CN103940991B - 猪沙门氏菌耐热毒素快速检测试纸条 - Google Patents
猪沙门氏菌耐热毒素快速检测试纸条 Download PDFInfo
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Abstract
本发明涉及一种猪沙门氏菌耐热毒素检测试剂显示的器具,特别是涉及一种猪沙门氏菌耐热毒素快速检测试纸条,试纸条含有支撑层、反应试剂载体吸附层,支撑层为不吸水薄片条,反应试剂载体吸附层粘贴于支撑层上,从样品测试端依次为纤维层,猪沙门氏菌耐热毒素金标单抗或多抗纤维层,纤维素膜层,手柄端为吸水材料层;分别用猪沙门氏菌耐热毒素配对单抗或多抗或单抗溶液在纤维层上印制检测印迹“<b>|</b>”、“<b>/</b>”或“<b>\</b>”,分别用羊(兔)抗小鼠或猪IgG的多抗或用SPA溶液在纤维素膜层上印制对照印迹“<b>|</b>”、“<b>/</b>”或“<b>\</b>”。该检测试纸条,特异性强,敏感性高,检测结果显示形象、直观、准确,无需仪器设备,无需专业检测人员,费用低,操作简便、快速,可大大降低劳动强度,缩短检测时间,能在饲养场,肉类加工厂,出入境检验检疫局等场所进行现场检测,易于推广应用。
Description
一、技术领域
本发明是一种涉及猪沙门氏菌耐热毒素的检测试剂显示器具,特别是涉及一种可检测猪沙门氏菌耐热毒素的检测试纸条。
二、技术背景
沙门氏菌是重要的人畜共患病的病原体,在当今世界由其引起的食物中毒病例约占食物中毒病例的4o%,常有人类因沙门氏菌食物中毒而死亡的病例报道,引起人类的疾病主要有伤寒、副伤寒、胃肠炎和败血症。近几年来,该病发生频率仍呈上升趋势,由于人类食物中毒主要来源于畜禽及其制品。因此快速准确检测畜禽之中沙门氏菌阳性感染者已成为当务之急。
猪沙门氏菌病主要发生于4个月龄以内的断乳仔猪。成年猪和哺乳猪很少发病。细菌可通过病猪或带菌猪的粪便、污染的水源和饲料等经消化道感染健康猪。鼠类也可传播本病。沙门氏菌分布广,能从各种途径传入猪群。许多猪可借本身的抵抗力将其消灭或免疫,有些则成为带菌猪。但如饲养管理不当、气候突变或长途运输等,猪的抵抗力普遍下降时,或病菌几经通过猪体,毒力增高也可造成本病的暴发。1~4月龄仔猪易感性较高。半岁以上猪的免疫***已逐步完善,感染后很少发病,但在相当一段时间内带有本菌。在应激因素作用下,尤其是在发生猪瘟时,往往发生本病的并发和继发感染。本病一年四季均可发生,多雨潮湿季节更易发,在猪群中一般散发或呈地方流行。环境污秽、潮湿、棚舍拥挤、粪便堆积、饲料和饮水供应不及时等应激因素易促进本病的发生。
临床上分急性(败血)型和慢性(坏死性肠炎)型。前者多见于断乳前后的仔猪。表现体温升高至41~42℃,精神不振,食欲废绝,不愿行动,呼吸困难,腹泻、呕吐,耳根、胸前和腹下、下肢呈现紫斑或暗红。多以死亡告终。后者,通常以下痢为主要症状,粪便呈粥状,排出灰白色或黄绿色恶臭水样粪,有时混血液和坏死组织碎片。食欲降低,渴欲增加。由于持续下痢,日渐消瘦、衰弱,最后衰竭而死。在发生猪瘟时,往往并发或继发该病。
剖检:急性败血型,在耳、腹、四肢内侧皮肤有出血斑点,各脏器浆膜、喉头、膀胱粘膜和肾脏均有出血点,脾脏肿大,边缘钝,呈特征性的蓝色。***肿胀出血,胃肠粘膜呈卡他性炎症。慢性型病死猪尸体消瘦,盲肠、结肠粘膜呈局灶性或弥漫性增厚,被覆有灰黄色干酪样伪膜,除去伪膜形成溃疡,溃疡边缘多无堤状***。脾稍肿大,肝有时可见黄灰色坏死小点,肺有慢性卡他性炎症,含有黄色干酪样结节。
