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Multiplex PCR and quality control of Epinotia aporema granulovirus production

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Abstract

A specific multiplex PCR was developed for the rapid and highly sensitive quality control of the viral DNA during Epinotia aporema granulovirus (EpapGV) production. At the beginning of this work only 2.3% of the EpapGV genomic sequence was known. In order to increase the availability of specific information, the terminal sequences of the inserts of several selected clones of EpapGV genomic libraries were determined. These data comprised 8.4% of the total DNA sequence and corresponded to regions distributed throughout the genome. Based on the small fraction of known sequence available a set of 32 primers was designed, using information theory to set the basis for this study. Each pair of designed primers was initially tested in individual PCRs to assess the correct size of the expected product and the sensitivity of the amplification. The specificity was verified in multiplex PCRs, using alternatively 1–3 sets of selected 5–6 primer pairs and EpapGV DNA preparations from different sources and degrees of purity. The results indicate that the multiplex PCR could be used for quality control in the bioinsecticide production, as well as in other applications such as the detection of latent infections in E. aporema colonies, and studies related to virus distribution, vertical transmission, host range, or persistence in the field.

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Acknowledgements

This work was supported by grants from BID-SECYT-CONICET to VR and ASC, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC BA) and Universidad Nacional de La Plata (UNLP) to VR; Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, FONCyT) to VR and PDG, and Programas Prioritarios de Investigación (Universidad Nacional de Quilmes) to PDG. PDG and VR hold a research career award from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina). ASC is a staff researcher at IMYZA-INTA (Instituto de Microbiología y Zoología Agrícola, Instituto Nacional de Tecnología Agropecuaria, Argentina).

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  1. Maria Alejandra Manzán

    Present address: Center for Integrative Toxicology, Michigan State University, 313 National Food Safety and Toxicology Building, East Lansing, MI, 48824, USA

Authors and Affiliations

  1. Instituto de Biotecnología y Biología Molecular (IBBM-UNLP-CONICET-CCT La Plata), Facultad de Ciencias Exactas, Universidad Nacional de La Plata – Consejo Nacional de Investigaciones Científicas y Técnicas, Calle 49 y 115, 1900, La Plata, Argentina

    Maria Alejandra Manzán, Marina Elizabeth Biedma & Víctor Romanowski

  2. LIGBCM (Laboratorio de Ingeniería Genética y Biología Celular y Molecular) Departamento Ciencia y Tecnología, Universidad Nacional de Quilmes. Roque Saenz Peña 352, 1876, Bernal, Argentina

    Ernesto Mario Aljinovic, Pablo Daniel Ghiringhelli & Víctor Romanowski

  3. IMYZA (Instituto de Microbiología y Zoología Agrícola), Instituto Nacional de Tecnología Agropecuaria, CC 25, 1712, Castelar, Argentina

    Alicia Sciocco-Cap

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  1. Maria Alejandra Manzán
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Correspondence to Pablo Daniel Ghiringhelli.

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Manzán, M.A., Aljinovic, E.M., Biedma, M.E. et al. Multiplex PCR and quality control of Epinotia aporema granulovirus production. Virus Genes 37, 203–211 (2008). https://doi.org/10.1007/s11262-008-0256-7

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