Development of a multiplex RT-PCR assay for the routine detection of seven RNA viruses in Apis mellifera

Abstract

Colony losses in apiaries are frequently one of the most important problems in beekeeping. Colony loss is multifactorial with many reported disorders, Colony Collapse Disorder (CCD), is an increasingly recognised phenomenon which is thought to be caused by many pathogens, including viruses. The aim of this study was to develop a multiplex RT-PCR (mRT-PCR) test to obtain faster results in routine diagnostic laboratories for seven crucial bee viruses. Specific primers for seven RNA viruses, including Israeli acute bee paralysis virus (IAPV), deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), black queen cell virus (BQCV), kashmir bee virus (KBV) and chronic paralysis virus (CBPV), were used for testing procedure. The mRT-PCR assay can amplify seven plasmid DNA fragments from the pooled viral genomes and it was shown to be sensitive because virus copy numbers were detected to be 10⁴ copies/μl when log₁₀ serial dilutions were performed for the optimized mRT- PCR method. It is concluded that, mRT-PCR test can be used in routine analysis because this assay can perform specific, sensitive and reliable results also achieves economic gains and time due to detecting seven viral agents simultaneously.

Keywords: Apis mellifera; Diagnosis; Honey bee viruses; multiplex RT-PCR.