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Transboundary and emerging diseases2025; 2025; 1852368; doi: 10.1155/tbed/1852368

Real-Time Reverse Transcription Multienzyme Isothermal Rapid Amplification for Rapid Detection of African Horse Sickness Virus.

Abstract: African horse sickness (AHS) is an acute infectious disease of equids caused by the AHS virus (AHSV), which can cause up to 90% mortality in naive horses. Reliable and rapid diagnosis is crucial for the surveillance and control of AHSV. As one of the AHSV detection methods recommended by World Organization for Animal Health (WOAH), the RT-qPCR assay has the drawbacks such as complex operation, expensive instruments, and long detecting time, which limit its application in simple laboratories or outdoors. In this study, a real-time reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) assay was established to detect AHSV. Primers and exo-probes were designed, synthesized, and screened based on the conserved regions of the AHSV gene. A series of experiments were conducted to evaluate the performances of the established real-time RT-MIRA for detecting AHSV. The valid testing results showed that this method was highly specific for the detection of AHSV, without exhibiting any cross-reactivity towards other equine viruses or other Orbivirus; its limit of detection (LOD) was 10 copies/μL, which was consistent with that of RT-qPCR, meaning it had good sensitivity for detecting AHSV. Furthermore, the real-time RT-MIRA for AHSV performed good repeatability, and its standard curve exhibited good linearity with a correlation coefficient of  = 0.9898, which indicated that the established method could be used for the quantitative detection of ASHV. As no AHS infection cases have been reported in China, 120 simulated clinical samples were tested by the real-time RT-MIRA and RT-qPCR for AHSV, which results showed there was a significant correlation between the two assays, with a value of 0.966 and an value of 0.9576. Parallel detection of 396 equine blood samples and 1760 Culicoides by this method and the RT-qPCR showed that all samples were negative for AHSV. Furthermore, the results of the real-time RT-MIRA could be judged by naked eyes under a portable equipment with blue light (480 nm). In conclusion, the real-time RT-MIRA for AHSV was specific and sensitive and had the advantages of convenient operation, visualization, no need for special equipment, and could be a reliable tool for rapid screening and detection of AHSV in field or border ports.
Copyright © 2025 Chaohua Huang et al. Transboundary and Emerging Diseases published by John Wiley & Sons Ltd.
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Publication Date: 2025-01-13 PubMed ID: 40302746PubMed Central: PMC12016855DOI: 10.1155/tbed/1852368Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research article presents the development and evaluation of a real-time reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) assay for the rapid and sensitive detection of African horse sickness virus (AHSV), aimed at providing a simpler and faster alternative to the conventional RT-qPCR method.

Background and Importance

  • African Horse Sickness (AHS): AHS is a highly lethal viral disease affecting equids (horses, donkeys, mules), with mortality rates up to 90% in horses that have not been previously exposed.
  • Need for Rapid Diagnosis: Quick and reliable detection of AHSV is critical for controlling outbreaks and preventing the spread of the disease.
  • Current Standard (RT-qPCR): The World Organization for Animal Health endorses RT-qPCR for AHSV detection, but it is hindered by complicated procedures, expensive equipment, and lengthy processing, making it impractical for routine use in basic labs or on-site diagnosis.

Objective of the Study

  • To develop an alternative diagnostic tool—real-time RT-MIRA—for AHSV detection that is rapid, sensitive, specific, and easier to perform without requiring costly instruments.

Methodology

  • Primer and Probe Design: Researchers designed and synthesized primers and exo-probes targeting conserved regions of the AHSV genome, ensuring broad detection capability across virus variants.
  • Assay Development: The RT-MIRA assay uses isothermal amplification, enabling it to run at a constant temperature, simplifying the process compared to temperature cycling needed in PCR.
  • Experimental Evaluation: Multiple tests were conducted to assess specificity (ability to detect AHSV without cross-reacting with other equine viruses or related Orbiviruses), sensitivity, repeatability, and quantitative capabilities.

