Source: https://patents.google.com/patent/CN106290842A/en
Timestamp: 2019-04-21 08:36:45+00:00

Document:
The invention relates to the field of biological detection, especially to a colloidal-gold detection kit for Vibrio cholera O139, and a preparation method and application thereof. The colloidal-gold detection kit for Vibrio cholera O139 comprises a test strip; the test strip comprises a base plate and further comprises a sample pad, a colloidal-gold pad, a cellulose nitrate membrane and a water-absorbing pad which are located on the surface of the base plate and successively arranged from a sample introduction end; the colloidal-gold pad comprises a monoclonal antibody against Vibrio cholera O139; the cellulose nitrate membrane is coated with a detection line and a control line; and the monoclonal antibody against Vibrio cholera O139 on the colloidal-gold pad is labeled with colloidal gold. The colloidal-gold detection kit for Vibrio cholera O139 provided by the invention detects Vibrio cholera O139 by using colloidal gold immunochromatography techniques for the first, has both sensitivity and specificity and has the advantages of rapid and simple operation, accurate results, economic performance, etc.
 本发明涉及生物检测领域，特别是涉及一种霍乱弧菌0139胶体金检测试剂盒及其制备方法和用途。  The present invention relates to the field of biological detection, particularly to a V. cholerae 0139 colloidal gold detection kit and its preparation method and uses.
 霍乱弧菌（Vibrio cholerae)是霍乱的病原体，根据其0抗原的不同可分为200多个血清群，。  V. cholera (Vibrio cholerae) is the causative agent of cholera, according to their 0 antigen can be divided into a plurality of serogroups 200,. 在霍乱弧菌的200多个血清群中，只有01群和0139群霍乱弧菌能引起霍乱。 In more than 200 serogroups of V. cholerae, only 01 groups and 0139 Vibrio cholerae can cause cholera. 霍乱是一种古老且流行广泛的烈性传染病之一。 One highly infectious diseases as cholera is an ancient and widespread. 人类是霍乱弧菌的唯一易感者。 Man is the only susceptible Vibrio cholerae. 霍乱弧菌进入消化道到达小肠，在肠粘膜表面吸附并迅速繁殖。 Vibrio cholerae into the digestive tract to reach the small intestine, and multiply rapidly adsorbed on the surface of the intestinal mucosa. 产生的霍乱肠毒素作用于小肠粘膜， 引起小肠液过度的分泌。 Cholera toxin acts on the small intestine, small intestine caused by excessive secretion. 曾在世界上引起多次大流行，主要表现为上吐下泻，导致脱水和代谢性酸中毒、循环衰竭，死亡率甚高。 Has caused a pandemic in the world several times, mainly for diarrhea and vomiting, leading to dehydration and metabolic acidosis, circulatory failure, high mortality rate. 属于国际检疫传染病。 Is an international quarantine infectious diseases.
 霍乱是我国法定的甲类传染病，国家要求在接诊24h内报出检验结果。  Cholera is our statutory Class infectious diseases, countries require test results reported in the admissions 24h. 因此，及早鉴定出霍乱弧菌对控制该病的流行和治疗有极其重要的意义。 Therefore, early identification of Vibrio cholerae has extremely important implications for the treatment and control of epidemic disease. 目前霍乱弧菌的快速诊断方法如快速制动试验和免疫荧光菌球试验等效果不理想，主要因为患者粪便中的霍乱弧菌时多时少，最少时检查结果呈阴性。 Rapid diagnostic method currently Vibrio cholerae as fast brake test and immunofluorescence test and other bacteria ball is not satisfactory, mainly due to uneven when Vibrio cholerae in the stools of patients, at least when the test results were negative.
 鉴于以上所述现有技术的缺点，本发明的目的在于提供一种霍乱弧菌0139胶体金检测试剂盒及其制备方法和用途，用于解决现有技术中的问题。  In view of the foregoing disadvantages of the prior art, an object of the present invention is to provide a V. cholerae 0139 colloidal gold detection kit and its preparation method and use, for solving the problems of the prior art.
 本发明的第一方面，提供一种霍乱弧菌0139胶体金检测试剂盒，包括试纸卡，所述试纸卡包括底板、及位于底板表面的从加样端开始依次排列的样品垫、金标垫、硝酸纤维素膜和吸水垫，所述金标垫上包含抗霍乱弧菌0139型单克隆抗体，所述硝酸纤维素膜上包被有检测线和质控线，所述金标垫上的抗霍乱弧菌0139型单克隆抗体采用胶体金标记。  The first aspect of the present invention, there is provided a V. cholerae 0139 colloidal gold detection kits, including test strip card, said card comprising a base paper, and starting from the bottom plate surface sequentially arranged loading end of the sample pad, gold labeling pad, a nitrocellulose membrane, and an absorbent pad, said pad comprising a gold-labeled anti-Vibrio cholerae 0139 monoclonal antibody, the nitrocellulose membrane coated with test and control lines, the gold-labeled pad Vibrio cholerae 0139 anti-monoclonal antibody using colloidal gold labeled.
