非洲猪瘟病毒实时荧光LAMP检测方法的建立与应用

Establishment and Application of Real-time Fluorescent LAMP for Detection of African Swine Fever Virus

关键词:  非洲猪瘟病毒  实时  荧光环介导等温扩增

Key words:  African Swine Fever Virus  Real-time  Fluorescent LAMP

摘要:

为建立一种快速简便、灵敏度高、准确性好的非洲猪瘟病毒恒温快速检测方法,本研究根据ASFV P72基因保守区域设计特异性引物,通过优化引物浓度、反应温度、反应时间,建立了一种非洲猪瘟病毒实时荧光LAMP快速检测方法。结果表明,该方法在63℃条件下扩增40 min,该方法灵敏度高,最低可检测限为10 copies/μL；特异性良好,与猪伪狂犬病毒、猪瘟病毒、美洲型猪繁殖与呼吸综合征病毒、欧洲型猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒无交叉反应；重复性好,变异系数低于5%。对70份临床样本检测,4份血液样品和9份脾脏样品检测阳性,与荧光定量PCR检测结果符合率为100%。该方法灵敏度高、特异性强、重复性好、符合率高,适用于非洲猪瘟现场快速诊断。

Abstract:

In order to develop a simple, highly-sensitive and accurate loop-mediated isothermal nucleic acid amplification (LAMP) for rapid detection of African Swine Fever Virus, specific primers were designed based on the highly conserved region of P72 gene of ASFV. By optimizing primer concentration, temperature and time, the real-time fluorescent LAMP for rapid detection of ASFV was established. The results mainfested that the real-time fluorescent LAMP assay was performed in 63℃ for 40min, the assay was capable of detecting 10 copies/μL, and no cross-reaction with PRV, CSFV, PRRSV(A), PRRSV(E), PEDV. A total of 70 clinical samples including blood, tissue and feed collected were examined by this method. The result showed that 4 blood samples and 9 spleen samples were positive, and the agreement of positive rate between real-time fluorescent LAMP and qPCR was 100%. Overall, the real-time fluorescent LAMP in this study is rapid, simple and effective and this method therefore suitable for clinical samples in field.