U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Laurel, MD, United States.
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Laurel, MD, United States.
J Microbiol Methods. 2020 Sep;176:106009. doi: 10.1016/j.mimet.2020.106009. Epub 2020 Jul 21.
Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.
大多数用于弯曲菌快速鉴定的方法都是为了检测耐热型空肠弯曲菌（C. jejuni）和大肠弯曲菌（C. coli）。越来越多的热敏弯曲菌物种现在被认为是新兴的人类病原体。目前还缺乏快速筛选这些新兴物种的方法。环介导等温扩增（LAMP）是一种核酸扩增方法，可实现快速且具有成本效益的细菌检测。针对 C. jejuni、C. coli、C. lari、C. upsaliensis、C. ureolyticus、C. fetus、C. gracilis、C. rectus 和 C. concisus 的 16S rRNA 序列设计了简并引物。在 68°C 下使用 WarmStart®比色 LAMP 试剂对 ATCC 参考菌株进行等温扩增 30 分钟。阳性反应表现为粉红色变为黄色；使用限制性内切酶消化（RsaI）确认对弯曲菌的特异性。开发的 LAMP 反应针对参考菌株具有特异性，这是通过排他性面板得到确认的，该面板包括其他肠道病原体，包括大肠杆菌、沙门氏菌、志贺氏菌、幽门螺杆菌和弯曲杆菌。该方法还评估了用于检测人工污染新鲜菠菜样本中的初级富集培养基中的 C. jejuni、C. coli 和 C. lari。LAMP 方法为现场监测和追溯分析中快速筛选致病性弯曲菌属提供了一种选择。
