致病性弧菌通常为革兰氏阴性菌,广泛分布于河口、港湾及近海水域[1-2]。目前已确认12种弧菌具有致病性[3-5],其中以副溶血性弧菌(Vibrio parahaemolyticus)、创伤弧菌(Vibrio vulnificus)、霍乱弧菌(Vibrio cholerae)、溶藻弧菌(Vibrio alginolyticus)和拟态弧菌(Vibrio mimicry)最为常见[6]。食用被致病性弧菌污染的水产品会引起食物中毒,出现恶心呕吐、腹痛腹泻、发热等急性肠胃炎症状,严重者会出现脱水、休克昏迷甚至死亡;可能还会出现伤口感染和原发性败血病等症状[7-10]。近年来,致病性弧菌引发的食品安全事件频发[11-12],探究快速、灵敏、准确的检测方法研究尤为必要。致病性弧菌的传统检测方法是培养法,但易出现假阳性,且检测周期长、灵敏度低,难以达到快速监测的目的。免疫检测方法基于特异性抗体,利用抗原-抗体特异性结合,能够靶向识别致病性弧菌,达到弧菌快速检测的目的,能够有效提高检测效率,且在灵敏度、特异性、现场适用性、样品前处理简便性等方面具有优势。该文综述了酶联免疫吸附法、免疫荧光法、免疫层析法、免疫磁珠分离法和免疫传感器法等方法在致病性弧菌检测上的应用,并对其发展趋势进行展望,为致病性弧菌检测方法研究的融合与创新提供参考。
1 致病性弧菌抗体及其制备
免疫分析方法构建的关键在于高质量抗体的制备。致病性弧菌抗体的效价、亲和力、特异性决定了抗原-抗体免疫识别反应的灵敏度及专一性。用于致病性弧菌的抗体主要有多克隆抗体、单克隆抗体和基因工程抗体[13]。其特点和应用见表1。
表1 致病性弧菌的抗体种类及其应用
Table 1 Types of antibodies against pathogenic Vibrio and their applications
类型制备流程优点缺点应用于检测致病性弧菌的种类多克隆抗体抗原制备、动物免疫、血清分离、纯化鉴定制备周期短、成本低、灵敏度高、免疫反应强特异性差、生产批次差异、存在交叉反应副溶血性弧菌[14-15]霍乱弧菌[16]创伤弧菌[17]单克隆抗体抗原制备、动物免疫、细胞融合、筛选鉴定、克隆培养效价高、特异性强、容易纯化或标记、重复性好步骤复杂、成本高、耗时长溶藻弧菌[18]创伤弧菌[19]副溶血性弧菌[20]霍乱弧菌[21]基因工程抗体基因克隆、动物免疫、表达载体构建、转染与表达、抗体表达纯化、抗体鉴定亲和力高、交叉反应性低稳定性好、免疫原性低获得成本高、制备步骤繁琐、表达系统限制创伤弧菌[22]副溶血性弧菌[23-24]霍乱弧菌[25]
多克隆抗体(polyclonal antibody,pAb)通常以重组或天然蛋白作为抗原,通过免疫动物诱导其体内B细胞发生应答,进而产生可特异性识别同一抗原上多个不同表位的抗体混合物。该类型抗体一般通过免疫动物血清采集、免疫球蛋白分离及特异性亲和纯化等步骤制备而得[26]。生产多克隆抗体常用的免疫动物为兔子、有蹄类动物(绵羊、山羊、马、猪等)、啮齿类动物以及鸡等[27]。其中,兔的耐受性强且血量大,取血相对其他动物更容易,制备所得的抗体量相对较大,其敏感性高、亲和力好、结构简单且更稳定[28]。尽管pAb能够识别抗原上的多个表位,但由于其本质是源于不同B细胞克隆的抗体混合物,pAb也存在特异性较低、易于发生交叉反应以及假阳性率较高等局限性。这些特性在一定程度上限制了其在某些免疫检测应用中的可靠性和重复性。
单克隆抗体(monoclonal antibody,mAb)是由单一B细胞克隆增殖和分化后所分泌的高度均一的抗体分子群,其可特异性识别并结合抗原的某一个特定表位。mAb通常采用杂交瘤技术制备。通过将单个具有分泌特异性抗体能力的杂交瘤细胞扩增为细胞群,可获得效价高、特异性强、可重复生产的抗体,因而在免疫检测领域应用极为广泛。然而,该技术也面临制备流程复杂、周期冗长、特性优异的单抗筛选难度大等难点[29]。
基因工程抗体又称重组抗体,是指通过重组DNA及蛋白质工程技术,对抗体编码基因定向遗传操作和重新装配,并转染至特定表达系统中生成的抗体分子。基因工程抗体种类繁多,包括纳米抗体(nanobody,Nb)、单域抗体(single domain antibody,sdAb)、抗原结合片段(fragment antigen-binding,Fab)、可变区片段(variable fragment,Fv)、单链抗体(single-chain fragment variable,ScFv)以及嵌合抗体(chimeric antibody,cAb)等(图1)。基因工程抗体可以在无需免疫的情况下,利用哺乳动物细胞系、细菌以及酵母等异源表达系统实现大规模生产,降低成本,且其具有良好的一致性、高重复性和较好的再现性[30-31]。
图1 不同种类抗体的结构示意图
Fig.1 Structural diagrams of different antibody types
注:嵌合抗体通常由鼠源抗体的可变区和人源抗体的恒定区组成。
2 免疫分析方法
2.1 酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)