 优选的，所述金标垫上的抗霍乱弧菌0139型单克隆抗体采用150nm胶体金标记， 具有信号受背景干扰小，检测灵敏度高，结果重复性好的优点。  Preferably, the gold-labeled anti-pad 0139 Vibrio cholerae monoclonal antibody colloidal gold labeled using 150nm, has the advantage of small signal receiving background interference, high sensitivity, good repeatability.
 优选的，所述底板为PVC底板。  Preferably, the bottom plate is PVC.
 优选的，所述硝酸纤维素膜上，检测线位于离加样端较近一侧，质控线位于离加样端较远一侧。  Preferably, the cellulose nitrate film, the detection line is located close to the side away from the loading end, the control line is located farther from the loading side end.
 优选的，所述检测线上包被有抗霍乱弧菌0139型单克隆抗体。  Preferably, the detection line 0139 is coated with monoclonal antibodies against Vibrio cholerae. 所述金标垫上的抗霍乱弧菌0139型单克隆抗体与检测线上的抗霍乱弧菌0139型单克隆抗体可以是相同的抗体，也可以是不同的抗体。 0139 monoclonal antibodies against Vibrio Vibrio cholerae monoclonal antibody 0139 with the gold-labeled test line pad against cholera antibody may be the same or may be different antibodies.
 优选的，质控线上包被羊抗鼠抗体。  Preferably, the quality control line coated with goat anti-mouse antibody.
 优选的，所述样品垫采用缓冲液处理，所述缓冲液选自PBS缓冲液、TriS-HCl缓冲液、甘氨酸缓冲液、硼酸盐缓冲液和柠檬酸-磷酸盐缓冲液中的一种或多种的组合，缓冲液的浓度为50-100mM。  Preferably, the buffer treated sample pads using the selected buffer PBS buffer, TriS-HCl buffer, glycine buffer, borate buffer and citrate - phosphate buffer a combination of one or more of, the buffer concentration is 50-100mM.
 优选的，所述缓冲液还包括反应增强剂，所述反应增强剂选自PEG4000、PEG6000、 PEG8000和PEG20000中的任意一种，所述反应增强剂的浓度为10~50g/L。  Preferably, the buffer solution further comprises a reaction enhancer, said enhancer is selected from any one reaction PEG4000, PEG6000, PEG8000 and PEG20000, said enhancer reaction concentration of 10 ~ 50g / L.
 优选的，所述缓冲溶液还包括表面活性剂，所述表面活性剂选自S-19TWEEN 20、 S-20TWEEN 80、S-13TRIT0N X-45、S-14TRIT0N X-100、S-15TRIT0N X305 中的任意一种或多种的组合，所述表面活性剂的浓度为20~40g/L。  Preferably, the buffer solution further comprises a surfactant, the surfactant is selected from S-19TWEEN 20, S-20TWEEN 80, S-13TRIT0N X-45, S-14TRIT0N X-100, S-15TRIT0N X305 in any one or more thereof, the concentration of the surfactant is 20 ~ 40g / L.
 优选的，为了使得试剂盒具有更佳的灵敏度和特异性，本发明所述的金标垫在预处理时所使用的预处理缓冲液包括下列组分：果糖、氢氧化铝、葡萄糖、氯化钠和甘氨酸， 且果糖、氢氧化铝、葡萄糖、氯化钾和甘氨酸的总浓度为4. 5 - 6. 5g/L，缓冲液的pH值为7. 3 - 7. 5。  Preferably, in order that the kit has a better sensitivity and specificity, the gold conjugate pad of the present invention is used in pretreatment pretreatment buffer comprises the following components: fructose, aluminum hydroxide, glucose , glycine and sodium chloride, and the total concentration of fructose, aluminum hydroxide, glucose, potassium chloride and glycine to 4. 5 - 6. 5g / L, pH buffer is 7.3 - 7.5.
 果糖I. 0-2. Og/L ;氢氧化错0• 25-0. 75g/L ;葡萄糖1.0 -L 5g/L ;氯化钠0• 25-0. 5g/L ;甘氨酸2. 0-2. 25g/L。 .  fructose I. 0-2 Og / L; wrong hydroxide 0 • 25-0 75g / L;. Glucose 1.0 -L 5g / L;. NaCl 0 • 25-0 5g / L; Glycine 2 . 0-2. 25g / L.
 所述预处理缓冲液的溶剂为水。  The pretreatment buffer in the solvent is water.
 所述预处理的具体步骤为：将金标垫在预处理液中浸泡1. 5~2h，取出放于36~ 38°C烘干。  In particular for the pretreatment step: The gold labeling pad soaked in the pretreatment liquid 1. 5 ~ 2h, put out at 36 ~ 38 ° C drying.
 所述预处理缓冲液可使用本领域各种常用的pH调节剂进行pH值的调节。  The pretreatment buffer in the art may be used various conventional pH adjusting agent to adjust the pH.
