猪流行性腹泻病毒RT-LAMP可视化检测方法的建立

Development of HNB-based Visual RT-LAMP Method for Detection of Porcine Epidemic Diarrhea Virus

为建立一种适用于现场快速检测的猪流行性腹泻病毒(PEDV)LAMP技术,基于羟基萘酚蓝(HNB)的可视化显色特点,根据PEDV M基因编码区序列,设计合成1套引物,通过反应物浓度和反应条件优化,建立了可闭管检测的PEDV RT-LAMP检测方法。特异性和灵敏度试验结果显示,建立的RT-LAMP检测技术快速、灵敏、特异,可于1 h内检出0.2 mL 0.1 TCID_(50)/mL的病毒RNA,与实时荧光RT-PCR检测方法灵敏度一致,与猪瘟病毒、猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型以及猪链球菌2型核酸不发生交叉反应。利用该方法对187份送检的粪拭子及病死猪组织样品进行应用检测,检出阳性样品9份,与荧光定量RT-PCR方法检测结果一致。试验结果表明,所建立的方法快速、特异,重复性满足要求,适用于送检样品的PEDV快速检测。

In order to establish a RT-LAMP method for rapid detection of porcine epidemic diarrhea virus(PEDV), based on color reaction of HNB and according to M gene sequence of PEDV,a set of primers were designed and synthesized. By optimizing the reaction concentration and conditions,the RT-LAMP method was developed. Specificity and sensitivity results showed the established method was fast,sensitive and specific. It could detect PEDV-RNA extracted from 0.2 mL virus suspension(0.1 TCID50/mL),which was consistent with the real-time RT-PCR method. No cross reactions were observed with the nucleic acids of classical swine fever virus(CSFV),pseudorabies virus (PRV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV-2) and Streptococcus suis type 2. Using the RT-LAMP method,a total of 9 positive samples were detected from 187 samples of fecal swabs and dead pigs submitted,which was also consistent with the results of real-time RT-PCR. As a conclusion,the established method was rapid,specific and repeatable,and it was suitable for rapid detection of PEDV in submitted tissue samples.