近几年来,该病的发生频率呈上升趋势,给养猪业带来了严重危害。因此,寻找到一种操作简便而又快速和特异的检测方法,无疑在猪沙门氏菌感染诊断和流行病学检测等方面有着重要意义。目前沙门氏菌及耐热毒常用的检测方法有以下几种:
(1)细菌分离培养:将病料或增菌培养液直接划线接种在选择培养基(S·S和亚硫酸铋琼脂)上,经37℃18~24小时培养后,如形成无色较小、边缘整齐半透明,有的因产生硫化氢(H2S)形成中心带黑色菌落时,将此可疑菌落接种于三糖铁培养基斜面,置37℃培养18~24小时。观察底层葡萄糖产酸或产酸产气,产生硫化氢变棕黑色,上层斜面乳糖不分解、不变色,则可初步判定为沙门氏菌。
(2)显微镜检查:取被检材料(肝、脾、肾、肠系膜***)制成涂片,自然干燥,用革兰氏染色法染色镜检,如看到两端钝圆或卵圆形,不运动,不形成芽胞和荚膜的革兰氏阴性小杆菌,则初步认为是沙门氏菌。
(3)免疫荧光抗体试验:取经增菌培养的被检样品一铂耳,制成薄的涂片,晾干后,用固定液(乙醇60毫升,三氯甲烷30毫升,甲醛10毫升混合)固定5~10分,再用95%乙醇浸洗后晾干。再将沙门氏菌荧光抗体滴加标本涂片上,放湿盒内,在37℃经30分后取出,用0.01MPH9.0磷酸盐缓冲液冲去多余的荧光抗体;再用相同的磷酸盐缓冲液浸洗10分,然后用蒸馏水冲洗、晾干,再滴加PH9.0碳酸盐缓冲甘油后,加盖玻片封片、镜检。
(4)酶联免疫吸附试验:酶联免疫吸附试验是将菌体抗原或耐热毒素抗体吸附于固相载体,在载体上进行免疫酶染色,底物显色后,用肉眼或分光光度计判定结果。沙门氏菌表面的脂多糖分子上的抗原、鞭毛抗原和外膜蛋白抗原等在沙门氏菌感染诊断中发挥了重要作用。利用这些菌体蛋白或耐热毒素的单克隆抗体建立的直接ELISA、夹心ELISA方法都能有效地检测沙门氏菌及耐热毒。
(5)聚合酶链式反应(PCR)技术:PCR技术具有高灵敏度、高特异性等优点,多重PCR方法已被广泛运用于沙门氏菌耐热毒素及病原菌的鉴定和流行病学调查。
上述检测方法需要专业人员在实验室操作,操作繁琐,检测费时费力;而且需要昂贵的仪器设备,如PCR仪、酶标仪等,对非专业人员而言,上述检测方法很难完成。虽然上述方法特异敏感,但无法实现现场快速检测或诊断。本发明,研究一种简便快速、实时在线检测试纸,对控制食物中毒和消灭此疾病意义重大。
三、发明内容
本发明的目的是为了克服现有技术中检测猪沙门氏菌耐热毒素存在的缺点,提供一种特异、敏感、简便快速检测猪沙门氏菌耐热毒素的方法,研制出检测猪沙门氏菌耐热毒素的检测试纸条。
本发明的技术方案是:提供一种猪沙门氏菌耐热毒素的检测试纸条,该试纸条含有支撑层和吸附层,支撑层为不吸水的薄片层,吸附层附着在支撑层上,吸附层从测试端依次为样品吸附纤维层、金标抗体纤维层、纤维素膜层和手柄端的吸水材料层,在纤维素膜层上设有检测印迹和对照印迹;金标抗体纤维层吸附有纳米级金颗粒标记的抗沙门氏菌耐热毒素的单克隆抗体,检测印迹用抗沙门氏菌耐热毒素的配对单抗印制,对照印迹用羊或兔抗小鼠IgG的多克隆抗体;或金标抗体纤维层吸附有纳米级金颗粒标记的抗沙门氏菌耐热毒素的多克隆抗体,检测印迹用抗沙门氏菌耐热毒素的单抗制备,对照印迹用金黄色葡萄球菌A蛋白(SPA)或抗猪IgG多抗制备。
