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Detection of Black Queen Cell Virus via Quantitative Real-Time PCR
The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators. Recently, pathogens and parasites affect the life span and fecundity of their host and been isolated from B. terristris. In order to detect viral infection in the field populations of B. terristris, we collected adults and isolated total RNA for reverse transcriptase-polymerase chain reaction (PCR). The PCR primers specific for several viruses such as deformed wing virus, Israel acute paralysis virus, Kashmir bee virus and black queen cell virus (BQCV) were newly designed and applied to gene amplification for cloning. Only BQCV was successfully amplified and sequenced, which suggests that BQCV may mainly infects the examined field population of B. terristris. To detect of capsid protein gene of BQCV, 4 selected regions were analyzed by primary PCR and 1 region was successfully amplified, which was further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that BQCV was detected at concentrations as low as 0.1ng/μl total RNA. This result suggests that the 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. terristris.
