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Background <p>The neglected tropical arbovirus,&#xa0;Mayaro Virus (MAYV), causes intense febrile illness, similar to&#xa0;Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV) . MAYV also shares mosquito vectors with Yellow fever virus (YFV) (<i>Haemagogus janthinomys</i>), making it prone to underdiagnosis and underreported cases. Moreover, MAYA, along with Venezuelan equine encephalitis virus (VEEV) and&#xa0;Oropouche virus, is&#xa0;considered a&#xa0;leading candidate&#xa0;for the&#xa0;next arboviral&#xa0;epidemic.&#xa0;Therefore, accurate&#xa0;diagnosis of MAYV is essential for identifying infected individuals and effectively managing outbreaks.</p> Objective <p>This study aimed to detect MAYV using reverse transcription-recombinase polymerase amplification (RT-RPA), a nucleic acid amplification technique that operates at a single constant temperature, coupled with the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system for end-point detection. RPA primers were designed to target the conserved regions of the MAYV nonstructural protein 1 (nsP1) gene, and the RPA reaction conditions were optimized as follows: temperature, time, and magnesium acetate concentration.</p> Results <p>Optimized MAYV RT-RPA assays resulted in, successful amplification within 20&#xa0;min at 40&#xa0;°C, with a limit of detection (LOD) at 10<sup>5</sup> viral copies without cross-reactivity with the mosquito-borne viruses YFV and Chikungunya virus (CHIKV). CRISPR/Cas12a assays were then performed at the same temperature for 15&#xa0;min. The LOD of the integrated RT-RPA/CRISPR system was 10<sup>3</sup> viral copies, with high specificity and no cross-reactivity against YFV or CHIKV. Excluding reverse transcription, MAYV can be detected as early as 35&#xa0;min using the combined MAYV RT-RPA and CRISPR/Cas12a assays.</p> Conclusion <p>We developed an RT-RPA/CRISPR assay for sensitive and specific MAYV detection, which is a potential point-of-care (POC) diagnostic tool for resource-limited settings lacking traditional molecular methods.</p>

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Integrating RT-RPA/CRISPR system for the end-point visual detection of Mayaro virus

  • 期刊论文

  • 发表日期：

    2025年7月18日

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  • Christine Ardelle Marquez,
  • Gna Ahn,
  • Woo-Ri Shin,
  • Eung Je Lee,
  • Chan-Hee Lee,
  • Hye-Kwon Kim,
  • Yang-Hoon Kim,
  • Ji-Young Ahn

摘要

Background

The neglected tropical arbovirus, Mayaro Virus (MAYV), causes intense febrile illness, similar to Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV) . MAYV also shares mosquito vectors with Yellow fever virus (YFV) (Haemagogus janthinomys), making it prone to underdiagnosis and underreported cases. Moreover, MAYA, along with Venezuelan equine encephalitis virus (VEEV) and Oropouche virus, is considered a leading candidate for the next arboviral epidemic. Therefore, accurate diagnosis of MAYV is essential for identifying infected individuals and effectively managing outbreaks.

Objective

This study aimed to detect MAYV using reverse transcription-recombinase polymerase amplification (RT-RPA), a nucleic acid amplification technique that operates at a single constant temperature, coupled with the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system for end-point detection. RPA primers were designed to target the conserved regions of the MAYV nonstructural protein 1 (nsP1) gene, and the RPA reaction conditions were optimized as follows: temperature, time, and magnesium acetate concentration.

Results

Optimized MAYV RT-RPA assays resulted in, successful amplification within 20 min at 40 °C, with a limit of detection (LOD) at 105 viral copies without cross-reactivity with the mosquito-borne viruses YFV and Chikungunya virus (CHIKV). CRISPR/Cas12a assays were then performed at the same temperature for 15 min. The LOD of the integrated RT-RPA/CRISPR system was 103 viral copies, with high specificity and no cross-reactivity against YFV or CHIKV. Excluding reverse transcription, MAYV can be detected as early as 35 min using the combined MAYV RT-RPA and CRISPR/Cas12a assays.

Conclusion

We developed an RT-RPA/CRISPR assay for sensitive and specific MAYV detection, which is a potential point-of-care (POC) diagnostic tool for resource-limited settings lacking traditional molecular methods.