ELISA是将抗原-抗体反应的特异性和酶催化反应的高效性相结合而开发的一种方法。致病性弧菌的检测通常采用夹心ELISA(图2)[32]。弧菌首先被固相载体上的特异性抗体捕获,形成抗原-抗体复合物,再加入辣根过氧化物酶(horseradish peroxidase,HRP)等标记抗体,形成抗体-抗原-抗体“三明治”,再加入3,3′,5,5′-四甲基联苯胺(3,3′,5,5′-tetramethylbiphenyl,TMB)等显色底物,产生可测量的颜色变化,以此判断目标弧菌的定量检测。
图2 夹心ELISA原理图
Fig.2 Sandwich ELISA schematic diagram
肖益群等[33]建立了能够特异性检测创伤弧菌的间接ELISA快速检测方法。该方法检测创伤弧菌pAb效价高达5.12×105,与其他37株鳗鲡病原菌均没有交叉反应,特异性强。曾静等[34]建立了检测创伤弧菌的双抗体夹心ELISA方法,检出限为103 CFU/mL,与非目标菌没有交叉反应,特异性高。职通瑞等[35]建立了溶藻弧菌快速检测的间接ELISA方法,检出限为104 CFU/mL,并具有高特异性。程晋霞等[36]成功制备了霍乱弧菌mAb,并将其应用于双抗体夹心ELISA。该方法的检出限达到103 CFU/mL,与非霍乱弧菌没有交叉反应,在基质添加试验中,检出限为1 CFU/g。ELISA方法操作简单、实用性强,但其灵敏度相对较低,且检测结果和重复性易受样品基质干扰。为提高灵敏度和准确度,可通过筛选高亲和力抗体、引入信号放大系统以及采用高催化效率的新型酶标记物等策略进行方法改进[37-39]。
2.2 免疫荧光法(immunofluorescence assay,IFA)
IFA是利用抗原-抗体特异性结合的反应,并用荧光染料作为示踪物来标记目标菌。IFA与ELISA相似,主要区别在于原理、标记物不同。如图3所示,IFA的原理是抗体与致病性弧菌结合形成复合物,荧光染料标记的二抗与其结合发出荧光,根据荧光信号强弱进行定性、定量分析[40]。许多物质都可产生荧光现象,能够明显产生荧光的并能作为染料使用的有机化合物被称为免疫荧光染料。目前,常用的荧光染料有异硫氰酸荧光素(fluorescein isothiocyanate,FITC)、丫啶橙(acridine orange,AO)、四甲基异硫氰酸罗丹明(tetraethyl rhodamine isothiocyanate,TRITC)、四乙基罗丹明(rhodamine,RIB200)等[41]。
图3 IFA原理图
Fig.3 IFA schematic diagram
于光等[42]建立了检测鱼贝类中副溶血性弧菌的免疫荧光法。该方法检出限为105 CFU/mL,特异性强,对市售351份海产鱼贝类样品中副溶血性弧菌定性检测结果与常规培养法无显著性差异。CHEN等[43]建立了以FITC标记二抗检测海蛎中副溶血性弧菌的免疫荧光法。该方法以2种外膜蛋白为抗原制备抗体,经18 h预增菌后该方法可检出副溶血性弧菌低至1.7 CFU/g的样品。WANG等[44]建立了检测霍乱弧菌O1和O139菌株的免疫荧光法。该方法对146份河口水样的增菌液的检测灵敏度显著高于常规培养法,检测2种菌株的检出限为103 CFU/mL,并且具有较高的特异性。
尽管免疫荧光法可快速、准确检测致病性弧菌,但仍然存在一些局限,如非特异性染色、信号稳定性差及操作复杂等[45]。为解决以上问题,可通过筛选高特异性抗体、选择高稳定荧光标记物、优化试剂体系,并引入自动化平台,从而在提升灵敏度和特异性的同时,实现对低浓度靶标的高效、精准检测[46]。
2.3 免疫层析法(immunochromatographic assay,ICA)
ICA结合了层析分离和免疫识别反应相结合的技术原理,具备操作简便、响应快速和成本低廉等优势。该方法无需精密仪器和专业人员操作即可实现对结果的可视化判读[47]。致病性弧菌检测通常采用夹心ICA,如图4所示[48],将捕获抗体和第二抗体分别包被于硝酸纤维素膜(nitrocellulose membrane,NC)膜上的检测线(T线)和控制线(C线),标记材料的检测抗体预先固定在结合垫上。当待测样品滴加到样品垫上,它会通过毛细管作用流动至结合垫,如含有目标弧菌,会先与标记抗体发生免疫反应,形成复合物,进一步流动在T线处被捕获抗体特异性识别,形成“标记抗体-目标弧菌-捕获抗体”复合物,从而在T线处产生肉眼可见或仪器可检的条带(阳性),反之无条带(阴性),而未被T线拦截的标记抗体被C线捕获,也会产生条带用于判断结果的有效性。
图4 夹心ICA原理图
Fig.4 Sandwich ICA schematic diagram
DONG等[49]针对弧菌的关键毒力因子制备单克隆抗体,通过抗体亚类检测、亲和力检测和最佳标记效率等试验,建立胶体金免疫层析法。该方法特异性好,灵敏度高,检出限为400 ng/mL。吴美娇等[50]基于双抗体夹心原理检测鲜虾及白蛤等海产品中的副溶血性弧菌,建立了胶体金免疫层析法。该方法检出限为4.77×103 CFU/mL。SAKATA等[51]建立了检测副溶血性弧菌的胶体金免疫层析法,该方法在8.5 h 内检测出牡蛎样品中的副溶血性弧菌,检出限为1.1～22 CFU/25 g。DING等[52]利用2株高亲和力单克隆抗体,建立了检测副溶血性弧菌的胶体金免疫层析法。该方法在10 min内可检出50 μL液体培养中的副溶血性弧菌,其最低检出限为500 CFU/mL,与16种菌株无交叉反应,特异性强。AuNPs、磁纳米粒子和彩色乳胶微球等作为免疫标记材料建立的方法能够高效完成弧菌的现场快速筛查。此外,荧光微球、量子点和上转换荧光材料等荧光型材料具备高灵敏、多通道、抗干扰、可定量的优点[53-54]。将其作为免疫标记材料可取代传统ICA的胶体金,来提高检测性能。CHEN等[55]基于CdTe@SiO2纳米复合材料作为红色荧光信号标记,建立了荧光免疫层析法。该方法最低检出限为3.95×10-3 CFU/mL,检测范围为2.51×10-4～1.40×10-8 CFU/mL,且具有良好的特异性和回收率。
