A qPCR assay for the quantification of selected genotypic variants of spodoptera frugiperda multiple nucleopolyhedrovirus (Baculoviridae)

Molina-Ruiz, Cindy S.; Zamora-Briseño, Jesús Alejandro; Simón de Goñi, Oihane; Lasa, Rodrigo; Williams, Trevor

A qPCR assay for the quantification of selected genotypic variants of spodoptera frugiperda multiple nucleopolyhedrovirus (Baculoviridae)

dc.contributor.author	Molina-Ruiz, Cindy S.
dc.contributor.author	Zamora-Briseño, Jesús Alejandro
dc.contributor.author	Simón de Goñi, Oihane
dc.contributor.author	Lasa, Rodrigo
dc.contributor.author	Williams, Trevor
dc.contributor.department	Institute for Multidisciplinary Research in Applied Biology - IMAB	en
dc.date.accessioned	2024-10-22T13:34:01Z
dc.date.available	2024-10-22T13:34:01Z
dc.date.issued	2024-05-20
dc.date.updated	2024-10-22T13:18:28Z
dc.description.abstract	Alphabaculoviruses are lethal dsDNA viruses of Lepidoptera that have high genetic diversity and are transmitted in aggregates within proteinaceous occlusion bodies. This mode of transmission has implications for their efficacy as biological insecticides. A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV-NIC) comprising nine genotypic variants has been the subject of considerable study due to the influence of variant interactions on the insecticidal properties of mixed-variant occlusion bodies. As part of a systematic study on the replication and transmission of variant mixtures, a tool for the accurate quantification of a selection of genotypic variants was developed based on the quantitative PCR technique (qPCR). First, primer pairs were designed around a region of high variability in four variants named SfNic-A, SfNic-B, SfNic-C and SfNic-E to produce amplicons of 103–150 bp. Then, using cloned purified amplicons as standards, amplification was demonstrated over a dynamic range of 108–101 copies of each target. The assay was efficient (mean ± SD: 98.5 ± 0.8%), reproducible, as shown by low inter- and intra-assay coefficients of variation (<5%), and specific to the target variants (99.7–100% specificity across variants). The quantification method was validated on mixtures of genotype-specific amplicons and demonstrated accurate quantification. Finally, mixtures of the four variants were quantified based on mixtures of budded virions and mixtures of DNA extracted from occlusion-derived virions. In both cases, mixed-variant preparations compared favorably to total viral genome numbers by quantification of the polyhedrin (polh) gene that is present in all variants. This technique should prove invaluable in elucidating the influence of variant diversity on the transmission and insecticidal characteristics of this pathogen.	en
dc.format.mimetype	application/pdf	en
dc.identifier.citation	Molina-Ruiz, C. S., Zamora-Briseño, J. A., Simón, O., Lasa, R., Williams, T. (2024) A qPCR assay for the quantification of selected genotypic variants of spodoptera frugiperda multiple nucleopolyhedrovirus (Baculoviridae). Viruses, 16(6), 1-13. https://doi.org/10.3390/v16060881.
dc.identifier.doi	10.3390/v16060881
dc.identifier.issn	1999-4915
dc.identifier.uri	https://academica-e.unavarra.es/handle/2454/52352
dc.language.iso	eng
dc.publisher	MDPI
dc.relation.ispartof	Viruses 2024, 16(6), 881
dc.relation.publisherversion	https://doi.org/10.3390/v16060881
dc.rights	© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.
dc.rights.accessRights	info:eu-repo/semantics/openAccess
dc.rights.uri	https://creativecommons.org/licenses/by/4.0/
dc.subject	Alphabaculovirus	en
dc.subject	Copy number determination	en
dc.subject	Efficiency	en
dc.subject	Fall armyworm	en
dc.subject	Genotypic diversity	en
dc.title	A qPCR assay for the quantification of selected genotypic variants of spodoptera frugiperda multiple nucleopolyhedrovirus (Baculoviridae)	en
dc.type	info:eu-repo/semantics/article
dc.type.version	info:eu-repo/semantics/publishedVersion
dspace.entity.type	Publication
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