Establishment and preliminary application of a real-time RT-PCR assay with TaqMan probe for detecting acute bee paralysis virus

Abstract:

[Background] Acute bee paralysis virus (ABPV) is a highly virulent bee virus that causes the death of honeybee and colony exhaustion. [Objective] This experiment was conducted to develop a rapid and sensitive real-time RT-PCR method for the detection of ABPV. [Methods] The primers and probe were designed according to the conservative sequence of capsid protein gene of ABPV registered in GenBank. By optimizing the reaction conditions such as primers, probe concentration, and alternating temperature, a detection method for ABPV based on TaqMan probes was successfully established, and the sensitivity, specificity and stability of the method were tested. [Results] The real-time RT-PCR assay showed a good linear relationship between 9.8×10¹-9.8×10⁸ copies/μL, linear correlation coefficient R² was 0.998, and amplification efficiency was 103.8%. The sensitivity limit of the method was 9.8 copies/μL, and there was no cross-reaction with other honey bee viruses, which represents showing high sensitivity and specificity. The coefficient of variation (CV) for intra-assay and inter-assay repeatability were 0.19%-0.80% and 0.57%-1.07% respectively. In 70 samples of honey bee collected from Fujian regions in 2018 and 2019, the ABPV detection rate was 2.86%. [Conclusion] The ABPV real-time RT-PCR detection method can be used for laboratory testing, epidemiological investigation and epidemic monitoring.