ICA作为即时检验技术之一,尤其适用于弧菌的现场检测。但该方法研究需要考虑一些关键问题:1)夹心ICA一般需要捕获抗体和检测抗体,抗体验证配对需要耗费大量筛选时间;2)抗体和纳米材料(量子点、磁性微球等)偶联需要有机交联剂,可能会影响抗体活性,还增加了实验成本和操作复杂性;3)标记抗体耐受能力有限,易受外界条件干扰,不利于维持检测体系的稳定性与准确性[56-57]。
2.4 免疫磁珠分离法(immunomagnetic separation assay,IMS)
IMS是利用表面偶联抗体的免疫磁性颗粒(immunomagnetic particles,IMPs),与样品中目标抗原(如细菌、细胞、蛋白等)发生特异性结合后,利用外部磁场可实现目标组分的定向分离与富集。其原理如图5所示[58],将适量的IMPs与样品充分混合,IMPs表面偶联的特异性抗体会与目标弧菌发生免疫识别并结合,形成“IMPs-致病性弧菌”复合物。在磁场的作用下,磁珠结合的靶标复合物可定向移动,吸附于试管壁,且不受其他组分的干扰,弃去试管内的非目标细菌即可得到靶标物质。将该方法与ELISA、IFA、ICA、PCR等检测技术联用,可有效消除基质干扰、提高检测效率。
图5 IMS原理图
Fig.5 IMS schematic diagram
LIU等[59]利用多克隆抗体和免疫磁性颗粒建立了磁性纳米探针标记免疫层析试纸检测副溶血性弧菌。该方法通过快速磁分离特性,与样品富集和预孵育过程相结合,线性范围为1.4×106～2.26×104 CFU/mL,检出限为4.73×103 CFU/mL,可裸眼观察到副溶血性弧菌5.65×104 CFU/mL。ZHAO等[60]利用链霉亲和素标记的磁性纳米颗粒和生物素标记的多克隆抗体制备了免疫磁珠,并优化了最佳抗体用量、孵育时间、免疫磁分离时间、分离温度等条件,建立了一种免疫磁性分离和实时聚合酶链式反应相结合的方法。该方法检出限为18.5 CFU/g,灵敏度高、特异性强、简单快速,并且优于基于常规聚合酶链式反应(polymerase chain reaction,PCR)测定。ZHAI等[61]基于免疫磁性纳米颗粒和量子点形成磁性量子点,与抗体偶联制备信号探针,建立了一种快速灵敏的副溶血性弧菌检测方法。该检测方法耗时150 min,检出限为102 CFU/mL,检测范围为102～106 CFU/mL。LUN等[62]建立了PCR与IMS结合检测副溶血性弧菌方法。该方法采用PCR筛选副溶血性弧菌,免疫磁珠包被抗体在60 min内对副溶血性弧菌检测,捕获效率高达90%,对纯培养物的检出限为10 CFU/mL。
综上所述,IMS方法作为一种高效、快速的样品前处理技术,能够有效代替传统的选择性前增菌培养过程。该方法与高灵敏检测技术联用时,可显著缩短检测周期、并提高检测灵敏度和特异性[63]。但需通过持续优化其反应体系,最大程度提高实际样品中痕量靶标的富集效率[64-65]。
2.5 免疫传感器
免疫传感器是一种基于抗原-抗体特异性识别原理的分析装置,其通过将免疫反应的高特异性与传感元件的高灵敏度相结合,实现对目标物的精确检测。原理如图6所示。当固定于传感器的抗体捕获到致病性弧菌后发生免疫识别,会引起电学、光学和磁学等物理化学信号的改变,这些变化通过换能器将其转换为可定量读取的信号,加之传输与处理系统进行分析,实现对目标致病性弧菌的检测[66]。目前电化学免疫传感器和光学免疫传感器较为常见。
图6 免疫传感器原理图
Fig.6 Immunosensor schematic diagram
电化学免疫传感器是一类将特异性免疫识别反应与电化学信号转换机制相结合的生物传感装置,其通过安培法、阻抗法或电导法等电化学换能机制,将抗原-抗体结合事件转化为可定量测量的电信号,从而实现对目标分析物的高灵敏度检测[67]。目前检测致病性弧菌的电化学免疫传感器的修饰材料有AuNPs、石墨烯、氧化钒纳米粒子等[68],可以提高传感器的灵敏度和实用性。郝寿辰等[69]采用巯基乙酸-硒化镉(TGA-CdSe)量子点敏化TiO2作为光敏材料,通过组装构建成的光电化学传感器。该方法光电流与副溶血性弧菌浓度成线性关系,检测范围为102～107 CFU/mL,检出限为50 CFU/mL。SONG等[70]基于检测抗体固定在氧化石墨烯载体上作为检测单元,捕获抗体固定在Fe3O4颗粒作为捕获单元,通过捕获单元、副溶血性弧菌和检测器单元之间的免疫反应来检测副溶血性弧菌。该方法线性范围为102～108 CFU/mL,检出限为33 CFU/mL,且在海水和海产品中具有高选择性和良好的回收率。
