猪传染性胃肠炎病毒可视化RT-LAMP检测方法的建立

Establishment of a visual RT-LAMP detection assay for TGEV

通过对比猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)主要流行毒株的M基因序列,选择其高度保守的区域设计LAMP引物,以羟基萘酚蓝(HNB)为指示剂,建立了1种快速检测TGEV的可视化环介导等温扩增方法。结果显示,采用建立的方法在恒温水浴锅中约1 h即可完成检测;用该方法检测猪流行性腹泻病毒,猪轮状病毒,猪繁殖与呼吸综合征病毒,猪细小病毒,猪圆环病毒,均不发生交叉反应;以pET-21b-M质粒为模板评价所建立方法的敏感性,最低检测限为3.7×10^3个拷贝,比普通PCR方法高2个数量级;在反应前向反应体系中加入HNB,反应结束后阳性反应管颜色从紫罗兰色变为天蓝色,阴性反应管颜色无变化,且阴阳性管颜色差异明显。用建立的方法对收集的42份临床腹泻样品进行检测,共检测到21份样品阳性。为快速检测TGEV提供方法。

Transmissible gastroenteritis virus(TGEV) is a viral pathogen which causes intestinal diseases in pigs.In this study,a visual Loop-mediated isothermal amplification method for TGEV detection was established using hydroxyl naphthol blue(HNB) as indicator.M gene sequences of major epidemic TGEV strain was compared and then the specific primers for Loop-mediated isothermal amplification(LAMP) were designed according to the highly conserved region of TGEV M gene.Using the established LAMP method,TGEV detection could be completed in a constant temperature within 1 h,and there was no cross reaction to porcine epidemic diarrhea virus(PEDV),porcine rotavirus(PoRV),porcine parvovirus(PPV),porcine circovirus type 2(PCV2) and porcine reproductive and respiratory syndrome virus(PRRSV).Its sensitivity was evaluated and it was found that the lowest detectable number of pET-21 b-M plasmid copies was 3.7×10^3 per microliter.It was two orders of magnitude higher than conventional PCR.By adding HNB before the reaction,the color of the positive tube could be visually observed changing from purple to blue,however the color of negative reaction tube did not change.The color difference between negative and positive tubes was obvious.42 clinical samples from swine farms were assayed with the established LAMP method,the results showed that 21 samples were positive.The establishment of this method provides a practical method for the rapid diagnosis of TGE in basic-level clinical units.