Efficient detection of African Swine Fever Virus using minimal equipment through a LAMP PCR method.

Autor:	Bohorquez JA; Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, United States., Lanka S; Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, United States., Rosell R; WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Barcelona, Spain.; Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain.; IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain.; Departament d'Acció Climàtica, Alimentació i Agenda Rural, Generalitat de Catalunya, Barcelona, Spain., Pérez-Simó M; WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Barcelona, Spain.; Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain.; IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain., Alberch M; WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Barcelona, Spain.; Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain.; IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain., Rodriguez F; Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain.; IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain., Ganges L; WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Barcelona, Spain.; Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain.; IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Barcelona, Spain., Maddox CW; Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, United States.; Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, United States.
Jazyk:	angličtina
Zdroj:	Frontiers in cellular and infection microbiology [Front Cell Infect Microbiol] 2023 Jan 27; Vol. 13, pp. 1114772. Date of Electronic Publication: 2023 Jan 27 (Print Publication: 2023).
DOI:	10.3389/fcimb.2023.1114772
Abstrakt:	African swine fever virus (ASFV) currently represents the biggest threat to the porcine industry worldwide, with high economic impact and severe animal health and welfare concerns. Outbreaks have occurred in Europe and Asia since ASFV was reintroduced into the continent in 2007 and, in 2021, ASFV was detected in the Caribbean, raising alarm about the reemergence of the virus in the Americas. Given the lack of vaccines against ASFV, control of the virus relies on molecular surveillance, which can be delayed due to the need for sample shipment to specialized laboratories. Isothermal PCR techniques, such as LAMP, have become increasingly attractive as point-of-care diagnostic tools given the minimal material expense, equipment, and training required. The present study aimed to develop a LAMP assay for the detection of ASFV. Four LAMP primer sets were designed, based on a consensus sequence for the ASFV p72 gene, and were tested using a synthetic plasmid containing the cloned ASFV p72 target gene as a positive control. Two primer sets, were selected for further validation, given their very short time for amplification. Both primer sets showed thermal stability, amplifying the ASFV DNA at temperatures between 60-70°C and proved to have an analytical limit of detection as low as one ASFV-plasmid DNA copy/µL, using both fluorometric and colorimetric methods. The selected primers did not yield false positive or cross reactive results with other common swine pathogens, showing high specificity. Testing of DNA-spiked samples showed that LAMP amplification was not affected by the nature of the matrices, including oral fluids, tonsils, blood, or rectal swabs. The primer sets were able to detect the two more prevalent ASFV genotypes in the field. Taken together, the results show that ASFV-LAMP-BG2 and ASFV-LAMP-BG3 would be a useful tool for rapid, highly sensitive on-site diagnostic testing. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2023 Bohorquez, Lanka, Rosell, Pérez-Simó, Alberch, Rodriguez, Ganges and Maddox.)
Databáze:	MEDLINE
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