Department of Life Science, College of Natural Science, Kyonggi University, Suwon 443-760, Republic of Korea.
J Virol Methods. 2012 Jan;179(1):195-200. doi: 10.1016/j.jviromet.2011.10.014. Epub 2011 Nov 3.
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.
建立了一种实时逆转录聚合酶链反应（qRT-PCR）检测方法，用于快速、灵敏地检测 sacbrood 病毒（SBV）基因组，并应用于蜜蜂样本。使用含有部分 SBV 基因组的质粒 DNA 进行连续稀释，qRT-PCR 检测可检测到低至 1×10(2)拷贝/μl（相关系数>0.99）的病毒，而传统 RT-PCR 检测可检测到 1×10(3)拷贝/μl。作为一种快速检测方法，使用具有 6μl 微腔体积和快速出色的加热/冷却速率的 GenSpector TMC-1000 硅基芯片式热循环仪进行超快速实时 PCR（URRT-PCR）。使用该方法，在 40 个 PCR 循环后 17 分钟内检测到 10(3)拷贝的 pBX-SBV3.8 克隆，包括熔点分析。为了减少 SBV 的检测时间，尝试了从蜜蜂样本中合成 SBV 基因组的 cDNA，并将其用作 URRT-PCR 检测的模板。结果表明，5 分钟的反应时间足以合成用于 SBV URRT-PCR 检测的 cDNA 模板。本研究描述了一种新型的基于 PCR 的方法，能够在 22 分钟内检测环境样本中的 RNA 病毒，包括实时逆转录、PCR 检测和熔点分析。
