Trial application of PCR techniques in detection of Bombyx mori nucleopolyhedrovirus from cocoon harvesters

Abstract

To clarify the contamination status of Bombyx mori nucleopolyhedrovirus (BmNPV) on cocoon harvesters, usability of PCR techniques in epizootiological research for the silkworm virus was investigated by using BmNPV-specific primers based on the major capsid Bp 39 gene region reported by Lu and Iatrou (1996). In the first stage PCR using a primer pair, Bp 39-1F and -2R, sensitivity and reproducibility of the primer pair to the BmNPV from occlusion bodies (OBs) were restricted in the amplification of the viral DNA from 102 OBs per 20μl of PCR reaction mixture. Application of nested-PCR using a primer pair, Bp 39-3F and -4R after the first PCR, enhanced sensitivity and reproducibility of the amplification of the target sequence of Bp 39 gene region from the viral DNA from 1-10 OBs. Using dust samples from cocoon harvesters collected by gauze wiping method devised in this study and applying the above PCR techniques, contamination status of BmNPV on six cocoon harvesters of sericultural farms was clearly distinguished. This technique could be applicable in estimating the BmNPV dispersal and contamination status on various structural objects of sericultural farms in the future research.