光学免疫传感器是借助光学材料对抗体或抗原进行功能化修饰,通过光学信号的变化来实现目标菌的检测。目前,常见的光学免疫传感器主要包括比色型[71]、荧光型[72]、表面等离子体共振型和表面增强拉曼散射免疫传感器等[73]。WU等[74]建立了AuNPs比色法检测副溶血性弧菌的免疫传感器。该方法检出限低至10 CFU/mL,纯培养计算检出限低至5.6 CFU/mL,且与其他弧菌种及非弧菌种无交叉反应。WANG等[75]建立了一种AuNPs聚集的比色免疫传感器。该方法可在100 min完成副溶血性弧菌的检测,检出限为103 CFU/mL。ZHANG等[76]基于负载抗体和Fe3O4纳米颗粒的氧化石墨烯,建立了一种用于检测副溶血性弧菌的低场核磁共振/比色双模式均相免疫传感器。该方法在最佳实验条件下检出限为60 CFU/mL,具有良好的选择性、稳定性、精密度、准确度和一致性。
3 总结与展望
随着人们对食品安全要求的不断提高,致病性弧菌的检测方法得到了迅速发展。免疫分析方法凭借其快速、灵敏、特异以及可实现现场高通量检测的优势,在致病性弧菌检测领域正发挥着日益重要的作用,检测范围已覆盖海水、贝类、虾蟹等复杂基质(表2)。
表2 免疫分析方法在致病性弧菌检测中的应用
Table 2 Application of immunoassay methods in pathogenic Vibrio detection
类别具体方法检测弧菌种类线性范围/(CFU/mL)检出限/(CFU/mL)检测样品检测时间/min参考文献ELISA纳米抗体/夹心ELISA副溶血性弧菌—8.8×105虾180[77]ELISA斑点-ELISA创伤弧菌105～1071虾、牡蛎—[78]IFA金纳米团簇与对硝基酚之间的内过滤效应副溶血性弧菌103～1075×102鲫鱼30[79]IFA免疫荧光聚集法霍乱弧菌—103河水—[44]ICACdTe@SiO2-pAb/荧光测流免疫法创伤弧菌2.51×104～1.40×1083.95×103生理盐水、海水、虾15[55]ICA纸基夹心免疫副溶血性弧菌—6.05×105牡蛎—[80]IMS磁珠夹心ELISA副溶血性弧菌10～10510牡蛎40[81]IMS免疫磁珠分离-胶体金免疫层析试纸副溶血性弧菌—10-1海水55[82]免疫传感器电化学/荧光副溶血性弧菌10～1086虾、蟹、鱼50[83]免疫传感器低场核磁共振磁性生物传感器副溶血性弧菌5～1.0×1064海水、鱼50[84]免疫传感器局部表面等离子体共振霍乱弧菌—10稻叶菌株60[85]
注：“—”表示文献未注明。
本文系统归纳了致病性弧菌的免疫分析方法,并分析了各类方法的特点及优势。免疫分析方法研究仍面临两大挑战:1)检测灵敏度和特异性有待进一步提升,以应对复杂食品基质的干扰并精准检出低浓度目标弧菌;2)当食品中污染的致病性弧菌在食品加工过程中经过高温高压、消毒剂、防腐剂以及贮藏等处理后处于休眠或存活但无法在常规实验室条件下生长和繁殖的状态,该状态会导致菌体表面抗原表位会发生显著改变,抗原与抗体结合效率下降,易出现假阴性现象,同时表面抗原表达量减少使得低浓度弧菌难以被检出,从而造成漏检,成为潜在隐患。
因此,发展超灵敏、准确且具备多目标检测能力的免疫分析方法已成为重要趋势。首先,开发高性能信号探针与标记策略,采用发光纳米材料(如量子点、上转换纳米颗粒)、纳米酶或磁性纳米颗粒作为标记以增强信号强度,并利用金属有机框架(metal-organic frameworks,MOFs)、共价有机框架(covalent organic frameworks,COFs)及介孔二氧化硅纳米颗粒(mesoporous silica nanoparticles,MSNs)等新型多孔材料作为标记载体,提升探针稳定性与检测灵敏度。其次,革新抗体设计策略以精准识别存活困难或处于休眠状态的弧菌,通过优化半抗原构象、免疫佐剂及高通量筛选技术,制备高亲和力单克隆抗体,并深入挖掘目标弧菌的特异性表面标志物以开发新型抗体,从根本上降低漏检概率。最后,推动多技术融合,将免疫分析与微流控芯片、智能传感终端及分子生物学技术相结合,构建集样本预处理、信号放大与检测于一体的智能化系统,以克服单项技术的局限性,全面提升检测性能。
[1] LAVERTY A L, PRIMPKE S, LORENZ C, et al.Bacterial biofilms colonizing plastics in estuarine waters, with an emphasis on Vibrio spp.and their antibacterial resistance[J].PLoS One, 2020, 15(8):e0237704.
[2] FROELICH B A, DAINES D A.In hot water:Effects of climate change on Vibrio-human interactions[J].Environmental Microbiology, 2020, 22(10):4101-4111.
[3] BAKER-AUSTIN C, OLIVER J D, ALAM M, et al.Vibrio spp. infections[J].Nature Reviews Disease Primers, 2018, 4(1):5.
[4] ROBERT-PILLOT A, COPIN S, HIMBER C, et al.Occurrence of the three major Vibrio species pathogenic for human in seafood products consumed in France using real-time PCR[J].International Journal of Food Microbiology, 2014, 189:75-81.
