Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, United States.
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, United States.
J Microbiol Methods. 2013 Aug;94(2):125-132. doi: 10.1016/j.mimet.2013.05.009. Epub 2013 May 16.
Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams. In particular, oyster hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis since 2006. Accurate detection of vibrios, including V. tubiashii, is critical to the hatcheries in order to allow for rapid remediation efforts. The current methods for detection of Vibrio spp. are not ideal for use at the hatchery. Plating samples require time and is not sensitive to extracelluar pathogenic products, such as the secreted zinc-metalloprotease, VtpA. Other sensitive methods to detect bacteria, such as qPCR, require a high level of laboratory skills and expensive supplies that are prohibitive for use at hatchery sites. Thus, hatcheries would benefit from a sensitive, simple method to detect V. tubiashii and its secreted toxin. Here, we describe the development of two inexpensive and highly specific tests for the shellfish-toxic zinc-metalloprotease secreted by V. tubiashii: enzyme-linked immunoassays (ELISA) and a lateral flow immunoassay (dipstick assay). Both technologies rely on a set of monoclonal antibodies used in a sandwich format, with the capture antibody recognizing a different epitope than the detection antibody on the mature VtpA protein. Both assays are quantitative and give colorimetric readouts. The sandwich ELISA was sensitive when VtpA was diluted into PBS, but was markedly less sensitive in conditions that correlate with the environment of hatchery-derived samples, such as in the presence of seawater, algae, or oyster larvae. In contrast, the dipstick assay remained very sensitive in the presence of these contaminants, is less work-intensive, and much more rapid, making this format the preferred assay method for detecting VtpA on site in a hatchery or environmental setting.
创伤弧菌已被证实与贝类物种（包括牡蛎、象拔蚌和蛤）的疾病爆发有关。特别是，自 2006 年以来，太平洋西北地区的牡蛎孵化场一直受到间歇性弧菌病的困扰。准确检测弧菌，包括创伤弧菌，对于孵化场至关重要，以便能够快速进行补救措施。目前用于检测弧菌属的方法并不理想，不适合在孵化场使用。平板样品需要时间，并且对外源致病性产物（如分泌的锌金属蛋白酶 VtpA）不敏感。其他用于检测细菌的敏感方法，如 qPCR，需要高水平的实验室技能和昂贵的耗材，这对于孵化场来说是不可行的。因此，孵化场将受益于一种敏感、简单的方法来检测创伤弧菌及其分泌的毒素。在这里，我们描述了两种用于检测创伤弧菌分泌的贝类毒素锌金属蛋白酶的廉价且高度特异性的测试方法：酶联免疫吸附测定（ELISA）和横向流动免疫测定（侧流免疫测定）。这两种技术都依赖于一组用于三明治格式的单克隆抗体，其中捕获抗体识别成熟 VtpA 蛋白上的检测抗体不同的表位。两种检测方法都是定量的，并给出比色读数。当 VtpA 稀释到 PBS 中时，夹心 ELISA 很敏感，但在与孵化场样本环境相关的条件下，如在海水中、藻类或牡蛎幼虫存在的情况下，敏感性明显降低。相比之下，在存在这些污染物的情况下，侧流免疫测定仍然非常敏感，工作量较小，速度更快，因此这种格式是在孵化场或环境环境中现场检测 VtpA 的首选检测方法。
