A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef.
A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef.
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DOI：
10.1371/journal.pone.0148092
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Rasooly R
中科院分区：
文献类型：
--
作者：
He X;Kong Q;Patfield S;Skinner C;Rasooly R
志贺毒素(STX)是所有产生志贺毒素的大肠杆菌(STEC)的共同毒力因子，可导致包括出血性结肠炎和溶血性尿毒症综合征(HUS)在内的多种疾病。尽管有几种商业试剂盒可用于检测STEC生产的STX，但没有一种试剂盒能够识别STX的所有亚型，包括STX1的三个亚型和STX2的七个亚型。研制了抗STX1和STX2的新的单抗和多克隆抗体。利用新开发的抗体库建立了一种能够检测所有已知STX1和STX2亚型的通用夹心ELISA法。为了准确监测每一种Stxs亚型的检测敏感性，我们创建了重组类毒素，并将其用作ELISA的标准。由于所结合的抗体具有很高的亲和力，在磷酸盐缓冲盐水(PBS)中对Stx1a和Stx2a不同亚型的检测限在10至50pg/mL之间，因此该方法具有很高的灵敏度。该方法还能够根据培养液上清液或琼脂平板上的单个菌落产生STX来鉴定STEC，而无需在液体介质中进行长时间的浓缩。当应用于磨碎的牛肉样本时，这种新开发的ELISA法能够区分添加了单个细菌细胞的牛肉样本。建立了一种高灵敏度、通用性强的STX1和STX2亚型检测方法。由于检测范围窄，避免了假阴性结果，大大改进了目前的技术。这项研究中开发的检测方法可用于迅速检测新出现的血清型，并在食品供应链的早期阶段对绞碎牛肉样本进行STEC污染筛查，从而避免可能的召回。
Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2. New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell. A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.