4.6 Article

Development and Characterization of Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of Monkeypox Virus

期刊

VIRUSES-BASEL
卷 14, 期 10, 页码 -

出版社

MDPI
DOI: 10.3390/v14102112

关键词

monkeypox virus; nucleic acid detection; recombinase polymerase amplification assay; CRISPR-Cas12a; rapid detection system; lateral flow

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资金

  1. Ministry of Science and Technology of China [2021YFC0863400, 2022YFE0114700]
  2. Alliance of International Scientific Organizations [ANSO-CR-SP-2020-02]
  3. Shanghai Municipal Science and Technology Major Project [2019SHZDZX02]
  4. Institut Pasteur
  5. Fondation Merieux
  6. Chinese Academy of Sciences
  7. International Affairs Department of the Institut Pasteur of Paris
  8. Agence National de la Recherche (Grant AFRIPOX)

向作者/读者索取更多资源

Monkeypox, caused by the monkeypox virus, is a zoonotic disease mainly found in West and Central Africa. Recent outbreaks in multiple countries worldwide have raised concerns about its global spread. The development of rapid diagnostic tests using recombinase-based isothermal amplification assays provides a promising solution for early detection and control of monkeypox outbreaks.
Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), in which outbreaks mainly occurred in West and Central Africa, with only sporadic spillovers to countries outside Africa due to international travel or close contact with wildlife. During May 2022, multiple countries in Europe, North and South America, Australia, Asia, and Africa reported near-simultaneous outbreaks of MPXV, the first time that patient clusters were reported over such a large geographical area. Cases have no known epidemiological links to MPXV-endemic countries in West or Central Africa. Real-time PCR is currently the gold standard for MPXV diagnostics, but it requires trained laboratory personnel and specialized equipment, and results can only be obtained after several hours. A rapid and simple-to-operate point-of-care diagnostic test for MPXV is crucial for limiting its spread and controlling outbreaks. Here, three recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of MPXV isolates were developed. These three assays target the MPXV G2R gene, and the limit of detection for these systems is approximately 10(0) copies of DNA per reaction. The assays were found to be specific and non-cross reactive against other pox viruses, such as vaccinia virus, and the results can be visualized within 20-30 min. The assays were validated with DNA extracted from 19 clinical samples from suspected or confirmed MPXV patients from Central Africa, and found to be consistent with findings from traditional qPCR. These results provide a solid platform for the early diagnosis of potential MPXV cases, and will help with the control and prevention of current and future outbreaks.

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