【摘要】 为建立一种快速简便、灵敏度高、准确性好的犬细小病毒环介导等温扩增(LAMP)可视化检测方法,本研究根据犬细小病毒(CPV)的VP2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,建立了犬细小病毒LAMP可视化快速检测方法。结果显示,该方法在63℃条件下恒温扩增60 min具有良好的敏感性和特异性,其最低检测限为10拷贝/μL,与犬瘟热病毒、犬冠状病毒、狂犬病病毒、犬流感病毒均无交叉反应。对200份临床样品进行检测,检出阳性样品21份,与实时荧光定量PCR检测结果符合率为100%。该方法无需特殊设备,肉眼即可直接观察检测结果,具有高效、简便、特异性好、灵敏度高等优点,适用于犬细小病毒现场快速诊断。更多还原

【Abstract】 In order to develop a simple,highly-sensitive and accurate loop-mediated isothermal nucleic acid amplification(LAMP) for rapid detection of canine parvovirus,specific primers were designed based on the highly conserved region of VP2 gene of CPV. By optimizing primer concentration,temperature and time,the visual LAMP for rapid detection of CPV was established. The results of specificity and sensitivity test showed that the sensitivity was 10 copies/μL,and there was no cross reaction with canine distemper virus,canine coronavirus,rabies virus and canine influenza virus,isothermal amplification within 60 minute at 63 ℃. The method was used to detect 200 clinical samples and 21 positive samples were detected. The agreement of positive rate between real-time fluorescent LAMP and qPCR was 100%. Moreover,the results could be observed with naked eyes without special equipment. In a word,it is a suitable method for the rapid detection in the field,with the advantages of good characteristics,high sensitivity,high efficient and convenience.更多还原