Detection of three honeybee viruses simultaneously by a single Multiplex Reverse Transcriptase PCR

A single multiplex reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed for the simultaneous detection of three honeybee viruses: acute bee paralysis virus (ABPV), sacbrood virus (SBV) and black queen cell virus (BQCV). Unique PCR primers were designed from the complete genome sequence to amplify fragments of 900 bp from ABPV, 434 bp from SBV and 316 bp from BQCV. Individual bee pupae homogenates or total RNA extracted from these crude extracts were used in the RT-PCR amplification. Sequence analysis of the fragments amplified revealed nucleotide sequence identities between 97 and 98% for each virus against its reference strain. In a blind test, samples containing various combinations of ABPV, SBV and BQCV were successfully identified. Furthermore, field samples of honeybee pupae were screened for viral infections, and evidence of virus inapparent infection as well as virus co-infection were found.