{"title":"LAMP Assay Coupled with a <i>Pyrococcus furiosus</i> Argonaute System for the Rapid Detection of Porcine Epidemic Diarrhea Virus.","authors":"Zhaorong Yu, Ying Shao, Yu Zhang, Fanyu Cheng, Peng Fang, Jian Tu, Xiangjun Song, Kezong Qi, Zhenyu Wang","doi":"10.1021/acssynbio.4c00446","DOIUrl":null,"url":null,"abstract":"<p><p>Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and <i>Pyrococcus furiosus</i> Argonaute (<i>Pf</i>Ago) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and <i>Pf</i>Ago gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-<i>Pf</i>Ago system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-<i>Pf</i>Ago system can provide a new solution for the development of a portable, visual PEDV testing platform.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7000,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Synthetic Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acssynbio.4c00446","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and PfAgo gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-PfAgo system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-PfAgo system can provide a new solution for the development of a portable, visual PEDV testing platform.
期刊介绍：
The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism.
Topics may include, but are not limited to:
Design and optimization of genetic systems
Genetic circuit design and their principles for their organization into programs
Computational methods to aid the design of genetic systems
Experimental methods to quantify genetic parts, circuits, and metabolic fluxes
Genetic parts libraries: their creation, analysis, and ontological representation
Protein engineering including computational design
Metabolic engineering and cellular manufacturing, including biomass conversion
Natural product access, engineering, and production
Creative and innovative applications of cellular programming
Medical applications, tissue engineering, and the programming of therapeutic cells
Minimal cell design and construction
Genomics and genome replacement strategies
Viral engineering
Automated and robotic assembly platforms for synthetic biology
DNA synthesis methodologies
Metagenomics and synthetic metagenomic analysis
Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction
Gene optimization
Methods for genome-scale measurements of transcription and metabolomics
Systems biology and methods to integrate multiple data sources
in vitro and cell-free synthetic biology and molecular programming
Nucleic acid engineering.
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