传感器类型
侧流层析试纸条
检测对象
霍乱弧菌O1血清群 (Vibrio cholerae serogroup O1)、霍乱弧菌O139血清群 (Vibrio cholerae serogroup O139);样品基质:碱性蛋白胨水 (APW) 富集培养物/临床与环境菌株培养物
检测原理
将试纸条浸入含霍乱弧菌的APW培养物后,液体经毛细作用向吸收垫迁移,同时使结合垫上干燥的金标抗体复溶。样品中O1 LPS与金标抗O1 LPS mAb结合,O139 LPS与金标抗O139 LPS mAb结合;形成的免疫复合物随液流迁移至硝酸纤维素膜相应检测线,并被预印的抗O1或抗O139捕获抗体固定。过量金标抗体被质控线山羊抗小鼠IgG捕获。40 nm胶体金在检测线聚集,因表面等离子共振产生红色条带,肉眼判读。抗原浓度越高,金标复合物越多,条带越明显;低于可见阈值则不显色。
检测灵敏度
LOD: 10^8 CFU/ml (O1)、10^7 CFU/ml (O139);富集后最低接种量: 10^2 CFU/ml (O1和O139)
效应效果
试纸条在马来西亚国家评价中对O1和O139检测线均显示100%敏感性和100%特异性。印度国际评价中,O1检测线71株O1菌株中66株阳性,总体敏感性93–100%,特异性100%;O139检测线30株O139菌株全部阳性,2株非O139菌株出现假阳性,总体敏感性100%,特异性98–100%。未出现无效结果,且对非O1/非O139霍乱弧菌及多种肠道菌基本无交叉反应。试纸条密封室温保存21周后仍可肉眼判读,检测线强度略弱但无显著损失,适合资源有限地区快速筛查和疫情监测。
传感器的构成
- 塑料背衬:plastic adhesive backing,承载样品垫、结合垫、硝酸纤维素膜和吸收垫并固定试纸条
- 样品垫:cellulose fibre sample pad (CFSP173000),接收APW样品并引导液体迁移
- 结合垫:glass fibre conjugate pad (GFCP083000),干燥固定胶体金-抗O1 LPS mAb和胶体金-抗O139 LPS mAb复合物
- 硝酸纤维素膜:laminated nitrocellulose membrane card (HF135MC100),印迹抗O1 LPS mAb、抗O139 LPS mAb和山羊抗小鼠IgG (H&L)作为检测/质控捕获抗体
- 吸收垫:absorbent pad,吸收液体并维持毛细流动
- 识别元件:anti-O1 LPS mAb (6G1E12F10, IgG3)和anti-O139 LPS mAb (9A11D6, IgG1),分别识别O1和O139 LPS
- 信号标记物:colloidal gold nanoparticles (40 nm)与抗LPS mAb偶联,聚集后通过表面等离子共振产生红色线
- 封闭剂:western blocking reagent、BSA、Tween-20和sucrose,减少非特异性结合并稳定金标复合物
中文摘要
霍乱是由摄入被污染食物或水引起的急性肠道传染病,流行和大流行霍乱仅与霍乱弧菌O1和O139血清群相关。传统培养鉴定耗时长,PCR检测需要专业设备和人员,难以在资源有限地区快速应用。本研究开发并评价了一种干试剂一步法侧流层析生物传感器,用于同时检测碱性蛋白胨水(APW)富集培养物中的霍乱弧菌O1和O139血清群。该试纸条以胶体金纳米颗粒为信号标记,用针对脂多糖(LPS)的血清群特异性单克隆抗体进行功能化,形成双检测线。检测基于免疫层析原理:样品中抗原与金标抗体结合后,被硝酸纤维素膜上相应捕获抗体捕获,胶体金聚集产生肉眼可见红色线。干试剂试纸条操作简便、结果快速、无需冷链,适合资源有限地区的霍乱快速筛查和疫情监测。
英文摘要
Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.