Development of a real-time RT-PCR assay with TaqMan probe for specific detection of acute bee paralysis virus

Real-time polymerase chain reaction (real-time PCR) is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), whereby the probe is designed to bind within the amplified PCR fragment.A new real-time reverse transcription and polymerase chain reaction (real time RT-PCR) assay with TaqMan probe for specific detection of acute bee paralysis virus was designed. The assay was optimized to be highly sensitive and analytically specific and tested with a selection of genetically diverse ABPV strains originating from Slovenia, the United Kingdom (UK), Hungary and Germany. The detection limit of the assay and sensitivity comparisons with conventional RT-PCR were analyzed and this assay can detect a minimum of 44 copies of ABPV/reaction and is 230 times more sensitive than conventional RT-PCR. In addition, the assay is highly reproducible, with an average slope of standard curve made of ten-fold dilutions of standard copies/reaction −3.479 ± 0.19 and an average slope of standard curve made of ten-fold dilutions of RNA −3.409 ± 0.18.

► A specific real-time assay with TaqMan probe for ABPV detection was developed.
► The assay discriminates ABPV from other honeybee viruses.
► An average efficiency with ten-fold serial dilutions of RNA is 96.8%.
► An average efficiency with ten-fold serial dilutions of cloned ABPV RNA is 94.2%.
► The limit of detection of the assay is 44 copies/reaction.