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Novel sensitive isothermal-based diagnostic technique for the detection of African swine fever virus

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Abstract

A rapid, simple, and sensitive diagnostic technique for the detection of African swine fever virus (ASFV) nucleic acid was developed for testing clinical samples in the field or resource-constrained settings. In the current study, the saltatory rolling-circle amplification (SRCA) technique was used for the first time to detect ASFV. The technique was developed using World Organization for Animal Health (WOAH)-approved primers targeting the p72 gene of the ASFV genome. The assay can be performed within 90 minutes at an isothermal temperature of 58°C without a requirement for sophisticated instrumentation. The results can be interpreted by examination with the naked eye with the aid of SYBR Green dye. This assay exhibited 100% specificity, producing amplicons only from ASFV-positive samples, and there was no cross-reactivity with other pathogenic viruses and bacteria of pigs that were tested. The lower limits of detection of SRCA, endpoint PCR, and real-time PCR assays were 48.4 copies/µL, 4.84 × 103 copies/µL, and 4.84 × 103 copies/µL, respectively. Thus, the newly developed SRCA assay was found to be 100 times more sensitive than endpoint and real-time PCR assays. Clinical tissue samples obtained from ASFV-infected domestic pigs and other clinical samples collected during 2020-22 from animals with suspected ASFV infection were tested using the SRCA assay, and a 100% accuracy rate, negative predictive value, and positive predictive value were demonstrated. The results indicate that the SRCA assay is a simple yet sensitive method for the detection of ASFV that may improve the diagnostic capacity of field laboratories, especially during outbreaks. This novel diagnostic technique is completely compliant with the World Health Organization’s “ASSURED” criteria advocated for disease diagnosis, as it is affordable, specific, sensitive, user-friendly, rapid and robust, equipment-free, and deliverable. Therefore, this SRCA assay may be preferable to other complex molecular techniques for diagnosing African swine fever.

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Acknowledgments

This research received funding from the Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/PR39032/ADV/90/285/2020). The authors are also grateful to the director of ICAR RC NEH, Umiam, Meghalaya, for providing the necessary facilities. We also thank the Animal Husbandry & Veterinary Department, Meghalaya, for sharing samples and data. The authors are thankful to Dr. K. Srinivas for his help in revising the manuscript.

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    Authors and Affiliations

    1. Division of Animal and Fisheries Sciences, ICAR RC for NEH Region, Umiam, Meghalaya, India

      A. Arun Prince Milton, Samir Das, Sabia Khan, K. M. Momin, C. B. Prasad, Sandeep Ghatak & Arnab Sen

    2. Animal Husbandry & Veterinary Department, Meghalaya, India

      H. Kylla

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    1. A. Arun Prince Milton
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    2. Samir Das
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    Contributions

    AAPM, conceptualization, methodology, investigation, writing – original draft. SD, investigation, formal analysis. SK, investigation. KMM, investigation. CBP, investigation. HK, sample and data collection. SG, formal analysis, writing – review and editing. AS, supervision, writing – review and editing

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    Correspondence to Sandeep Ghatak or Arnab Sen.

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    The authors declare that they have no competing interests.

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    This research was approved by Institutional Animal Ethics Committee, ICAR RC NEH, Meghalaya.

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    Communicated by William G Dundon

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    A. Arun Prince Milton and Samir Das contributed equally.

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    Milton, A.P., Das, S., Khan, S. et al. Novel sensitive isothermal-based diagnostic technique for the detection of African swine fever virus. Arch Virol 168, 79 (2023). https://doi.org/10.1007/s00705-023-05702-z

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