检测印迹用抗沙门氏菌耐热毒素的配对单抗制备即用沙门氏菌耐热毒素的配对单抗溶液制备;检测印迹用抗沙门氏菌耐热毒素的多克隆抗体制备即为用沙门氏菌耐热毒素的多克隆抗体制备。
支撑层用不吸水的硬质塑胶片条或硬纸条制成;测试端样品吸附纤维层用玻璃棉制成;金标抗体纤维层用玻璃棉和金标抗体制成,金标抗体可以是单抗或多克隆抗体。
纤维素膜层用硝酸纤维素膜、或纯纤维素膜、或羧化纤维素膜、或聚偏二氟乙烯PVDF纤维素膜制成。
吸水材料层用吸水纸制成。
检测印迹和对照印迹为直线式、或斜线式,纤维素膜层上含有一条检测印迹和一条对照印迹,检测印迹和对照印迹的排列形式为“||”、“//”、“\\”中的任一种。
试纸条吸附层上面含有一层保护层,保护层附着在吸附层上,在测试端样品吸附纤维层、金标抗体纤维层及吸水材料层上覆盖有保护膜,在测试端样品吸附纤维层与金标抗体纤维层交界处对应的保护膜上印制有样品标记线,该标记线偏向测试端样品吸附纤维层一侧处约0.5cm处。
根据需要,选择上述金标抗体纤维层、检测印迹和对照印迹排列形式中一种形式。
本发明的积极有益效果:
1.检测特异性强、敏感性高:本发明检测试纸条以纳米级金颗粒标记高亲和力特异性单克隆抗体或特异性多克隆抗体为基础而制成,金标抗体中金颗粒与抗体分子之间无共价键形成,二者通过异性电荷间的范德华力相结合,金颗粒不影响单克隆抗体或多克隆抗体的特异性和结合力,并且具有较高的标记率。本发明检测试纸条具有较高的特异性和敏感性,可检测到纳克级沙门氏菌耐热毒素。
2.操作简便、快速:使用本发明试纸条检测时无需附加任何其它仪器和试剂,只需将其测试端***待检的样品液中30秒左右,然后在1-5分钟内即可判定检测结果。
3.检测结果直观、准确:本发明试纸条以是否显示棕红色的检测线和对照线作为判定阳性和阴性结果的依据,即只在纤维素膜的对照线印记处显示一条棕红色对照线C,而在检测线印记处无棕红色条带显示,表示被检测的沙门氏菌耐热毒素为阴性结果;在纤维素膜的对照线印记处显示一条棕红色对照线C,在检测印迹处显示一条棕红色条带T,则表示被检测的沙门氏菌耐热毒素为阳性结果。无论阳性结果或阴性结果对照线C均应显示,当对照线C不显示时,说明试纸条失效。
4.检测费用降低:使用本发明检测试纸条,不需其它仪器及试剂,节省了仪器、设备和附加试剂费用;非专业人员也可随时实时在线检测,无需支付专家诊断检查费及其相关费用,可极大的降低检测成本的投入,降低检测费用。
5.使用范围广:本发明检测试纸条操作简单,即“傻瓜式”操作,而且携带方便、易保存,可满足不同单位和不同层次人员的需要,包括专业化验、海关检疫、卫生防疫、质量监测、畜产品加工、集约化养殖到个体养殖等,具有广阔的市场前景和社会效益。
四、附图说明:
图1一种猪沙门氏菌耐热毒素的检测试纸条的侧视结构示意图
图2一种猪沙门氏菌耐热毒素的检测试纸条的俯视结构示意图
五、具体实施方式:
以下实施例仅为了进一步说明本发明,并不限制本发明的内容。猪沙门氏菌耐热毒素的检测试纸条的制备,需要制备抗沙门氏菌耐热毒素的单克隆抗体和多克隆抗体,用于制备检测印迹和金标抗体纤维层;同时需要制备羊或兔抗鼠IgG抗体,羊或兔抗猪IgG抗体,用于制备对照印迹。
1.羊(兔)抗鼠或抗猪IgG抗体的制备:
