Multienzyme isothermal rapid amplification (MIRA)-LFD assay for rapid detection of PEDV and PoRVA

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods Pub Date : 2025-10-10 DOI:10.1016/j.mimet.2025.107289

Yicong Fan , Jianxin Wang , Xianyu Bian , Wei Wang , Jinzhu Zhou , Xuehan Zhang , Mi Hu , Min Sun , Shanshan Yang , Yongxiang Zhao , Baochao Fan , Bin Li , Junming Zhou

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引用次数: 0

Abstract

Porcine epidemic diarrhea virus (PEDV) and porcine rotavirus A (PoRVA), as primary pathogens of viral diarrhea in piglets, lead to substantial economic losses in global swine production. Existing detection approaches—including virus isolation, ELISA, and PCR—are limited by prolonged turnaround times, dependence on specialized equipment, or low sensitivity, which hinder their utility for rapid on-site diagnosis. In this study, we established RT-MIRA-LFD, a rapid, sensitive, and visually interpretable on-site detection platform for PEDV and PoRVA, leveraging multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD). Forward and reverse primers targeting conserved regions of the PEDV N and PoRVA VP6 genes were designed with 5′-6-FAM and 5′-biotin modifications, respectively, enabling seamless integration of MIRA amplification and LFD readout. Sensitivity testing revealed detection limits of 20 copies/μL (PEDV) and 4.5 copies/μL (PoRVA) for plasmid standards. Both assays showed no cross-reactivity with TGEV, PDCoV, CSFV, or other common swine pathogens, confirming high specificity. Clinical validation with 50 anal swab samples demonstrated 98 % (PEDV) and 96 % (PoRVA) concordance with RT-qPCR, supported by Kappa values of 0.92 and 0.96. The entire workflow completes within 30 min, bypasses complex instrumentation, and allows direct visual interpretation, dramatically enhancing field-deployable detection efficiency. This RT-MIRA-LFD platform offers a practical solution for early diagnosis, outbreak monitoring, and targeted control of PEDV and PoRVA, especially in resource-limited settings, thereby supporting efforts to reduce economic impacts on swine operations.

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多酶等温快速扩增(MIRA)-LFD法快速检测PEDV和PoRVA。

猪流行性腹泻病毒（PEDV）和猪轮状病毒A （PoRVA）作为仔猪病毒性腹泻的主要病原体，给全球养猪生产造成了巨大的经济损失。现有的检测方法——包括病毒分离、ELISA和pcr——由于周转时间长、依赖专用设备或灵敏度低而限制了它们在现场快速诊断中的应用。在本研究中，我们利用多酶等温快速扩增（MIRA）和侧流试纸（LFD），建立了RT-MIRA-LFD，一个快速、灵敏、视觉可解释的PEDV和PoRVA现场检测平台。针对PEDV N和PoRVA VP6基因保守区域分别设计了5'-6-FAM和5'-生物素修饰的正向引物和反向引物，实现了MIRA扩增和LFD读取的无缝整合。灵敏度测试显示，质粒标准品的检出限为20 copies/μL （PEDV）和4.5 copies/μL （PoRVA）。两种检测均显示与TGEV、PDCoV、CSFV或其他常见猪病原体无交叉反应，证实了高特异性。50份肛门拭子样本的临床验证显示，与RT-qPCR的一致性分别为98% % （PEDV）和96% % (PoRVA)， Kappa值分别为0.92和0.96。整个工作流程在30 min内完成，绕过复杂的仪器，并允许直接可视化解释，极大地提高了现场部署的检测效率。这种RT-MIRA-LFD平台为PEDV和PoRVA的早期诊断、疫情监测和有针对性的控制提供了实用的解决方案，特别是在资源有限的情况下，从而支持减少对养猪业的经济影响。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of microbiological methods 生物-生化研究方法

CiteScore

4.30

自引率

4.50%

发文量

151

审稿时长

29 days

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.