Multiplex PCR and quality control of Epinotia aporema granulovirus production

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.

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus, on A. gemmatalis (Lepidoptera: Noctuidae) larvae

“…DNA samples were prepared as described by Manzan et al (2008) from virus-killed larvae or control larvae. The presence of AgMNPV-SF and/or EpapGV viral DNA in the treated larvae, was determined using specific primers designed to amplify the AgMNPV polyhedrin ORF (Arana et al, 2001) and EpapGV granulin ORF (Parola et al, 2002).…”

Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

“…A formulation was assayed under controlled greenhouse conditions resulting in 80% larval mortality with a dose equivalent to 2 × 10 12 OBs/hectare. Quality control procedures of the viral product were developed [ 167 , 168 ] and permission has been granted for experimental field studies.…”

Quality Control of Baculoviral Bioinsecticide Production

“…This technique provides results composed of a set of enzymatic amplified fragments that are characteristic for a viral species (when primers were designed completely specific), or for a phylogenetic group (when primers derived from multiple alignments of orthologous sequences). With regard to trials designed to particular viruses, it should be noted the work developed for EpapGV (Manzán et al, 2008). Meanwhile, for the detection of groups of related viruses are not many references.…”