title: Development of a real-time RT-PCR assay with TaqMan probe for specific detection of acute bee paralysis virus.
abstract: 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.
authors.affiliation: University of Ljubljana, Veterinary Faculty, Institute of Microbiology and Parasitology, Gerbičeva 60, 1000 Ljubljana, Slovenia. urska.jamnikar@vf.uni-lj.si
authors.forename: Urška
authors.initials: U
authors.lastname: Jamnikar Ciglenečki
authors.forename: Ivan
authors.initials: I
authors.lastname: Toplak
mesh_terms: D000818:Animals; D001516:Bees / Q000821:virology*; D056629:Dicistroviridae / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D015345:Oligonucleotide Probes