Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada; Canadian Research Institute for Food Safety, 43 McGilvray Street, Guelph, ON, N1G 2W1, Canada.
Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada.
Food Microbiol. 2019 Dec;84:103236. doi: 10.1016/j.fm.2019.05.020. Epub 2019 May 30.
Hepatitis A virus (HAV) continues to be the leading cause of viral hepatitis. HAV outbreaks have been linked to the consumption of milk, but methods for HAV detection in milk are very limited. We developed a method to concentrate HAV in milk using protamine-coated iron oxide (FeO) magnetic nanoparticles (PMNPs). In this study, protamine was covalently coated on the surface of the MNPs (20-30 nm) by a three-step chemical reaction. The successful linkage of protamine to the MNPs was confirmed by Fourier transform infrared spectroscopy (FTIR), zeta potential, and transmission electron microscopy (TEM). When used for concentrating HAV from 40 mL of milk, 50 μL of PMNPs were added to the sample and mixed for 20 min by gentle rotation, followed by a magnet capture for 30 min. The captured PMNPs were washed with glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and HAV RNA was extracted using the QIAamp MinElute Virus Spin Kit and quantified by real-time RT-PCR. The method showed a detection limit of 8.3 × 10 PFU of HAV in milk. The whole concentration procedure could be completed in approximately 50 min. The developed method was simple, inexpensive, and easy-to-perform.
甲型肝炎病毒 (HAV) 仍然是病毒性肝炎的主要病因。甲型肝炎病毒爆发与饮用牛奶有关，但牛奶中甲肝病毒的检测方法非常有限。我们开发了一种使用鱼精蛋白包裹的氧化铁 (FeO) 磁性纳米颗粒 (PMNPs) 浓缩牛奶中甲肝病毒的方法。在这项研究中，通过三步化学反应将鱼精蛋白共价连接到 MNPs（20-30nm）表面。傅里叶变换红外光谱 (FTIR)、Zeta 电位和透射电子显微镜 (TEM) 证实了鱼精蛋白与 MNPs 的成功连接。当用于从 40mL 牛奶中浓缩 HAV 时，将 50µL PMNPs 添加到样品中，通过轻轻旋转混合 20 分钟，然后用磁铁捕获 30 分钟。捕获的 PMNPs 用甘氨酸缓冲液（0.05M 甘氨酸、0.14M NaCl、0.2%（v/v）Tween 20、pH9.0）洗涤，并使用 QIAamp MinElute 病毒旋转试剂盒提取 HAV RNA，并通过实时 RT-PCR 定量。该方法在牛奶中的检测限为 8.3×10PFU 的 HAV。整个浓缩过程大约需要 50 分钟。所开发的方法简单、廉价且易于实施。
