牛传染性鼻气管炎可视化LAMP检测方法的建立和应用

Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus

摘要:

根据牛传染性鼻气管炎病毒（IBRV）gB基因保守序列（GenBank Accession No. DQ006857.1），利用Primer Explorer V4软件设计3对LAMP引物，通过反应体系的优化、敏感性试验和特异性试验，建立IBRV的LAMP检测方法，并对393份临床样本进行了检测应用。结果显示，建立的IBRV LAMP方法在65 ℃、50 min条件下可扩增出LAMP特征性梯状条带，并可通过颜色变化判定结果。该方法可以检测到10 copies/μL的质粒DNA，与nested-PCR方法的敏感性相当，比PCR方法敏感1 000倍，且与牛病毒性腹泻病毒、猪伪狂犬病毒、水泡口炎病毒等均无交叉反应。应用该方法检测301份鼻腔拭子和92份血清样本，阳性率分别为87.6%和58.8%，表明鼻腔棉拭子更适用于IBRV的临床检测。文中建立的IBRV LAMP方法具有可视化、快速、特异、灵敏性强的优势，适合基层和现场对临床样本进行快速检测，为IBR的防控提供了技术支撑。

Abstract:

Three pairs of primers were designed according to the conserved region of IBRV gB gene published in GenBank(GenBank Accession No. DQ006857.1) using the software Primer Explorer V4. The loop mediated isothermal amplification (LAMP) assay was established by optimization of the reaction system and then evaluated through sensitivity and specificity tests. In total 393 clinical specimens were detected for IBRV using the established LAMP assay performed at 65℃ for 50 min, which produced a ladder-like pattern of amplification bands and the detection result could be judged by color change. The sensitivity of the assay was 10 copies/μL plasmid DNA which was 1000 times higher than that by PCR method and equivalent to nested-PCR. There was no cross-reactivity of the assay with bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and vesicular stomatitis virus (VSV). The positive rate of 301 nasal swabs and 92 serum specimens were 87.6% and 58.8%, respectively, which meant nasal swab specimen was more suitable for clinical IBRV detection by the method. The IBRV LAMP method established in this study has the advantages of visualization, quickness, specificity and sensitivity and be suitable for rapid detection of clinical IBRV detection on the spot.