以饱和硫酸铵法提取小鼠或猪血清中的IgG,取1份血清加2份PBS液(pH7.2)混匀,加等体积饱和硫酸铵液混匀,置4℃冰箱内2h,在4℃、10000r/min离心15min,弃上清液;以适量PBS液(pH7.2)溶解沉淀,加饱和硫酸铵液至其最终浓度为33%,置4℃冰箱内2h,在4℃、10000r/min条件下离心15min,弃上清液,以少量PBS液(pH7.2)溶解沉淀,置4℃冰箱内用PBS液(pH7.2)过夜透析,换液2~3次,在4℃、10000r/min条件下离心15min,收集上清液,以紫外分光光度计测定其蛋白浓度。以50μg~100μg(IgG)/kg体重经皮下或肌肉注射抗体阴性健康羊或家兔3~4次,末次免疫20天后,静脉采血,以ELISA测定其血清抗体效价在1:2000以上,心脏采血或颈动脉放血,收集其高免血清,以饱和硫酸铵法提取羊(兔)抗小鼠或猪的IgG(其提取方法与上述提取小鼠血清IgG相同,不再重述),用于制备本发明试纸条的对照印迹。
2.沙门氏菌耐热毒素单克隆抗体(Mi)的制备:
每只用50μg~100μg沙门氏菌耐热毒素抗原免疫Balb/c系小鼠三次,每次间隔15~30d;第三次加强免疫后3~4d,将免疫小鼠眼球放血,拉颈致死,用75%酒精浸泡5~10min,无菌取其脾脏,剪碎并经100目尼龙网过滤,1000r/min离心10min,收集脾细胞;将1×108个脾细胞与2~5×107个NS0骨髓瘤细胞混合,1000r/min离心10min弃上清,将含有沉淀细胞的离心管置于37℃的水中,并缓缓加入0.7~1ml40%~50%PEG4000(pH8.5~9.0)作用1min,然后缓慢加入无血清1640培养基15ml,以终止PEG的作用,37℃水浴5~10min,1000r/min离心10min弃上清,将细胞沉淀重悬于HAT选择培养基中,并加入
96孔培养板(100μl~200μl/孔),置于37℃5%CO2培养箱中培养。培养7~10d后,以5μg~10μg/ml的纯化的病原体特异抗原包被96孔酶标板,以酶联免疫吸附试验(ELISA)检测杂交瘤的培养上清,挑取强阳性细胞克隆(OD450≥0.5),进行连续三次的有限稀释法克隆化,获得阳性杂交瘤细胞株,其染色体数为92~98,其分泌的单克隆抗体,能够特异识别沙门氏菌耐热毒素,而不与其它毒素发生交叉反应,亲和力常数达109~10,轻链亚型为к或λ,重链亚型为IgG1、IgG2a、IgG2b、IgG3;获得的配对单克隆抗体,用于制金标单抗体玻璃棉或检测印迹。
3.金标单抗玻璃棉的制备:
利用柠檬酸钠还原法制备纳米级金颗粒:即在50~100ml沸腾的0.01~0.05%氯金酸水溶液中加入2~4ml的0.5~2%柠檬酸三钠溶液,获得直径15nm左右的纳米级金颗粒。以0.1mol/L的K2CO3调金颗粒溶液的pH至8.5~9.5,以1:1000~1300的标记比将待标记的单克隆抗体加入pH8.5~9.5的金溶胶中,标记10min后,加20%PEG10000至最终浓度为0.05%,4℃、1500~3000r/min离心20min,除去未结合的金颗粒颗粒,4℃、15000r/min离心1h,弃上清,获金标抗体混合物后,用丙烯葡聚糖S-400柱层析,分离纯化金标抗体,获得的金标抗体。将1:100~500稀释的金标抗体,吸附于精制玻璃棉中,4℃低温真空干燥,制备金标单克隆抗体玻璃棉。
4.沙门氏菌耐热毒素多克隆抗体(Ci)的制备:
沙门氏菌耐热毒素多克隆抗体(Ci)的制备。采用沙门氏菌耐热毒素抗原多次免疫接种抗体阴性健康猪。末次免疫20天后静脉采血,以ELISA测定其血清抗体效价在1:2000以上,心脏采血或颈动脉放血,收集其高免血清,以饱和硫酸铵法提取血清中IgG抗体(方法与小鼠血清IgG的提取相同,不再重述)。
