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

• Published in: Journal of virological methods 2012-09, Vol.184 (1-2), p.63-68
• Main Authors: Ciglenečki, Urška Jamnikar, Toplak, Ivan
• Format: Article
• Language: English
• Subjects: Acute bee paralysis virus; Animals; Bees - virology; Biological and medical sciences; detection limit; Dicistroviridae - isolation & purification; DNA; dyes; Fundamental and applied biological sciences. Psychology; Honeybee; Limiting dilution; Microbiology; Oligonucleotide Probes - chemistry; Oligonucleotide Probes - genetics; quantitative polymerase chain reaction; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA; Sensitivity and Specificity; TaqMan; Techniques used in virology; Time Factors; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.05.010

► 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. 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.