Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus
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Author
Bohórquez, Jose Alejandro
Muñoz-Aguilera, Adriana
Lanka, Saraswathi
Alberch, Mònica
Maddox, Carol W.
Publication date
2024-04-15ISSN
2235-2988
Abstract
Background: 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.
Methods: 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.
Results: 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 crossreaction 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.
Conclusion: 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.
Document Type
Article
Document version
Published version
Language
English
Subject (CDU)
619 - Veterinary science
Pages
14
Publisher
Frontiers Media
Is part of
Frontiers in Cellular and Infection Microbiology
Citation
Bohórquez, JA, Muñoz-Aguilera A, Lanka S, Coronado L, Rosell R, Alberch M, Maddox CW and Ganges L. 2024. “Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus”. Frontiers in Cellular and Infection Microbiology 14: 1372166. doi: 10.3389/fcimb.2024.1372166
Grant agreement number
MICINN/Programa Estatal para impulsar la investigación científico-técnica y su transferencia/PID2021-125599OB-I00/ES/FACTORES DEL VIRUS Y DEL HOSPEDADOR COMO DIANAS PARA EL DISEÑO DE UNA NUEVA ESTRATEGIA DIVA FRENTE A LA PESTE PORCINA CLÁSICA/
Program
Sanitat Animal
This item appears in the following Collection(s)
- ARTICLES CIENTÍFICS [2559]
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