基于荧光显色的环介导等温扩增技术(LAMP)检测禽呼肠孤病毒

Fluorescent reagent in detection of avian reovirus by loop-mediated isothermal amplification(LAMP)

建立了一种基于荧光显色的禽呼肠孤病毒(ARV)环介导等温扩增(LAMP)检测方法。该检测方法使用针对ARV的p10基因8个区域的6条LAMP引物,能够在等温(63℃)条件下1h内完成反应,具有良好的敏感性和特异性,最低能够检出102个拷贝的病毒,敏感性比普通PCR高100倍,而对其他病毒检测结果为阴性。在荧光显色中分别应用SYBR GreenⅠ和钙黄绿素作为显色剂,其结果与琼脂糖凝胶电泳一致。在对80份临床样本的检测中,使用LAMP和PCR检测出阳性样本的数量分别为68份和55份。因此,基于荧光显色的LAMP方法可以应用于ARV的快速检测,具有在科研机构、基层实验室特别是检测现场推广和应用的潜力。

Abstraet：A novel detection method of avian reovirus by loop=mediated^isothermal amplification （LAMP） was reported using fluorescent reagent as the product judgment. Width one set of primers containing six special primers targeting p10 gene,the LAMP reaction could,complete within 1 hour at 63℃. Sensitivity and specificity of this method was tested. The detect limit of the assay was 102 copies virus, which was 100 times higher than traditional PCR. In： the fluorescent reagent test, SYBR Green I and calcein reagent were respectively used in LAMP product judgement,and the result was consistent with agarose gel electrophoresis. In the detection of 80 qlinical samples, 68 and 55 samples were positive by LAMP and PCR assays, respectively： Therefore, LAMP assay with fluorescent reagent provides a useful tool for fast detection of avian reo;cirus. Key words： ARV ; LAMP ; fluorescent reagent ; calcein