School of Chemical engineering & Technology, Tianjin University, Nankai District, Tianjin 300072, China.
Arch Virol. 2012 Aug;157(8):1463-9. doi: 10.1007/s00705-012-1322-y. Epub 2012 May 8.
A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0.0001 TCID(50), reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field.
已开发出一种基于环介导等温扩增 (LAMP) 的快速检测方法，用于检测山羊关节炎-脑炎 (CAEV) 前病毒 DNA。该 LAMP 检测法使用了一组针对位于 p25 基因区域内高度保守序列设计的五个引物。该检测法成功地从细胞培养物、全血样本和分离的 PBMC 中检测到 CAEV 前病毒 DNA。与阴性对照没有交叉反应。使用 Loopamp 实时浊度计监测扩增；记录浊度和相应时间。从 CAEV-Shanxi DNA 的扩增早在 17 分钟即可检测到，最大灵敏度为 0.0001 TCID(50)，在 32 分钟时达到。使用 AGID、PCR 和 LAMP 检测法对 68 份动物血液样本进行了检测，阳性率分别为 30.9%、33.8%和 47.1%。可以直接使用全血，无需分离 PBMC 和核酸提取，将整个过程时间缩短至大约 80 分钟。因此，LAMP 检测法提供了一种简单、快速、具有成本效益的方法，用于特异性和灵敏性地检测 CAEV 前病毒 DNA，应该有助于根除计划和流行病学研究。此外，LAMP 检测法可以在设备较差的实验室以及现场进行。
