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·论 著· [文章编号]1000-8861(2019)09-0811-07;[DOI]10.13431/j.cnki.immunol.j.20190127 粪便具核梭杆菌的免疫磁珠捕获联合实时荧光定量PCR检测方法的 建立及评价 顿国栋,童亚楠,卢晓雪,冯宇阳,叶新力,李 静,唐 彬,邓 铃,何晓奕,李 倩,毛旭虎* [摘 要] 目的 建立定量检测粪便中具核梭杆菌(Fusobacterium nucleatum,F. nucleatum)的免疫磁珠捕获联合实时荧光定 量 PCR(IMBs-qPCR)技术方法。方法 5×108CFU F. nucleatum(0.4%甲醛灭活)免疫新西兰兔制备多克隆抗体,饱和硫酸铵沉淀 法和Hi Trap Protein G HP亲和层析法纯化抗体;优化 MS 300免疫磁珠偶联多克隆抗体和捕获F. nucleatum的反应条件。根据 F. nucleatum特异性基因NusG设计引物和构建含目的基因的载体质粒,建立 qPCR 反应体系并绘制 qPCR 标准曲线。最后,在 不同浓度(50 CFU/200 mg~105CFU/200 mg)F. nucleatum的人粪便标本中,评价该方法的检测效能。结果 成功制备F. nucleatum 多克隆抗体,抗体ELISA 效价>320 000,盐析和亲和层析纯化抗体纯度分别为 60%和 85%;多克隆抗体偶联免疫磁珠时偶联缓 冲液最佳 pH 值为 5.0,最佳温度为 25 ℃,最佳偶联时间为 2.5 h,加入抗体最适量为 10 µg/mg,免疫磁珠最佳捕获温度为 37 ℃, 时间为 40 min;成功建立 qPCR 反应体系;IMBs-qPCR 和粪便全基因组 DNA 提取法直接检测模拟粪便标本最低检测限分别为 100 CFU/200 mg 和 400 CFU/200 mg,ROC 曲线中 AUC 分别为 0.948 和 0.878。结论 成功建立了 IMBs-qPCR 检测粪便中 F. nucleatum的方法,该方法具有简便快速、灵敏度高、特异性好等特点。 [关键词] 具核梭杆菌;结直肠癌;免疫磁珠;qPCR [中图分类号] R378.8 [文献标识码] A Establishment and evaluation of immunomagnetic bead and quantitative real-time PCR for detection of Fusobacterium nucleatum in feces DUN Guodong1 , TONG Ya’nan1 , LU Xiaoxue1 , FENG Yuyang1 , YE Xinli2 , LI Jing2 , TANG Bin1,2 , DENG Ling1 , HE Xiaoyi1 , LI Qian1 , MAO Xuhu1,* 1. Department of Clinical Microbiology and Immunology, College of Pharmacy and Medical Laboratory, Army Medical University, Chongqing 400038, China; 2. Digestive Diseases Center, Third Affiliated Hospital of Chongqing Medical University, Chongqing 401120, China *Corresponding Author: MAO Xuhu, E-mail: maoxh2012@hotmail.com [Abstract] This study was performed to establish a method which utilizes immunomagnetic beads and quantitative real-time PCR to detect Fusobacterium nucleatum (F. nucleatum) in stool rapidly and quantitatively. First, we treated F. nucleatum with 0.4% formaldehyde to prepare antigen and immunized New Zealand rabbits with 5×108CFU antigen to generate polyclonal antibody, which subjected to ELISA for titer detection. Then the polyclonal antibody was purified by saturated ammonium sulfate and Hi Trap Protein G HP, and the purity of the purified polyclonal antibody was detected by SDS-PAGE. The reaction conditions of MS300 immunomagnetic beads and polyclonal antibodies were optimized. Second, based on the specific gene of NusG in F. nucleatum genome, a pair of primers was designed and a qPCR reaction system was established. We constructed a 基金项目:国家自然科学基金青年基金(81501796) 作者单位:400038重庆,陆军军医大学药学与检验医学系临 床微生物与免疫学教研室(顿国栋,童亚楠,卢晓雪,冯宇阳, 唐 彬,邓 铃,何晓奕,李 倩,毛旭虎);401120,重庆医科 大学附属第三医院消化疾病中心(叶新力,李 静,唐 彬) *通信作者:毛旭虎,E-mail: maoxh2012@hotmail.com ·811· 免疫学杂志 