Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Services, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA.
Smiths Detection, 2202 Lakeside Boulevard, Edgewood, MD 21040, USA.
Toxins (Basel). 2024 Mar 14;16(3):148. doi: 10.3390/toxins16030148.
Shiga-toxin-producing (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1-0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.
产志贺毒素（STEC）可引起多种疾病，包括出血性结肠炎和溶血性尿毒综合征（HUS）。目前食品药品监督管理局（FDA）的食品安全检验服务（FSIS）检测方法用于 STEC 检测的方法是采用食品和药物管理局（FDA）细菌分析手册（BAM）协议，包括富集、细胞培养和基因组测序，需要时间完成，从而延迟诊断和治疗。我们希望开发一种快速、敏感且具有潜在便携性的检测方法，通过使用 CANARY（细胞分析和抗原风险与产量通知）B 细胞基于生物传感器技术检测志贺毒素（Stx）来识别 STEC。评估了五种潜在的生物传感器细胞系检测 Stx2 的能力。使用最佳生物传感器细胞系（T5）的结果表明，该生物传感器在 4°C 下用箔覆盖的测定缓冲液重新组成后稳定，在第 5 天之前的估计检测限（LOD）约为 0.1-0.2ng/mL，在第 10 天约为 0.4ng/mL。该检测方法可检测到广泛的 Stx2 亚型，包括 Stx2a、Stx2b、Stx2c、Stx2d 和 Stx2g，但与密切相关的 Stx1、蓖麻毒素或相思豆毒素不发生交叉反应。此外，该检测方法能够检测到在接种后 8 和 24 小时用丝裂霉素 C 培养的 STEC 培养上清液中的 Stx2。这些结果表明，本研究中开发的 STEC CANARY 生物传感器具有灵敏度高、重现性好、特异性强、快速（约 3 分钟）的特点，可能适用于环境和食品的监测，以保护公众健康。
