Liaoning Medical University, Department of Laboratory Animal Center, Jinzhou 121001, China.
J Virol Methods. 2011 Sep;176(1-2):115-9. doi: 10.1016/j.jviromet.2011.05.028. Epub 2011 May 27.
Chinese sacbrood virus (CSBV) has emerged as an important etiologic agent of honeybee infections and is lethal for individual bees, even causing the collapse of entire colonies. Although diagnostic methods for CSBV have been established in many clinical laboratories, application of these methods is largely restricted by the apparatus needed to carry out the reaction and by cost, therefore a simpler and less expensive diagnostic method for CSBV infection is required. In this study a simple and inexpensive system is described that is based on the loop-mediated isothermal amplification (LAMP) assay. The LAMP and the polymerase chain reaction (PCR) methods were compared for their ability to detect CSBV in 31 clinical samples, in purified CSBV-LNQY strains or to be able to discriminate between cDNA samples from other viruses. The detection limit of the LAMP method was 1pg, showing that LAMP is as sensitive as reverse transcriptase (RT)-PCR for CSBV detection. In addition, no DNA band from other related viruses samples was amplified by either method, suggesting that this LAMP assay is as specific as RT-PCR for CSBV detection. All 31 clinical samples that were LAMP assay-positive were also amplified by RT-PCR, however the LAMP assay was faster, more cost effective, and easier to perform as the target gene amplified rapidly, within 2h, and only a standard laboratory water bath or heat block was required for the reaction. The results demonstrate clearly that this LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of CSBV infection of bees.
中国囊状幼虫病毒（CSBV）已成为一种重要的蜜蜂感染病原，对个体蜜蜂具有致命性，甚至导致整个蜂群崩溃。尽管在许多临床实验室已经建立了 CSBV 的诊断方法，但由于反应所需的仪器和成本的限制，这些方法的应用受到了很大的限制，因此需要一种更简单、更经济的 CSBV 感染诊断方法。在本研究中，描述了一种简单而廉价的基于环介导等温扩增（LAMP）检测的系统。比较了 LAMP 和聚合酶链反应（PCR）方法在检测 31 份临床样本、纯化的 CSBV-LNQY 株或区分来自其他病毒的 cDNA 样本中的能力。LAMP 方法的检测限为 1pg，表明 LAMP 与 RT-PCR 一样灵敏，可用于检测 CSBV。此外，两种方法均未扩增来自其他相关病毒样本的 DNA 带，表明该 LAMP 检测方法与 RT-PCR 一样特异，可用于 CSBV 的检测。所有 31 份经 LAMP 检测呈阳性的临床样本均经 RT-PCR 扩增，但 LAMP 检测更快、更具成本效益，且更容易操作，因为目标基因在 2 小时内快速扩增，仅需标准实验室水浴或热块即可进行反应。结果清楚地表明，这种基于 LAMP 的检测方法是一种快速、敏感的蜜蜂 CSBV 感染诊断的有用工具。