金标多抗和金标多抗玻璃棉的制备,与金标单抗玻璃棉的制备方法相同,不再重述。详见具体实施方式中的内容3。
5.本发明检测试纸条检测原理
当本发明检测试纸条测试端***待检样品溶液后,待检溶液通过虹吸带动待检沙门氏菌耐热毒素进入金标抗体纤维层,并与其中的金标抗体(Mi或Ci)一起沿硝酸纤维素膜向手柄端扩散,最终渗入手柄端吸水材料层,扩散过程中金标抗体能够与相应的待检沙门氏菌耐热毒素结合,结合金标抗体的沙门氏菌耐热毒素能够被纤维素膜上检测印迹的配对单抗或多抗拦截,当样品液中含有被检沙门氏菌耐热毒素时,则出现一条棕红色的检测线;羊或兔抗鼠或抗猪IgG则可与相应的金标单抗或多抗结合,出现1条棕红色对照线。当待检样品液中不含沙门氏菌耐热毒素时,试纸条只显示出一条棕红色对照线;当纤维素膜上没有对照线显示时,则表明试纸条已失效。
6.本发明检测试纸条的检测操作方法
(1)检测样品的处理:取病猪病变组织或市售鲜肉,1:1~5加入生理盐水并用剪刀剪碎,浸出液为待检样品,病猪全血或血清加入生理盐水1:1~5稀释后为待检样品。
(2)检测操作:将本发明检测试纸条样品端***待检样品液中,***深度不超过标记线9,约30秒后取出试纸条,水平放置约1~5分钟,同时观察结果。
(3)结果判定:如果在检测试纸条纤维素膜上只显示出一条棕红色对照线C,表示检测结果为阴性,说明在被检样品中不含沙门氏菌耐热毒素;如果检测试纸条上的纤维素膜出现对照线C,检测印迹处出现一条检测线,表示检测结果为阳性,即在待检样品中含有沙门氏菌耐热毒素;如果纤维素膜上没有对照线C显示,则表明试纸条已失效。
实施例一:猪沙门氏菌耐热毒素的检测试纸条
参见图1和图2,图中1为支撑层,用硬质塑胶薄片条制成,2为测试端的样品吸附纤维层,用玻璃棉制成,3为金标抗体纤维层,吸附有纳米级金颗粒标记的抗沙门氏菌耐热毒素的单克隆抗体的玻璃棉,根据上述具体实施方式3中所述的制备方法制备其金标单抗玻璃棉,4为纤维素膜层,采用硝酸纤维素膜制成,5为吸水材料层,用吸水纸制成,将编号2、3、4、5各层从左端测试端至右粘贴在硬质塑胶薄片条1上,彼此之间交界处互相交叉重叠。在硝酸纤维素膜层4上,6为用抗沙门氏菌耐热毒素的配对单抗溶液印制的检测印迹T,7为用羊或兔抗鼠IgG溶液印制的对照印迹C,检测印迹和对照印迹为直线式、或斜线式,两种印迹带排列形成的组合形式为“||”、“//”、“\\”中的任一种。8-1为覆盖在测试端样品吸附纤维层2和金标抗体纤维层3上面的白色保护膜,在2和3交界处对应保护膜8-1位置上偏向于样品吸附纤维层2一侧0.5cm处印有标记线9,9的右端印有箭头及max字样,吸水材料层5(手柄端)上覆盖有其它颜色(如黄色)保护膜8-2。
待测样品溶液的制备及检测操作步骤,与具体实施方式6中的检测操作方法相同,不再重述。
实施例二:猪沙门氏菌耐热毒素的检测试纸条,与实施例一基本相同,不同之处在于:
金标抗体纤维层3用吸附有金颗粒标记的抗沙门氏菌耐热毒素的多克隆抗体的玻璃棉制成,根据上述具体实施方式3中所述的制备方法制备其金标多克隆抗体玻璃棉;在硝酸纤维素膜层4上,6为用抗沙门氏菌耐热毒素的单抗溶液印制的检测印迹T,7为用羊或兔抗猪的IgG溶液印制对照印迹C,两种印迹带排列形成的组合形式为“||”、“//”、“\\”中的任一种。其它包括检测样品制备、操作方法和结果判定等均与具体实施方式6中的操作方法相同,不再重述。
