{"title":"Highly sensitive detection of Staphylococcus aureus α-hemolysin protein (Hla or α-toxin) by apta-qPCR","authors":"Abolfazl Jahangiri , Samira Dahaghin , Ehsan Malekara , Raheleh Halabian , Mahdieh Mahboobi , Elham Behzadi , Hamid Sedighian","doi":"10.1016/j.mimet.2024.107084","DOIUrl":null,"url":null,"abstract":"<div><div>Alpha-toxin of <em>Staphylococcus aureus</em> belongs to the pore-forming toxin (PFT) family, which can lyse red and white blood cells. In addition to the existence of the <em>hla</em> gene in the majority of <em>S. aureus</em> strains (about 95 %), higher expression exhibits enhanced pathogenicity to the bacteria. Various methods, such as antibodies and aptamers, could serve to detect this toxin. In the current study, for the first time, an improved sandwich aptamer-antibody-based method was developed using specific murine polyclonal antibodies and a specific aptamer to detect a wide range of α-toxin levels. Denatured recombinant α-toxin was administered to mice to trigger the production of specific antibodies, which were subsequently purified from immune sera. These antibodies served as capturers in the designed apta-qPCR assay, with an aptamer employed as a detector. The results showed that spiked α-toxin in the undiluted serum samples could detect α-toxin between 300 and 0.5 ng/mL with no cross-reactivity. The coefficient of variation (CV) percent of intra- and inter-assays were 0.84 and 1.06, respectively. Since in the apta-qPCR assay, a combination of specific polyclonal antibodies as capture and specific aptamer along with real-time PCR (qPCR) sensitivity is used, this robust method could be used in diagnostic laboratories to detect various levels of the toxin in human serum samples.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"229 ","pages":"Article 107084"},"PeriodicalIF":1.7000,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of microbiological methods","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0167701224001969","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Alpha-toxin of Staphylococcus aureus belongs to the pore-forming toxin (PFT) family, which can lyse red and white blood cells. In addition to the existence of the hla gene in the majority of S. aureus strains (about 95 %), higher expression exhibits enhanced pathogenicity to the bacteria. Various methods, such as antibodies and aptamers, could serve to detect this toxin. In the current study, for the first time, an improved sandwich aptamer-antibody-based method was developed using specific murine polyclonal antibodies and a specific aptamer to detect a wide range of α-toxin levels. Denatured recombinant α-toxin was administered to mice to trigger the production of specific antibodies, which were subsequently purified from immune sera. These antibodies served as capturers in the designed apta-qPCR assay, with an aptamer employed as a detector. The results showed that spiked α-toxin in the undiluted serum samples could detect α-toxin between 300 and 0.5 ng/mL with no cross-reactivity. The coefficient of variation (CV) percent of intra- and inter-assays were 0.84 and 1.06, respectively. Since in the apta-qPCR assay, a combination of specific polyclonal antibodies as capture and specific aptamer along with real-time PCR (qPCR) sensitivity is used, this robust method could be used in diagnostic laboratories to detect various levels of the toxin in human serum samples.
期刊介绍：
The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach.
All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.
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