Multiplex PCR and quality control of Epinotia aporema granulovirus production.
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.
Manzán
Maria Alejandra
MA
Instituto de Biotecnología y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata-Consejo Nacional de Investigaciones Científicas y Técnicas, Calle 49 y 115, 1900, La Plata, Argentina. alemanza@msu.edu
Aljinovic
Ernesto Mario
EM
Biedma
Marina Elizabeth
ME
Sciocco-Cap
Alicia
A
Ghiringhelli
Pablo Daniel
PD
Romanowski
Víctor
V
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D025722:Granulovirus / Q000235:genetics / Q000502:physiology*; D009036:Moths / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014776:Virus Cultivation; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral