Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 3, 166 28 Prague, Czech Republic.
Biosens Bioelectron. 2011 Feb 15;26(6):2828-34. doi: 10.1016/j.bios.2010.10.001. Epub 2010 Dec 18.
Members of the genus Cronobacter are opportunistic pathogens formerly known as Enterobacter sakazakii, which induce severe meningitis and sepsis in neonates and infants, with a high fatality rate. In this work, a simple and rapid immunochromatographic strip test for the detection of this pathogen was developed. Following the shortened bacteria cultivation and isolation of DNA, a specific gene sequence targeting 16S rRNA from Cronobacter spp. was amplified by PCR using 5'-end labelled specific primers. The PCR product, amplicon labelled with digoxigenin on one side and biotin on the other side, was directly added to the immunochromatographic strip test, composed of nitrocellulose membrane with bound antibody against digoxigenin in the test line. The visualization was mediated by colloidal carbon conjugated to neutravidin, and the appearance of grey/black line was indicative of the presence of specific amplicon. Colour intensity of the test line in pathogen-positive assay was visually distinguishable from that of negative sample within 10 min. The visual detection limit of PCR product was 8 ng. The specificity of the developed method was confirmed by standard microbiological techniques. Whole detection procedure with the incorporated immunostrip was applied to analysis of infant formulae samples, contaminated with less than 10 cells of Cronobacter spp. per 10 g. The results from immunochromatographic test indicated the absolute agreement with those from standard microbiological methods. Moreover, the developed procedure considerably reduced the total analysis time to 16 h whereas the reference microbiological method needs 6-7 days.
阪崎克罗诺杆菌属的成员是机会致病菌，以前称为阴沟肠杆菌，会在新生儿和婴儿中引起严重的脑膜炎和败血症，死亡率很高。在这项工作中，开发了一种简单快速的免疫层析条检测方法。在缩短细菌培养和 DNA 分离后，通过 PCR 使用 5'-端标记的特异性引物扩增针对 Cronobacter spp. 的 16S rRNA 的特定基因序列。将一侧标记有地高辛、另一侧标记有生物素的 PCR 产物直接添加到免疫层析条测试中，该测试由带有针对地高辛的抗体的硝酸纤维素膜组成，测试线。可视化通过与亲和素结合的胶体碳介导，出现灰色/黑色线表示存在特定的扩增子。在 10 分钟内，阳性检测中测试线的颜色强度可以与阴性样本明显区分开来。PCR 产物的目视检测限为 8ng。通过标准微生物技术确认了所开发方法的特异性。将包含免疫条的整个检测过程应用于婴儿配方奶粉样品的分析，这些样品每 10 克中含有少于 10 个 Cronobacter spp. 细胞。免疫层析测试的结果与标准微生物方法的结果完全一致。此外，所开发的方法将总分析时间大大缩短至 16 小时，而参考微生物方法需要 6-7 天。