Claims (7)
1.一种检测猪沙门氏菌耐热毒素的检测试纸条,该试纸条含有支撑层和吸附层,支撑层为不吸水的薄片层,吸附层附着在支撑层上,吸附层从测试端依次为样品吸附纤维层、金标抗体纤维层、纤维素膜层和手柄端的吸水材料层,在纤维素膜层上制备有检测印迹和对照印迹,其特征是金标抗体纤维层吸附有纳米级金颗粒标记的抗猪沙门氏菌耐热毒素单克隆抗体,检测印迹用抗猪沙门氏菌耐热毒素的配对单抗或多克隆抗体印制,对照印迹用羊或兔抗小鼠IgG的多克隆抗体或金黄色葡萄球菌A蛋白印制;或金标抗体纤维层吸附有纳米级金颗粒标记的抗猪沙门氏菌耐热毒素的多克隆抗体,检测印迹用抗猪沙门氏菌耐热毒素的单抗制备,对照印迹用金黄色葡萄球菌A蛋白或抗猪IgG多抗制备;
其中,抗猪沙门氏菌耐热毒素单克隆抗体的制备方法如下:
用50μg~100μg沙门氏菌耐热毒素抗原免疫Balb/c系小鼠三次,每次间隔15~30d;第三次加强免疫后3~4d,将免疫小鼠眼球放血,拉颈致死,用75%酒精浸泡5~10min,无菌条件下取其脾脏,剪碎并经100目尼龙网过滤,1000r/min离心10min,收集脾细胞;将1×108个脾细胞与2~5×107个NS0骨髓瘤细胞混合,1000r/min离心10min弃上清,将含有沉淀细胞的离心管置于37℃的水中,并缓缓加入0.7~1ml40%~50%PEG4000作用1min,所述PEG4000pH为8.5~9.0,然后缓慢加入无血清1640培养基15ml,以终止PEG的作用,37℃水浴5~10min,1000r/min离心10min弃上清,将细胞沉淀重悬于HAT选择培养基中,并加入96孔培养板,100μl~200μl/孔,置于37℃5%CO2培养箱中培养;培养7~10d后,以5μg~10μg/ml的纯化的病原体特异抗原包被96孔酶标板,以酶联免疫吸附试验检测杂交瘤的培养上清,挑取OD450≥0.5的强阳性细胞克隆,进行连续三次的有限稀释法克隆化,获得阳性杂交瘤细胞株,其染色体数为92~98,其分泌的单克隆抗体能够特异识别沙门氏菌耐热毒素,而不与其它毒素发生交叉反应,亲和力常数达109~10,轻链亚型为к或λ,重链亚型为IgG1、IgG2a、IgG2b或IgG3。
2.根据权利要求1所述的试纸条,其特征是检测印迹用抗猪沙门氏菌耐热毒素的配对单抗制备即用抗猪沙门氏菌耐热毒素的配对单抗溶液制备。
3.根据权利要求1所述的试纸条,其特征是支撑层用不吸水的硬质塑胶片条或硬纸条制成;测试端样品吸附纤维层用玻璃棉制成;金标抗体纤维层用玻璃棉和金标抗体制成,金标抗体是单抗或多克隆抗体。
4.根据权利要求1所述的试纸条,其特征是纤维素膜层用硝酸纤维素膜、或纯纤维素膜、或羧化纤维素膜制成。
5.根据权利要求1所述的试纸条,其特征是吸水材料层用吸水纸制成。
6.根据权利要求1所述的试纸条,其特征是检测印迹和对照印迹为直线式、或斜线式,纤维素膜层上含有一条检测印迹和一条对照印迹,检测印迹和对照印迹的排列形式为“||”、“//”、“\\”中的任一种。
7.根据权利要求1所述的试纸条,其特征是在测试端样品吸附纤维层、金标抗体纤维层及吸水材料层上覆盖有保护膜,在测试端样品吸附纤维层与金标抗体纤维层交界处对应的保护膜上印制有样品标记线,该标记线偏向测试端样品吸附纤维层一侧约0.5cm处。
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