient operation. In addition, the CA-OL-CDA method holds potential in on-site detection for C. albicans using color shift by adopting the reaction mixture based on HNB.
期刊介绍：
Frontiers in Cellular and Infection Microbiology is a leading specialty journal, publishing rigorously peer-reviewed research across all pathogenic microorganisms and their interaction with their hosts. Chief Editor Yousef Abu Kwaik, University of Louisville is supported by an outstanding Editorial Board of international experts. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.
Frontiers in Cellular and Infection Microbiology includes research on bacteria, fungi, parasites, viruses, endosymbionts, prions and all microbial pathogens as well as the microbiota and its effect on health and disease in various hosts. The research approaches include molecular microbiology, cellular microbiology, gene regulation, proteomics, signal transduction, pathogenic evolution, genomics, structural biology, and virulence factors as well as model hosts. Areas of research to counteract infectious agents by the host include the host innate and adaptive immune responses as well as metabolic restrictions to various pathogenic microorganisms, vaccine design and development against various pathogenic microorganisms, and the mechanisms of antibiotic resistance and its countermeasures.
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