title: Rapid detection of sacbrood virus in honeybee using ultra-rapid real-time polymerase chain reaction.
abstract: A real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for the fast and highly sensitive detection of the sacbrood virus (SBV) genome and applied to honeybee samples. Using plasmid DNA containing a partial SBV genome and diluted serially, as few as 1×10(2)copies/μl (correlation co-efficiency >0.99) were detected by the qRT-PCR assay, whereas 1×10(3)copies/μl were detected by the conventional RT-PCR assay. As a rapid detection method, ultra-rapid real-time PCR (URRT-PCR) was carried out with a GenSpector TMC-1000 silicon-glass chip-based thermal cycler, which has a 6μl micro-chamber volume and a fast outstandingly heating/cooling rate. Using this method, 10(3)copies of pBX-SBV3.8 clone were detected within 17 min after 40 PCR cycles, including melting point analysis. To reduce the detection time for SBV, synthesis of the cDNA of the SBV genome from a honeybee sample was attempted for different reaction times and the cDNA was used as the template for URRT-PCR assays. The results indicated that a 5 min reaction time was sufficient to synthesize cDNA as the template for the SBV URRT-PCR assay. This study described a novel PCR-based method that is able to detect an RNA virus in environmental samples within 22 min, including reverse transcription, PCR detection and melting point analysis in real-time.
authors.affiliation: Department of Life Science, College of Natural Science, Kyonggi University, Suwon 443-760, Republic of Korea.
authors.forename: Mi-Sun
authors.initials: MS
authors.lastname: Yoo
authors.forename: Kim Cuc Nguyen
authors.initials: KC
authors.lastname: Thi
authors.forename: Phu
authors.initials: P
authors.lastname: Van Nguyen
authors.forename: Sang-Hoon
authors.initials: SH
authors.lastname: Han
authors.forename: Soon-Hwan
authors.initials: SH
authors.lastname: Kwon
authors.forename: Byoung-Su
authors.initials: BS
authors.lastname: Yoon
mesh_terms: D000818:Animals; D001516:Bees / Q000821:virology*; D004772:Entomology / Q000379:methods; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*