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.
Food Environ Virol. 2023 Jun;15(2):144-157. doi: 10.1007/s12560-022-09548-7. Epub 2023 Jan 14.
Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg concentration (P < 0.05), in addition to primer quality. The optimal Mg concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay (P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 10 PFU/15 g, 8.3 × 10 PFU/50 g, 8.3 × 10 PFU/5 g, and 8.3 × 10 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.
食源性甲型肝炎感染已被认为是全球公共卫生的主要威胁。甲型肝炎病毒 (HAV) 感染发病率的增加与食品产品在全球范围内的贸易增长有关。快速、灵敏地检测食品中的 HAV 对于暴发调查非常重要。实时 RT-PCR 迄今为止是最广泛用于检测 HAV 的方法。然而，该技术依赖于扩增子的荧光测定，需要复杂、昂贵的仪器和经过培训的人员，限制了其在资源有限环境中的应用。在这项研究中，开发了一种稳健、经济实惠且简单的检测方法，即逆转录环介导等温扩增 (RT-LAMP) 检测法与实时生物发光扩增检测法 (BART) 相结合，用于检测不同食品基质中的 HAV，包括葱、草莓、贻贝和牛奶。研究了一步法 RT-LAMP-BART 和两步法 RT-LAMP-BART 在不同食品基质中检测 HAV 的效率，并与实时 RT-PCR 进行了比较。一步法 RT-LAMP-BART 的灵敏度显著受 Mg 浓度的影响（P<0.05），除了引物质量。一步法 RT-LAMP-BART 的最佳 Mg 浓度为 2 mM，两步法 RT-LAMP-BART 的最佳 Mg 浓度为 4 mM。与盒式纯化引物相比，HPLC 纯化引物可显著提高 RT-LAMP-BART 检测法的灵敏度（P<0.05）。在检测不同食品基质中的 HAV 时，两步法 RT-LAMP-BART 的性能与实时 RT-PCR 相当，优于一步法 RT-LAMP-BART。两步法 RT-LAMP-BART 检测葱、草莓、贻贝和牛奶中 HAV 的检测限分别为 8.3×10 PFU/15 g、8.3×10 PFU/50 g、8.3×10 PFU/5 g 和 8.3×10 PFU/40 mL。开发的 RT-LAMP-BART 是一种用于食源性 HAV 检测的有效、简单、灵敏和稳健的方法。
