当前位置: X-MOL 学术J. Virol. Methods › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Rapid and simple colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Bovine alphaherpesvirus 1
Journal of Virological Methods ( IF 3.1 ) Pub Date : 2020-12-10 , DOI: 10.1016/j.jviromet.2020.114041
Deborah Peltzer 1 , Kurt Tobler 1 , Cornel Fraefel 1 , Madeleine Maley 2 , Claudia Bachofen 1
Affiliation  

As the causative agent of Infectious Bovine Rhinotracheitis (IBR) and Infectious Pustular Vulvovaginitis/Balanoposthitis (IPV/IPB), Bovine alphaherpesvirus 1 (BoHV-1) is responsible for high economic losses in the cattle industry worldwide. This study aimed to establish a fast, colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of viral DNA. Phenol red is used as pH-sensitive readout, relying on a distinct color change from pink to yellow in case of a positive reaction. LAMP reactions with different primers were compared and a newly designed set targeting the gene encoding the tegument protein V67 provided best results, enabling readout within 8−30 min. LAMP showed less cross-reactions with other ruminant alphaherpesviruses than qPCR but was 10-fold less sensitive. However, LAMP still detected down to 14 copies. The test performance was evaluated using 26 well-characterized nasal swabs from cattle with respiratory disease. All samples were correctly identified when using column-extracted DNA. Using a simple DNA precipitation method, only two weak-positive samples turned indeterminate. Combining this DNA precipitation with a makeshift water bath heated by a gastronomic immersion heater allowed successful application of the colorimetric LAMP assay under resource-limited conditions. This technique can therefore help in managing IBR/IPV outbreaks where sophisticated laboratory equipment is unavailable.



中文翻译:

用于检测牛 α 疱疹病毒 1 的快速且简单的比色环介导等温扩增 (LAMP) 分析

作为传染性牛鼻气管炎 (IBR) 和传染性脓疱性外阴阴道炎/龟头包皮炎 (IPV/IPB) 的病原体,牛 α 疱疹病毒 1 (BoHV-1) 是全球养牛业高额经济损失的原因。本研究旨在建立一种快速、比色环介导的等温扩增 (LAMP) 检测方法,用于检测病毒 DNA。酚红用作 pH 敏感读数,在阳性反应的情况下依赖于从粉红色到黄色的明显颜色变化。比较了不同引物的 LAMP 反应,新设计的一组靶向编码被膜蛋白 V67 的基因提供了最佳结果,能够在 8-30 分钟内读出。LAMP 与其他反刍动物 alphaherpesviruses 的交叉反应比 qPCR 少,但敏感性低 10 倍。然而,LAMP 仍然检测到低至 14 个拷贝。使用来自患有呼吸系统疾病的牛的 26 个特征明确的鼻拭子评估了测试性能。使用柱提取的 DNA 时,所有样品都被正确识别。使用简单的 DNA 沉淀方法,只有两个弱阳性样本变为不确定。将这种 DNA 沉淀与由美食浸入式加热器加热的临时水浴相结合,可以在资源有限的条件下成功应用比色灯检测。因此,该技术可以帮助在没有精密实验室设备的情况下管理 IBR/IPV 爆发。只有两个弱阳性样本变得不确定。将这种 DNA 沉淀与由美食浸入式加热器加热的临时水浴相结合,可以在资源有限的条件下成功应用比色灯检测。因此,该技术可以帮助在没有精密实验室设备的情况下管理 IBR/IPV 爆发。只有两个弱阳性样本变得不确定。将这种 DNA 沉淀与由美食浸入式加热器加热的临时水浴相结合,可以在资源有限的条件下成功应用比色灯检测。因此,该技术可以帮助在没有精密实验室设备的情况下管理 IBR/IPV 爆发。

更新日期:2020-12-21
down
wechat
bug