Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang 150001, China.
Virol J. 2011 Mar 8;8:108. doi: 10.1186/1743-422X-8-108.
Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease in young chickens caused by infectious bursal disease virus (IBDV). It causes huge economic losses to the poultry industry. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the detection and discrimination of IBDV.
In this study, we applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect IBDV in one simple step and further identified the very virulent strain from non-vvIBDVs with a simply post-amplification restriction enzyme analysis. Based on sequence analysis, a set of two inner, two outer and two loop primers were designed to target the VP5 gene and they showed great specificity with no cross reaction to the other common avian pathogens. The detection limit determined by both color change inspection and agarose gel electrophoresis was 28 copies viral RNA, which was almost as sensitive as a real-time RT-PCR previous developed in our laboratory. We also identified a unique Tfi I restriction site located exclusively in non-vvIBDVs, so very virulent strain could be distinguished from current vaccine strains. By screening a panel of clinical specimens, results showed that this method is high feasible in clinical settings, and it obtained results 100% correlated with real-time RT-PCR.
RT-LAMP is a rapid, simple and sensitive assay. In combination with the Tfi I restriction analysis, this method holds great promises not only in laboratory detection and discrimination of IBDV but also in large scale field and clinical studies.
传染性法氏囊病（IBD）是一种由传染性法氏囊病病毒（IBDV）引起的、高度传染性的雏鸡免疫抑制病。它给家禽业造成了巨大的经济损失。本研究的目的是开发一种环介导等温扩增（LAMP）方法，用于检测和区分 IBDV。
在本研究中，我们应用反转录环介导等温扩增（RT-LAMP）在一个简单的步骤中检测 IBDV，并通过简单的扩增后酶切分析从非超强毒 IBDV 中鉴定出超强毒株。基于序列分析，设计了一组两对内引物、两对外引物和两对环引物，以针对 VP5 基因，它们与其他常见的禽病原体没有交叉反应，具有很好的特异性。通过颜色变化检测和琼脂糖凝胶电泳确定的检测限为 28 拷贝病毒 RNA，这与我们实验室之前开发的实时 RT-PCR 几乎一样敏感。我们还鉴定了一个独特的 Tfi I 限制位点，该位点仅存在于非超强毒 IBDV 中，因此可以区分超强毒株和当前的疫苗株。通过对一组临床标本进行筛选，结果表明该方法在临床环境中具有高度可行性，并且与实时 RT-PCR 的结果完全相关。
RT-LAMP 是一种快速、简单和敏感的检测方法。结合 Tfi I 限制分析，该方法不仅有望用于实验室检测和区分 IBDV，而且有望用于大规模现场和临床研究。
