空肠弯曲杆菌是人细菌性胃肠炎的常见食源性致病菌之一,严重危害人类的健康,建立快速精准检测食源性空肠弯曲杆菌的方法,对于保障人们的健康和食品安全具有重要意义.本研究建立了一种可视化、低成本基于hipO基因的环介导等温扩增(LAMP)技术快速检测空肠弯曲菌的方法.该LAMP方法检测空肠弯曲菌CJFX01基因组DNA为阳性,而对肠出血性大肠杆菌O157:H7、鼠伤寒沙门菌、单增李斯特菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌、产气荚膜梭菌等食源性致病菌和胸膜肺炎放线杆菌Aha01、副猪嗜血杆菌Ahh01、多杀性巴氏杆菌Ahm03、猪链球菌Ahs02、猪丹毒丝菌Ahe01、支气管败血波氏杆菌Ahb01等猪源致病菌基因组DNA检测结果均为阴性,表明其具有良好的特异性.灵敏度的检测结果表明,LAMP检测空肠弯曲杆菌的检测限为5.4×101 CFU/mL,而基于F3/B3引物对常规PCR的检测限为5.4×106 CFU/mL,即LAMP检测空肠弯曲杆菌的灵敏度比常规PCR高100 000倍.本研究建立的LAMP检测方法具有快速、可视化、低成本、特异性好和灵敏度高等优点,适用于实验室和基层食品安全监管部门进行现场快速检测.
Campylobacter jejuni is one of the common food-borne pathogenic bacteria of human bacterial gastroenteritis,which seriously harms health.It is of great significance to develop a rapid and accurate method for detection of C.jejuni.In this study,a visual and low-cost loop mediated isothermal amplification(LAMP)method based on hipO gene was developed for rapid detection of C.jejuni.The LAMP detection was positive using the genomic DNA of C.jejuni CJFX01 as template but negative for enterohemorrhagic Escherichia coli O157:H7,Salmonella typhimurium,Listeria monocytogenes,Yersinia enterocolitica,Staphylococcus aureus,Clostridium perfringens and Actinobacillus pleuropneumoniae,Haemophilus parasuis,Pasteurella multocida,Streptococcus suis,Erysipelas suis or Bordetella bronchiseptica,indicating that it had good specificity.The detection limit of LAMP for C.jejuni was 5.4×101 CFU/mL while the detection limit of conventional PCR based on F3/B3 primers was 5.4×106 CFU/mL,indicating that the sensitivity of LAMP for detecting C.jejuni was 100,000 times higher than that of conventional PCR.The LAMP developed in this study had the advantages of rapid,visual,low cost,good specificity and high sensitivity,which might be suitable for rapid detection in laboratories and grass-roots food safety supervision departments.