Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu Province, China.
J Virol Methods. 2012 Feb;179(2):390-5. doi: 10.1016/j.jviromet.2011.11.027. Epub 2011 Dec 6.
The porcine boca-like virus (Pbo-likeV) was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS). In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Pbo-likeV. A set of four primers specific for six regions of Pbo-likeV VP1/2 genes was designed with the online software. The reaction temperature and time were optimized to 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change due to addition of fluorescent dye. The developed method was highly specific for detection of Pbo-likeV, and no cross-reaction was observed with other swine viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and classic swine fever virus (CSFV) found commonly in China. The lower detection limit of the LAMP assay was approximately 10 copies per reaction, and it was 100 times more sensitive than that of conventional PCR. Furthermore, the efficiency of LAMP for detection Pbo-likeV in clinical samples was comparable to PCR and sequencing. These results showed that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of Pbo-likeV, and the procedure of LAMP does not rely on any special equipment. It has capacity for the detection of Pbo-likeV both in the laboratory and on farms.
猪口蹄样病毒（Pbo-likeV）最近在瑞典断奶后多系统消耗综合征（PMWS）的猪中被发现。在本研究中，开发了一种环介导等温扩增（LAMP）检测方法，用于快速、特异和灵敏地检测 Pbo-likeV。使用在线软件设计了针对 Pbo-likeV VP1/2 基因六个区域的一组四个特异性引物。优化了反应温度和时间分别为 65°C 和 60 分钟。通过琼脂糖凝胶电泳或通过添加荧光染料观察颜色变化来检测 LAMP 产物。所开发的方法对 Pbo-likeV 的检测具有高度特异性，与在中国常见的其他猪病毒如猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒 2 型（PCV2）、猪细小病毒（PPV）和经典猪瘟病毒（CSFV）没有交叉反应。LAMP 检测的最低检测限约为 10 个拷贝/反应，比常规 PCR 灵敏 100 倍。此外，LAMP 检测临床样本中 Pbo-likeV 的效率与 PCR 和测序相当。这些结果表明，LAMP 检测方法是一种简单、快速、敏感和特异的检测 Pbo-likeV 的技术，并且 LAMP 程序不依赖于任何特殊设备。它具有在实验室和农场检测 Pbo-likeV 的能力。