[5] BONNIN-JUSSERAND M, COPIN S, LE BRIS C, et al.Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V.parahaemolyticus and V-vulnificus):Review of microbiological versus recent molecular detection methods in seafood products[J].Critical Reviews in Food Science and Nutrition, 2019, 59(4):597-610.
[6] YIN J F, WANG M Y, CHEN Y J, et al.Direct detection of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus from clinical and environmental samples by a multiplex touchdown polymerase chain reaction assay[J].Surgical Infections, 2018, 19(1):48-53.
[7] LENG F, LIN S L, WU W, et al.Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection:A case report and literature review[J].European Journal of Clinical Microbiology &Infectious Diseases, 2019, 38(11):1999-2004.
[8] MOK J S, RYU A, KWON J Y, et al.Abundance, antimicrobial resistance, and virulence of pathogenic Vibrio strains from molluscan shellfish farms along the Korean coast[J].Marine Pollution Bulletin, 2019, 149:110559.
[9] WU W, JING Z H, YU X Q, et al.Recent advances in screening aquatic products for Vibrio spp[J].TrAC Trends in Analytical Chemistry, 2019, 111:239-251.[10] VEZZULLI L, COLWELL R R, PRUZZO C.Ocean warming and spread of pathogenic Vibrios in the aquatic environment[J].Microbial Ecology, 2013, 65(4):817-825.
[11] BRAUGE T, MOUGIN J, ELLS T, et al.Sources and contamination routes of seafood with human pathogenic Vibrio spp.:A Farm-to-Fork approach[J].Comprehensive Reviews in Food Science and Food Safety, 2024, 23(1):e13283.
[12] TOROK V A, MAHBUB K R, GREY P A, et al.Survey of foodborne pathogenic Vibrio species in commercial Tasmanian bivalve shellfish and predictors of risk at harvest[J].International Journal of Food Microbiology, 2025, 430:111033.
[13] 谢雪钦, 刘舟.免疫学方法在食源性副溶血性弧菌检测中的应用研究进展[J].食品科学, 2017, 38(13):299-305.XIE X Q, LIU Z.Recent advances in application of immunological methods in detection of foodborne Vibiro parahaemolyticus[J].Food Science, 2017, 38(13):299-305.
[14] 朱慧, 李嘉文, 绳秀珍, 等.副溶血弧菌胶体金快速检测试纸的研制及应用[J].中国海洋大学学报(自然科学版), 2021, 51(3):24-33.ZHU H, LI J W, SHENG X Z, et al.Development of a competitive gold immunochromatographic test strip for rapid detection of Vibrio parahaemolyticus in fish[J].Periodical of Ocean University of China, 2021, 51(3):24-33.
[15] PATTIPEILOHY M, SUNDARI S.The immunogenicity of polyclonal antibody from induced Vibrio parahaemolyticus membrane by Elisa method[J].Journal of Physics:Conference Series, 2019, 1146(1):012032
[16] BAYAT M, KHABIRI A, HEMATI B.Development of IgY-based sandwich ELISA as a robust tool for rapid detection and discrimination of toxigenic Vibrio cholerae[J].Canadian Journal of Infectious Diseases &Medical Microbiology, 2018, 2018:4032531.
[17] WANG M Y, LIU X F, XIA J, et al.Vibrio vulnificus VvhA induces Th1 and Tfh cells to proliferate against Vibrio vulnificus in a mouse model of infection[J].Future Microbiology, 2017, 12:953-965.
[18] SITHIGORNGUL W, RENGPIPAT S, TANSIRISITTIKUL A, et al.Development of monoclonal antibodies for simple identification of Vibrio alginolyticus[J].Letters in Applied Microbiology, 2006, 43(4):436-442.
[19] MARTIN S J, SIEBELING R J.Identification of Vibrio vulnificus O serovars with antilipopolysaccharide monoclonal antibody[J].Journal of Clinical Microbiology, 1991, 29(8):1684-1688.
[20] WANGMAN P, CHAIVISUTHANGKURA P, TAENGCHAIYAPHUM S, et al.Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease[J].Journal of Fish Diseases, 2020, 43(2):207-214.
[21] ZHANG H Y, WANG Q, ZHUANG H X, et al.Development of recombinant antibodies and its application in immunomagnetic separation-based rapid detection of Vibrio cholerae in aquatic environments[J].Journal of Microbiology and Biotechnology, 2024, 34(11):2266-2278.
[22] 潘振, 陈杰敏, 胡成进.创伤弧菌溶细胞素噬菌体单链抗体库的构建[J].中华卫生应急电子杂志, 2021, 7(5):287-291.PAN Z, CHEN J M, HU C J.Construction of mouse-derived phage display library of scFv fragments against Vibrio vulnificus VvhA[J].Chinese Journal of Hygiene Rescue (Electronic Edition), 2021, 7(5):287-291.
[23] WANG Y Q, ZHANG L, WANG P, et al.Enhancing oriented immobilization efficiency:A one-for-two organism-bispecific nanobody scaffold for highly sensitive detection of foodborne pathogens[J].Analytical Chemistry, 2023, 95(46):17135-17142.
[24] SHAO S R, YANG H R, ZENG Q F, et al.Generation and evaluation of nanobodies specific for pirA and pirB from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease[J].Aquaculture, 2025, 602:742357.
[25] EBRAHIMI MOJARAD A, MOUSAVI GARGARIA S L.Aptamer-nanobody based ELASA for detection of Vibrio cholerae O1[J].Iranian Journal of Microbiology, 2020, 12(4):263-272.
