Rapid Detection of Black Queen Cell Virus from Honeybee using Reverse Transcription Real-Time Recombinase Polymerase Amplification (RT/RT RPA)

Abstract

Black Queen Cell Virus (BQCV) is one of pathogenic virus in honeybee which could be detected using reverse transcription real-time PCR (RT/RT-PCR). In this study, for rapid detection of BQCV, Recombinase Polymerase Amplification (RPA) was applied, and BQCV-specific reverse transcription real-time RPA (RT/RT-RPA) method was newly developed based on BQCV-specific RT/RT PCR. The Real-time RPA (RT-RPA) was performed at 37°C isothermal condition for 40 minutes. During the experiments, specific DNA amplifications were real-timely monitored using fluorescent detector. BQCV-specific DNA amplification could be detected from 3 min 26 sec after RPA reaction with specific DNA templates by RT-RPA, while 41 min 42 sec was required by qRTPCR with same quantities of initial templates. With generated cDNA from BQCV-infected honeybee, specific DNA amplification was recognized at 4 min 18 sec using RT-RPA, however, 66 min 5 sec was needed using Real-time PCR. Moreover, with reverse transcriptase and RPA solution, BQCVspecific DNA amplification could be detected at 8 min 36 sec from total RNA of BQCV-infected honeybee using one-step Reverse Transcription/Real-Time RPA (RT/RT-RPA).