School of Life Science, Shanghai University, Shanghai 200444, China.
Virol J. 2010 Aug 29;7:206. doi: 10.1186/1743-422X-7-206.
A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV) was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.
选择猪传染性胃肠炎冠状病毒（TGEV）核衣壳基因的保守核酸片段作为靶标，成功设计了 6 条特异性引物。通过在 60°C 孵育 1 小时，建立了环介导等温扩增（LAMP）检测 TGEV 的方法，并用 HphI 消化确认产物的特异性。在 LAMP 反应中加入 1×SYBR greenI 构建了高准确度的 TGEV 定量标准曲线。该研究建立的方法仅能检测到 TGEV，与其他病毒无交叉反应，表明其具有高度特异性。通过使用系列稀释样本作为模板，LAMP 的检测限约为 10pg RNA，比 PCR 敏感 10 倍，可与巢式 PCR 相媲美。