[26] 陈奇. 单核细胞增生性李斯特菌单克隆抗体、多克隆抗体及单域重链抗体的制备[D].南昌:南昌大学, 2014.CHEN Q.Preparation of anti-Listeria monocytogenes monoclonal antibody, polyclonal antibodies and single-domain heavy chain antibody[D].Nanchang:Nanchang University, 2014.
[27] NAKAZAWA M, MUKUMOTO M, MIYATAKE K.Production and Purification of Polyclonal Antibodies[M].High-Resolution Imaging of Cellular Proteins:Methods and Protocols.New York, NY:Springer New York.2016:49-59.
[28] 李世佳. 利用CRISPR技术敲除兔细胞HGPRT基因[D].福州:福州大学, 2018.LI S J.Knockout of HGPRT in rabbit cell by CRISPR/Cas9 technique[D].Fuzhou:Fuzhou University, 2018.
[29] QRIOUET Z, CHERRAH Y, SEFRIOUI H, et al.Monoclonal antibodies application in lateral flow immunochromatographic assays for drugs of abuse detection[J].Molecules, 2021, 26(4):1058
[30] 全美淋, 李飞, 刘金燕, 等.抗体技术在食源性致病菌快速检测中的研究进展[J].生物工程学报, 2024, 40(11):3985-4005.QUAN M L, LI F, LIU J Y, et al.Research progress of antibody technology in rapid detection of foodborne pathogens[J].Chinese Journal of Biotechnology, 2024, 40(11):3985-4005.
[31] HOLLIGER P, HUDSON P J.Engineered antibody fragments and the rise of single domains[J].Nature Biotechnology, 2005, 23(9):1126-1136.
[32] ZHAO Q, LU D, ZHANG G Y, et al.Recent improvements in enzyme-linked immunosorbent assays based on nanomaterials[J].Talanta, 2021, 223:121722.
[33] 肖益群, 翟少伟, 郭松林.鳗鲡源创伤弧菌的间接ELISA快速检测[J].集美大学学报(自然科学版), 2020, 25(2):81-87.XIAO Y Q, ZHAI S W, GUO S L.The indirect ELISA for rapid detection of Vibrio vulnificus from eels[J].Journal of Jimei University (Natural Science), 2020, 25(2):81-87.
[34] 曾静, 程晋霞, 张蕾, 等.创伤弧菌鞭毛蛋白单克隆抗体的制备及在食品检测中的应用[J].细胞与分子免疫学杂志, 2013, 29(10):1068-1071;1075.ZENG J, CHENG J X, ZHANG L, et al.Preparation of monoclonal antibodies against flagellin core protein of Vibrio vulnificus and its application in testing Vibrio vulnificus from food products[J].Chinese Journal of Cellular and Molecular Immunology, 2013, 29(10):1068-1071;1075.
[35] 职通瑞, 宋晓玲, 张晓静, 等.多抗间接ELISA方法的建立及其在海洋细菌快速检测中的应用[J].渔业科学进展, 2015, 36(2):71-76.ZHI T R, SONG X L, ZHANG X J, et al.Establishment of polyclonal antibody indirect enzyme-linked immunosorbent assay (ELISA) and the application in the rapid detection of marine bacteria[J].Progress in Fishery Sciences, 2015, 36(2):71-76.
[36] 程晋霞, 曾静, 张蕾, 等.霍乱弧菌鞭毛蛋白单克隆抗体的制备及双抗体夹心ELISA的建立[J].细胞与分子免疫学杂志, 2013, 29(11):1169-1173.CHENG J X, ZENG J, ZHANG L, et al.Preparation of monoclonal antibodies against flagellin core protein of Vibrio cholerae and its application in establishing double-antibody sandwich ELISA for testing Vibrio cholerae from food products[J].Chinese Journal of Cellular and Molecular Immunology, 2013, 29(11):1169-1173.
[37] HU Y Z, SUN Y, GU J X, et al.Selection of specific nanobodies to develop an immuno-assay detecting Staphylococcus aureus in milk[J].Food Chemistry, 2021, 353:129481.
[38] ZHANG C, LIU Z L, BAI M F, et al.An ultrasensitive sandwich chemiluminescent enzyme immunoassay based on phage-mediated double-nanobody for detection of Salmonella Typhimurium in food[J].Sensors and Actuators B:Chemical, 2022, 352:131058.
[39] WEI T X, DU D, ZHU M J, et al.An improved ultrasensitive enzyme-linked immunosorbent assay using hydrangea-like antibody-enzyme-inorganic three-in-one nanocomposites[J].ACS Applied Materials &Interfaces, 2016, 8(10):6329-6335.
[40] MONDAL S.Immunofluorescence Techniques[M].Manual of Histological Techniques, 2018.
[41] 王金雨, 刘志超, 赵晓东. 免疫荧光技术及其在水产动物疾病检测中的应用[J]. 江西水产科技, 2017(2): 37-39.WANG J Y, LIU Z C, ZHAO X D. Immunofluorescence technique and its application in disease detection of aquatic animals[J]. Jiangxi Fishery Science and Technology, 2017(2): 37-39.
[42] 于光, 陈贵连, 王文. 吖啶橙免疫荧光菌团法快速检测鱼贝类副溶血性弧菌的研究[J]. 兽医大学学报, 1991, 11(4): 365-368.YU G, CHEN G L, WANG W. Rapid detection of Vibrio parahaemoiyticus in sellfish by acridine orange immunofluorescent bacterial clumps method[J]. Chinese Journal of Veterinary Science, 1991, 11(4): 365-368.
[43] CHEN H C, CHANG T C.Rapid detection of Vibrio parahaemolyticus in oysters by immunofluorescence microscopy[J].International Journal of Food Microbiology, 1996, 29(2-3):311-319.
