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首页> 外文OA文献 >Sensitive Detection of Shiga Toxin 2 and Some of Its Variants in Environmental Samples by a Novel Immuno-PCR Assay▿
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Sensitive Detection of Shiga Toxin 2 and Some of Its Variants in Environmental Samples by a Novel Immuno-PCR Assay▿

机译：新型免疫PCR检测技术可灵敏地检测环境样品中的志贺毒素2及其某些变体

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Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.
机译：环境中产生志贺毒素的大肠杆菌（STEC）的报道很多。但是，由于样品中细菌的数量通常低于该方法的检测阈值，因此很难可靠地检测环境样品中的STEC。我们开发了一种新型且灵敏的免疫PCR（IPCR）检测试剂盒，用于检测志贺毒素2（Stx2）和Stx2变体。该测定法涉及在B亚基处对Stx2的免疫捕获，以及与识别Stx2 A亚基的检测抗体相连的DNA标记的实时PCR扩增。该测定的定性检测极限是在磷酸盐缓冲盐水（PBS）中为0.1 pg / ml，定量范围为10到100,000 pg / ml。 IPCR方法的灵敏度比PBS中的模拟常规酶联免疫吸附测定（ELISA）高10,000倍。尽管IPCR检测Stx2的灵敏度受到粪便，野猪结肠，土壤和流域水的环境样品基质的影响，但IPCR分析在23种富集的粪便，野猪结肠，土壤和流域培养物中的应用从环境中收集的样本显示，IPCR在所有15个样本中均检测到Stx2，这些样本通过实时PCR和培养方法显示为STEC阳性，证明了100％的敏感性和特异性。我们在这项研究中描述的三明治式IPCR的修饰将是一种灵敏且特异的筛选方法，用于评估环境中STEC的发生。

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