서양뒤영벌에서 Nosema ceranae와 Black Queen Cell Virus의 quantitative Real Time-polymerase chain reaction (qRT-PCR)을 이용한 진단
Nosema ceranae and Black Queen Cell Virus in Bombus terrestris, via quantitative Real Time-polymerase chain reaction (qRT-PCR) using the diagnostic
Bombus terrestris quantitative real-time PCR black queen cell virus Nosema ceranae Nosema apis diagnosis;
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The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators to the honeybee. Recently, as pathogens and parasites such as viruses, bacteria and mites affect the life span and fecundity of their host, the honeybee we well as B. terrestris also have been biologically damaged. There have been many reports on the detection for these pathogens described above, the diagnostic methodologies are not enough to be satisfactory. In this study, in order to detect the microsporidian pathogen, Nosema Spp. and black queen cell virus in the field populations of B. terristris, we collected adults and isolated their genomic DNA and totoal RNA for diagnostic PCR. The PCR primers specific for these two pathogens were newly designed and applied to gene amplification for cloning. The target genes, small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae and capsid proteins in BQCV, were successfully amplified and sequenced, indicating that N. ceranae and BQCV infects the examined field population of B. terristris. To optimize detection condition via quantitative real-time PCR, each gene was divided into several regions for rapid and specific gene amplification and detection. The qRT-PCR analyses demonstrated that SSU rRNA of N. ceranae and the capsid protein of BQCV were detected at concentrations as low as 0.85 ng/μl genomic DNA and 60 ng/μl total RNA. These results suggest that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae and BQCV infection in the field population as well as risk assessment of B. terrestris.