Key Findings

  • Specificity: The RT-MIRA assay detected AHSV specifically, showing no false positives from other viruses.
  • Sensitivity (Limit of Detection): The assay detected as few as 10 copies/μL of viral RNA, matching the sensitivity of the RT-qPCR standard.
  • Repeatability and Quantification: The assay showed consistent results across trials and exhibited strong linearity in standard curves (correlation coefficient r = 0.9898), indicating reliable quantitative detection of viral load.
  • Performance on Simulated Samples: Testing 120 simulated clinical samples showed a very high concordance with RT-qPCR results (correlation value r = 0.966, and agreement kappa = 0.9576).
  • Field Sample Testing: Using 396 equine blood samples and 1760 Culicoides midges collected in China (where AHS has not been reported), both RT-MIRA and RT-qPCR found no positive AHSV cases, demonstrating field applicability.
  • Visual Detection Feature: The assay results can be read visually with the aid of a portable blue light device (480 nm), facilitating immediate interpretation in field conditions without complex machinery.

Advantages of the RT-MIRA Assay

  • Rapid assay time with simplified isothermal amplification that does not require thermal cyclers.
  • Cost-effective compared to RT-qPCR, lowering barriers for widespread testing.
  • High sensitivity and specificity, providing reliable diagnostic results.
  • Visual readout capability enables easy field deployment and use in remote or basic settings.
  • Potentially useful for border inspections, outbreak surveillance, and routine screening where traditional PCR methods are unsuitable.

Conclusion

  • The established real-time RT-MIRA assay is a promising tool for the rapid, sensitive, and specific detection of African horse sickness virus.
  • Its ease of use, lack of requirement for specialized equipment, and capability for visual result interpretation make it practical for field use and resource-limited laboratories.
  • This method could improve disease surveillance and control efforts, especially in areas at risk for AHS outbreaks or requiring quick diagnostics at points of entry.

Cite This Article

APA
Huang C, Wang J, Ruan Z, Wu J, Lin Y, Cao C, Yang J, Weng Q, Jin Y, Chen P, Hua Q. (2025). Real-Time Reverse Transcription Multienzyme Isothermal Rapid Amplification for Rapid Detection of African Horse Sickness Virus. Transbound Emerg Dis, 2025, 1852368. https://doi.org/10.1155/tbed/1852368

Publication

Transboundary and emerging diseases
ISSN: 1865-1682
NlmUniqueID: 101319538
Country: Germany
Language: English
Volume: 2025
Pages: 1852368
PII: 1852368

Researcher Affiliations

Huang, Chaohua
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.
Wang, Jianchang
  • Laboratory of Animal Quarantine, Technology Center of Shijiazhuang Customs, Shijiazhuang 050051, Hebei Province, China.
Ruan, Zhouxi
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.
Wu, Jiang
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.
Lin, Yanxing
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.
Cao, Chenfu
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.
Yang, Junxing
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.
Weng, Qiaoyu
  • Department of Product Development, Shenzhen Biolove Technology Co., Ltd, Shenzhen 518120, China.
Jin, Ye
  • Department of Product Development, Hu Nan Project Bioscience Ltd, Changsha 410013, China.
Chen, Peng
  • Department of Product Development, Shenzhen Biolove Technology Co., Ltd, Shenzhen 518120, China.
Hua, Qunyi
  • Laboratory of Animal Quarantine, Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, Guangdong Province, China.

MeSH Terms

  • Animals
  • African Horse Sickness Virus / isolation & purification
  • African Horse Sickness Virus / genetics
  • Horses
  • African Horse Sickness / diagnosis
  • African Horse Sickness / virology
  • Nucleic Acid Amplification Techniques / veterinary
  • Nucleic Acid Amplification Techniques / methods
  • Sensitivity and Specificity
  • Real-Time Polymerase Chain Reaction / veterinary
  • Molecular Diagnostic Techniques / veterinary
  • Reverse Transcription

Conflict of Interest Statement

The authors declare no conflicts of interest.

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