2019年9月 第35卷 第9期 IMMUNOLOGICAL JOURNAL Vol. 35 No. 9 Sep. 2019 plasmid containing the target gene NusG with which to make a standard curve of qPCR. At last, simulated fecal specimens were made by manually adding different concentrations of F. nucleatum from 50 CFU/200 mg to 105 CFU/200 mg to human fecal specimens without F. nucleatum to identify the detection efficiency of this method. Data showed that the titer of the polyclonal antibody was more than 320 000. The purity of the polyclonal antibody purified by salting out and affinity methods were 60% and 85%, respectively. When the polyclonal antibody was conjugated to MS300 magnetic beads, the optimal pH of the coupling buffer is 6.0, the optimum temperature is 25 ℃ , the optimal coupling time is 2.5 h, and the optimal polyclonal antibody amount is 10 µg for 1 mg immunomagnetic beads. The optimal conditions for the capture of F. nucleatum are 37 ℃ and 40 min. The minimum detection limit of the construced IMBs-qPCR for simulated fecal samples is 100 CFU/200 mg, and the AUC in ROC curve is 0.948, while the minimum detection limit of fecal whole genome extraction kit for simulated fecal samples is 400 CFU/200 mg, and the AUC in ROC curve is 0.878. In conclusion, we have successfully established a method for rapid detection of F. nucleatum in feces by IMBs-qPCR. This method has high sensitivity and good specificity in the detection of F. nucleatum in feces and is easy to operate. [Key words] Fusobacterium nucleatum; Colorectal cancer; Immunomagnetic beads; Quantitative real-time PCR
1 材料与方法 1.1 主要菌株、试剂和仪器 F. nucleatum标准菌株 ATCC 25586 购自美国菌种保藏中心(American type collection center,ATCC),FAB 细菌厌氧培养基(英 国LAB M公司),厌氧工作站 Concept-400(美国 BAKER),免疫磁珠 MS 300(日本JSR),2-(N吗啉) 乙磺酸水合物MES(美国 SIGMA),1 (3-二甲氨基 丙基)-3-乙基碳二亚胺盐酸盐 EDC(美国 SIGMA), 细菌基因组 DNA 提取试剂盒(北京天根公司),TB GreenTM Premix Ex TaqTM Ⅱ(大连TaKaRa),硫酸铵 (重庆川东化工),甲醛(武汉博士德公司),磁力分选架(深圳博尔熙公司),生物透析袋(上海生工生物工 程公司),实时荧光定量 PCR 仪 C1000TM Thermal Cycler(美国 BIO-RAD),电泳仪(美国 BIO-RAD), 凝胶扫描仪 Expression 11000 XL(日本精工爱普生 株式会社)
畜牧兽医学报 2023,54(2):766-778 ActaVeterinariaetZootechnicaSinica doi:10.11843/j.issn.0366-6964.2023.02.033 开放科学(资源服务) 标识码(OSID): 基于免疫磁珠净化间接竞争酶联免疫吸附 试验检测氟喹诺酮类药物 黄婧洁1,2,李 苗1,2,陈莹娴1,2,钟雅兰2,张婷婷2,姜廷超男2,李建成1,2* (1.中国农业大学,动物源性食品安全检测技术北京市重点实验室,北京 100193; 2.中国农业大学动物医学院,北京 100193)
1.2 主要仪器与设备 MultiskanFC 酶联免疫测定仪:美国 Thermo 公司;QuattroLC 超高效液相色谱仪串联质谱仪: 美国 Waters公司;Mag-12-1 磁分离架:深圳市博尔熙科技发展有限公司;BE-1200z旋转混合仪:海 门市其林贝尔仪器制造有限公司;PrimoR 台式高 速冷冻离心机:德国贺力氏台式高速冷冻离心机; PHSJ-3FpH 检测仪:上海雷磁仪器有限公司;WH- I型微型漩涡混合仪:上海仪电分析仪器有限公司; DF-101S集热式恒温加热磁力搅拌器:巩义市予华 仪器有限责任公司;RP508 恒温摇床:上海仪电分 析仪器有限公司。
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