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5×108CFUF.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HiTrap Protein G HP亲和层析法纯化抗体;优化MS300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(50CFU/200mg^10^5CFU/200mg)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>320000,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2.5h,加入抗体最适量为10μg/mg,免疫磁珠最佳捕获温度为37℃,时间为40min;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100CFU/200mg和400CFU/200mg,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。
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 5×10^8 CFU 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 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
作者
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
DUN Guodong;TONG Ya’nan;LU Xiaoxue;FENG Yuyang;YE Xinli;LI Jing;TANG Bin;DENG Ling;HE Xiaoyi;LI Qian;MAO Xuhu(Department of Clinical Microbiology and Immunology,College of Pharmacy and Medical Laboratory,Army Medical University,Chongqing 400038,China;Digestive Diseases Center,Third Affiliated Hospital of Chongqing Medical University,Chongqing 401120,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2019年第9期811-816,822,共7页
Immunological Journal
基金
国家自然科学基金青年基金(81501796)
关键词
具核梭杆菌
结直肠癌
免疫磁珠
qPCR
Fusobacterium nucleatum
Colorectal cancer
Immunomagnetic beads
Quantitative real-time PCR
