【摘要】 以羧基化修饰的磁珠和抗猪流行性腹泻病毒(PEDV)M蛋白双价纳米抗体偶联构建免疫磁珠(IMNBs-Ⅱ),利用PEDV细胞毒,验证了其PEDV捕获和富集功能,结合IMNBs-Ⅱ的特征和反转录荧光定量聚合酶链反应(RTqPCR)的检测优势,建立了针对PEDV的一步RT-qPCR检测方法。特异性分析发现,该方法与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪细小病毒和猪圆环病毒等均无交叉反应,表明其具有较好的特异性;灵敏性分析发现,与传统的RT-qPCR方法相比,基于IMNBs-Ⅱ的RT-qPCR方法的检测敏感性提高了10倍。对临床样品检测证实,基于IMNBs-Ⅱ的RT-qPCR方法适用于临床粪便和组织样品的快速、准确检测。本研究建立的方法有效避免了核酸提取过程中的污染问题、简化了试验操作步骤、节省了检测时间,为PEDV高效检测提供更好的选择。更多还原

【Abstract】 In this study,the carboxylated magnetic beads were coupled with bivalent nanobodies against porcine epidemic diarrhea virus(PEDV)M protein to construct immunomagnetic beads(IMNBs-Ⅱ),The capture and enrichment function of IMNBs-Ⅱ was verified by using PEDV propagated in Vero cells.A one-step RT-qPCR detection method for PEDV was established by combining the characteristics of IMNBs-Ⅱ with the detection advantages of reverse transcription fluorescence quantitative polymerase chain reaction(RT-qPCR).Specific analysis found that this method has no cross reactivity with swine fever virus,porcine reproductive and respiratory syndrome virus,porcine parvovirus,porcine circovirus,indicating that it has good specificity.Sensitivity analysis revealed that the detection sensitivity of the RT-qPCR based on IMNBs-Ⅱ was increased 10 times compared to traditional RT-qPCR methods.Detection of the clinical samples confirm that the RT qPCR method based on IMNBs-Ⅱ is suitable for rapid and accurate detection of clinical feces and tissue samples.The method established in this study effectively avoids contamination issues during nucleic acid extraction,simplifies experimental procedures,and saves detection time,which provides a method for efficient detection of PEDV.更多还原