[44] WANG D C, XU X B, DENG X L, et al.Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay[J].Applied and Environmental Microbiology, 2010, 76(16):5520-5525.
[45] 王殿夫, 丁宁, 刘洪伟, 等.致病性弧菌检测新技术研究进展[J].食品安全质量检测学报, 2024, 15(2):160-167.WANG D F, DING N, LIU H W, et al.Research progress in new technologies for detecting pathogenic Vibrio[J].Journal of Food Safety &Quality, 2024, 15(2):160-167.
[46] 张文红. 高灵敏度免疫荧光技术在动物免疫抗体检测中的精准性与可行性研究[J].智慧农业导刊, 2024, 4(7):39-42.ZHANG W H.Research on accuracy and feasibility of high-sensitivity immunofluorescence technique in animal immune antibody detection[J].Journal of Smart Agriculture, 2024, 4(7):39-42.
[47] GONDHALEKAR C, BIELA E, RAJWA B, et al.Detection of E.coli labeled with metal-conjugated antibodies using lateral-flow assay and laser-induced breakdown spectroscopy[J].Analytical and Bioanalytical Chemistry, 2020, 412(6):1291-1301.
[48] FANG J H, LI S Y, LIU X X, et al.Development of a test strip for rapid detection of Gymnodinium catenatum[J].Environmental Monitoring and Assessment, 2022, 195(1):83.
[49] DONG X, XIE J M, WANG L Y, et al.Development of monoclonal antibody against PirB and establishment of a colloidal gold immunochromatographic assay for the rapid detection of AHPND-causing Vibrio[J].Animals, 2024, 14(11):1600.
[50] 吴美娇, 吴有雪, 刘程, 等.基于组合胶体金纳米颗粒的免疫层析试纸条快速可视化检测海产品中副溶血性弧菌[J].工业微生物, 2021, 51(2):1-9.WU M J, WU Y X, LIU C, et al.Rapid visual detection of Vibrio parahaemolyticus in seafood by immunochromatographic strip based on composited colloidal gold nanoparticles[J].Industrial Microbiology, 2021, 51(2):1-9.
[51] SAKATA J, YONEKITA T, KAWATSU K.Development of a rapid immunochromatographic assay to detect contamination of raw oysters with enteropathogenic Vibrio parahaemolyticus[J].International Journal of Food Microbiology, 2018, 264:16-24.
[52] DING C C, LI J W, LIU X, et al.Development of colloidal gold-based immunochromatographic strip test using two monoclonal antibodies for detection of Vibrio parahaemolyticus[J].Journal of Food Safety, 2018, 38(4):e12468.
[53] CHEN X R, LENG Y K, HAO L W, et al.Self-assembled colloidal gold superparticles to enhance the sensitivity of lateral flow immunoassays with sandwich format[J].Theranostics, 2020, 10(8):3737-3748.
[54] 李霁薇. 高灵敏检测两种食源性致病菌的聚集诱导发光免疫层析试纸条的开发[D].南昌:南昌大学, 2024.LI J W.Development of highly sensitive immunochromatographic test strips based on aggregation-induced emission for the detection of two foodborne pathogen[D].Nanchang:Nanchang University, 2024.
[55] CHEN S P, JI L, XIE J P, et al.Silica-coated CdTe nanomaterial as high-affinity red fluorescence signal label for the detection of Vibrio vulnificus by lateral flow immunoassay[J].Food Control, 2025, 172:111190.
[56] BHARDWAJ N, BHARDWAJ S K, BHATT D, et al.Highly sensitive optical biosensing of Staphylococcus aureus with an antibody/metal-organic framework bioconjugate[J].Analytical Methods, 2019, 11(7):917-923.
[57] 苏娜, 明亮, 何静, 等.免疫检测技术在食源性致病菌中的应用研究进展[J].食品与发酵工业, 2024, 50(12):343-352.SU N, MING L, HE J, et al.Research progress on application of immunoassays in foodborne pathogens[J].Food and Fermentation Industries, 2024, 50(12):343-352.
[58] MAO Y, HUANG X L, XIONG S C, et al.Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce[J].Food Control, 2016, 59:601-608.
[59] LIU Y Y, ZHANG Z H, WANG Y L, et al.A highly sensitive and flexible magnetic nanoprobe labeled immunochromatographic assay platform for pathogen Vibrio parahaemolyticus[J].International Journal of Food Microbiology, 2015, 211:109-116.
[60] ZHAO L C, LYU X R, CAO X, et al.Improved quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic separation and PMAxx-qPCR[J].Food Control, 2020, 110:106962.
[61] ZHAI Y, MENG X J, LI L, et al.Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence[J].RSC Advances, 2021, 11(61):38638-38647.
[62] LUN J S, LIU D, LIU T K, et al.Evaluation of outer membrane protein U (OmpU) as a novel capture target of Vibrio parahaemolyticus and rapid detection of acute hepatopancreatic necrosis disease (AHPND) using PCR combined with immunomagnetic separation[J].Aquaculture, 2018, 485:225-232.
[63] 郭姜里, 杜娟, 李宗双, 等.免疫磁分离技术在食源性致病菌前处理中的应用进展[J].食品安全质量检测学报, 2023, 14(21):97-106.GUO J L, DU J, LI Z S, et al.Application of immunomagnetic separation technology in pretreatment of foodborne pathogenic bacteria[J].Journal of Food Safety &Quality, 2023, 14(21):97-106.
[64] 曹潇, 赵力超, 陈洵, 等.免疫磁分离技术在食源性致病菌快速检测中的研究进展[J].食品科学, 2019, 40(15):338-345.CAO X, ZHAO L C, CHEN X, et al.Progress in the application of immunomagnetic separation in rapid detection of foodborne pathogens[J].Food Science, 2019, 40(15):338-345.
