基于钙黄绿素显色的可视化LAMP检测猪流行性腹泻病毒的研究

针对猪流行性腹泻病毒(PEDV)的NP基因设计1套环介导等温扩增(LAMP)引物,在反应体系中添加钙黄绿素/氯化锰指示剂代替传统的反应后添加SYBR Green Ⅰ染料,建立了基于钙黄绿素的可视化LAMP检测PEDV的方法。该方法能在65 ℃ 1 h内特异性扩增PEDV,与猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)及大肠杆菌等均无交叉反应,检测下限为3.73 pg/μL,其结果可用肉眼判断,快捷方便。用该方法对龙岩学院动物医学研究所接诊的93份腹泻病例进行检测,结果表明,LAMP方法检测PEDV的阳性率为43.01%(40/93),高于普通PCR的检出率(38.7%,36/93)。本试验建立的可视化LAMP检测方法能用于PED诊断。

Detection of Porcine Epidemic Diarrhea Virus by Calcein-based Visual Loop-mediated Isothermal Amplification (LAMP) Assay

In order to establish a loop-mediated isothermal amplification assay (LAMP),a set of LAMP primers targeting to NP gene of porcine epidemic diarrhea virus (PEDV) were designed and synthesized.Calcein and manganese chloride were added as the indicator instead of SYBR Green Ⅰ after reaction.The LAMP could amplify PEDV specifically at 65 ℃ in 1 h with detection limit of 3.73 pg/μL and the result could be judged by naked eye.The LAMP didn''t react with CSFV,TGEV,PoRV and E.coli.93 clinical samples of PED suspected case collected from Veterinary Institute of Longyan University were submit to detect PEDV by LAMP,the result showed that the positive rate of the samples was 43.01%,which was higher than that (38.7%) of conventional PCR.The established visual LAMP could be used for diagnosing PED.