Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus
Bohórquez, Jose Alejandro (University of Illinois at Urbana-Champaign. Veterinary Diagnostic Laboratory)
Muñoz-Aguilera, Adriana (Instituto Colombiano Agropecuario (ICA))
Lanka, Saraswathi (University of Illinois at Urbana-Champaign. Veterinary Diagnostic Laboratory)
Coronado, Liani (Unitat mixta d'investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal)
Rosell, Rosa (Generalitat de Catalunya. Departament d'Acció Climàtica, Alimentació i Agenda Rural)
Alberch, Mònica (Unitat mixta d'investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal)
Maddox, Carol W. (University of Illinois at Urbana-Champaign. Department of Pathobiology)
Ganges, Llilianne (Unitat mixta d'investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal)

Data:	2024
Resum:	Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.
Ajuts:	Ministerio de Ciencia e Innovación PID2021-125599OB-100
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	CSFV ; LAMP ; Diagnostic ; Point of care ; Viral RNA ; Surveillance ; RT-qPCR
Publicat a:	Frontiers in cellular and infection microbiology, Vol. 14 (april 2024) , ISSN 2235-2988

DOI: 10.3389/fcimb.2024.1372166
PMID: 38686097

14 p, 8.2 MB

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Documents de recerca > Documents dels grups de recerca de la UAB > Centres i grups de recerca (producció científica) > Ciències de la salut i biociències > Centre de Recerca en Sanitat Animal (CReSA-IRTA)
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