[65] 林吉恒, 黄朱梁, 彭志兰, 等.免疫磁珠分离技术在食源性致病菌检测中的应用[J].食品安全质量检测学报, 2019, 10(18):5998-6005.LIN J H, HUANG Z L, PENG Z L, et al.Application of immunomagnetic beads separation techniques in detection of foodborne pathogenic bacteria[J].Journal of Food Safety &Quality, 2019, 10(18):5998-6005.
[66] HUANG H, LIU M H, WANG X S, et al.Label-free 3D Ag nanoflower-based electrochemical immunosensor for the detection of Escherichia coli O157:H7 pathogens[J].Nanoscale Research Letters, 2016, 11(1):507.
[67] YU J, STANKOVIC D, VIDIC J, et al.Recent advances in electrochemiluminescence immunosensing[J].Sensors &Diagnostics, 2024, 3(12):1887-1898.
[68] 杨兴东, 曲扬, 胡骁飞, 等.免疫分析方法在氧氟沙星残留检测中的研究进展[J].浙江农业科学, 2025, 66(3):742-749.YANG X D, QU Y, HU X F, et al.Research progress of immunoassay methods in the detection of ofloxacin residues[J].Journal of Zhejiang Agricultural Sciences, 2025, 66(3):742-749.
[69] 郝寿辰. 快速检测副溶血性弧菌光电化学免疫传感器的构建及研究[D].大连:大连工业大学, 2019.HAO S C.Construction and study of photoelectrochemical immunosensor for rapid detection of Vibrio parahaemolyticus[D].Dalian:Dalian University of Technology, 2019.
[70] SONG X X, XU L H, WU L, et al.Electrochemical immunosensor for detection of Vibrio parahaemolyticus based on faradaycage-type anodic stripping voltammetry[J].Journal of the Electrochemical Society, 2017, 164(13):B704-B709.
[71] MAHHEIDARI N, RASHIDIANI J, KOOSHKI H, et al.An effort to making a colorimitric nano-biosensor for Vibrio cholera detection[J].Current Nanoscience, 2020, 16(5):793-804.
[72] KOURTI D, ANGELOPOULOU M, PETROU P, et al.Optical immunosensors for bacteria detection in food matrices[J].Chemosensors, 2023, 11(8):430.
[73] YANG Y, LI G L, WANG P X, et al.Highly sensitive multiplex detection of foodborne pathogens using a SERS immunosensor combined with novel covalent organic frameworks based biologic interference-free Raman tags[J].Talanta, 2022, 243:123369.
[74] WU W, ZHOU M, HE H, et al.A sensitive aptasensor for the detection of Vibrio parahaemolyticus[J].Sensors and Actuators B:Chemical, 2018, 272:550-558.
[75] WANG P, YU G G, WEI J, et al.A single thiolated-phage displayed nanobody-based biosensor for label-free detection of foodborne pathogen[J].Journal of Hazardous Materials, 2023, 443:130157.
[76] ZHANG D Y, CHEN L, LIN H, et al.Well plate-based LF-NMR/colorimetric dual-mode homogeneous immunosensor for Vibrio parahaemolyticus detection[J].Food Chemistry, 2024, 436:137757.
[77] 王鹏. 副溶血性弧菌纳米抗体的研制及免疫分析方法研究[D].杨凌:西北农林科技大学, 2023.WANG P.The development of nanobody and construction of immunoassay towards Vibrio parahaemolyticus[D].Yangling:Northwest A&F University, 2023.
[78] WANGMAN P, SURASILP T, PENGSUK C, et al.Development of a species-specific monoclonal antibody for rapid detection and identification of foodborne pathogen Vibrio vulnificus[J].Journal of Food Safety, 2021, 41(6):e12939.
[79] YANG G X, WEI C H, TANG Y Y, et al.A novel fluorescence immunoassay based on inner filter effect and gold nanoclusters for Vibrio parahaemolyticus determination[J].Results in Chemistry, 2021, 3:100208.
[80] RODRIGUEZ-QUIJADA C, LYONS C, SANCHEZ-PURRA M, et al.Gold nanoparticle paper immunoassays for sensing the presence of Vibrio parahaemolyticus in oyster hemolymph[J].ACS Omega, 2023, 8(22):19494-19502.
[81] LIU Y S, ZHAO C, SONG X L, et al.Colorimetric immunoassay for rapid detection of Vibrio parahaemolyticus[J].Microchimica Acta, 2017, 184(12):4785-4792.
[82] ZHOU J, ZHANG C D, ZHANG X, et al.Immunomagnetic separation-based nanogold enhanced surface plasmon resonance and colloidal gold test strips for rapid detection of Vibrio parahaemolyticus[J].Archives of Microbiology, 2020, 202(5):1025-1033.
[83] WANG S, HU J H, XIAO S, et al.Fluorescent/electrochemical dual-signal response biosensing strategy mediated by DNAzyme-ferrocene-triggered click chemistry for simultaneous rapid screening and quantitative detection of Vibrio parahaemolyticus[J].Sensors and Actuators B:Chemical, 2023, 380:133393.
[84] ZHANG D Y, LIN H, CHEN L, et al.Cluster-bomb type magnetic biosensor for ultrasensitive detection of Vibrio parahaemolyticus based on low field nuclear magnetic resonance[J].Analytica Chimica Acta, 2023, 1248:340906.
[85] FARIDFAR G, ZEINODDINI M, AKBARZEDEHKOLAHI S, et al.Immunodiagnostic of Vibrio cholerae O1 using localized surface plasmon resonance (LSPR) biosensor[J].International Microbiology, 2021, 24(1):115-122.