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Diagnostic PCR for Black Queen Eell Virus from Bombus Terrestris Via Real-time PCR
The bumblebee, Bombus terrestris, plays an important role as one of alternative pollinators since the outbreak of honeybee colony collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites affecting the life span and fecundity of their host have been discovered in B. terrestris. In this study, in order to detect viral infection in B. terrestris, we collected B. terrestris adults and isolated total RNA for diagnostic PCR. The PCR primers specific for pathogenic viruses were newly designed and applied to gene amplification for cloning and detection. Capsid protein gene of black queen cell virus (BQCV) among examined viral genes was only successfully amplified from collected bumble bee adults and sequenced. To optimize the detection of capsid protein gene of BQCV, 4 regions in the capsid protein gene were selected and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis revealed that capsid protein gene was directly detected with not more than 200 ng total RNA. This result suggests that an optimized detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terrestris.
