Utilization of Nucleotide Probes in Elisa Procedure for the Quantitative Determination of Baculovirus Titer

Abstract

The present work is the development of a simple, rapid, and universally applicable titration method that involves the direct detection of the viral DNA‐binding protein (DBP) gene derived from Autographa californica nucleopolyhedrovirus (AcNPV DBP) as a target for quantitative titer determination of baculovirus and the use of biotin specific probes directed to viral DBP gene. The procedure entails the amplification of the AcNPV DBP gene by using the PCR technique in the presence of digoxigenin‐11‐dUTP from the negative control (non‐infected) and infected samples, and the synthesis of the specific biotin label nucleotide probes directed to the AcNPV DBP gene. These specific probes are then used in the Enzyme Linked Immunosorbent Assay (ELISA) using the immobilized streptavidin on polystyrene microtitration plates for the quantitative determination of baculovirus titer. The plot of O.D._λ=405 nm against the log of the titer (pfu/mL) generated a straight line. The linear range for titer determination of baculovirus was between 10² and 10⁵ pfu/ml⁻¹ for 50 µl of supernatant. Moreover, because of the 96% homology of sequences concerning DBP gene between Bombyx mori nucleopolyhedrovirus (BmNPV) and AcNPV, specific biotin label nucleotide probes obtained from this procedure could be used as a universal tool for titer determination of both viruses.

Keywords:

• Autographa californica nucleopolyhedrovirus
• DNA‐binding protein
• polymerase chain reaction
• enzyme linked immunosorbent assay
• quantitative determination
• baculovirus titer