 优选的，所述试剂盒还包括卡壳，所述卡壳包括背卡和上盖，所述背卡设有试纸卡卡槽，所述试纸卡嵌于所述试纸卡卡槽内，所述上盖设有测试窗和加样孔，所述测试窗的位置与所述检测线和质控线的位置相配合，所述加样孔的位置与所述样品垫的位置相配合。  Preferably, the kit further includes a card housing including a card housing and the upper cover backing card, the card is provided with a back strip card slot, the card is embedded in the paper test strip card slot, the said cover is provided with a window and processing the test wells, and the position detecting lines and the control line of the test window cooperates, the loading position and the position of the hole cooperates sample pad.
 更优选的，所述卡壳为塑料卡壳。  More preferably, the card housing is a plastic insert.
 优选的，所述检测试剂盒用于检测霍乱弧菌0139。  Preferably, the test kit for detection of Vibrio cholerae 0139.
 本发明所提供的霍乱弧菌0139胶体金检测试剂盒，可配套免疫定量分析仪器使用。  V. cholerae 0139 colloidal gold detection kit of the present invention is provided, supporting the immunoassay quantitative analysis instrument. 免疫分析仪器通过采集检测线（T)和质控线（C)条带荧光信号，计算T/C信号值。 Article immunoassay instruments with the detection by collecting the fluorescence signal line (T) and control line (C), calculated T / C signal value. 使用前先将不同标准品滴加到试纸卡上，分析处理建立定标曲线（T/C信号值与标准品真实值的关系），再将检测样品时获得的T/C值与标准曲线比较，即可获得检测样品中的霍乱弧菌0139抗原含量。 Before use different standards dropwise added first strip card, establishing analysis process (the relationship between T / C signal value and the true value of the standard) calibration curve, T / C values ​​obtained when the standard curve and then comparison test sample , 0139 can be obtained Vibrio cholerae antigen content in the test sample.
 3)将样品垫、步骤1)制备金标垫、步骤2)制备的硝酸纤维素膜、吸水垫依次粘贴在底板上，切裁制得检测试纸卡；最后将检测试纸卡装入卡壳制得检测试剂盒。  3) The sample pad, Step 1) Preparation of gold labeling pad, Step 2) Preparation of a nitrocellulose membrane, and an absorbent pad sequentially attached on the base plate is cut out to prepare test strip card; detection test card into the final jam detection kits prepared.
 果糖1. 0-2. Og/L ;氢氧化错0• 25-0. 75g/L ;葡萄糖1.0 -L 5g/L ;氯化钠0• 25-0. 5g/L ;甘氨酸2. 0-2. 25g/L。  Fructose 1. 0-2 Og / L; wrong hydroxide 0 • 25-0 75g / L;. Glucose 1.0 -L 5g / L;. NaCl 0 • 25-0 5g / L; Glycine 2 . 0-2. 25g / L.
 本发明第三方面提供所述霍乱弧菌0139胶体金检测试剂盒在霍乱弧菌0139检测领域的用途。  In a third aspect the present invention provides the use of V. cholerae 0139 colloidal gold detection kits in the field of detection of Vibrio cholerae 0139.
 本发明所提供的霍乱弧菌0139胶体金检测试剂盒首次将霍乱弧菌0139通过胶体金免疫层析技术进行检测，兼具高灵敏性和高特异性，能够快速检测霍乱弧菌0139。  The present invention provides a V. cholerae 0139 colloidal gold assay kit for the first time detected by V. cholerae 0139 GICA technology, both high sensitivity and specificity, can quickly detect Vibrio cholerae 0139 . 此外， 所述检测试剂盒具有操作快速简便、结果准确、经济适用等优点。 Furthermore, the rapid detection kit having a simple, accurate, economical and other advantages.
 以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。  Hereinafter, an embodiment of the present invention by certain specific examples, those skilled in the art disclosed in this specification may readily understand the content of other advantages and effects of the present invention. 本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。 The present invention may also be implemented or applied through other different specific embodiments, the details of the specification may be carried out in various modified or changed without departing from the spirit of the invention based on various concepts and applications.
 在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式"一个"、"一"和"这个"包括复数形式。  Before further description of the specific embodiments of the invention, it should be understood that the scope of the present invention is not limited to the following particular specific embodiments; also to be understood that the terminology used in the embodiment of the present invention is for describing particular embodiment, not intended to limit the scope of the present invention; the present invention as claimed in the specification and claims, unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" include plural forms.
 当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。  When numerical ranges are given embodiments, it should be understood that the present invention unless otherwise indicated, between two end points and two end points of each range of values ​​of one of the numbers can be selected. 除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。 Unless defined otherwise, the same meaning in the present invention all technical and scientific terms used in this technical field is generally understood in the art. 除实施例中使用的具体方法、设备、 材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。 In addition to a specific method, apparatus, materials used in the embodiment, the control according to the present technical field and described in the prior art in the art of the present invention, the method may also be used with the embodiment of the present invention, equipment, materials any methods, devices and materials similar or equivalent to the prior art to implement the invention.
 3)将样品垫、步骤1)制备金标垫、步骤2)制备的硝酸纤维素膜、吸水垫依次粘贴在PVC底板上，切裁制得宽3-5mm的检测试纸卡；最后将检测试纸卡装入卡壳制得检测试剂盒。  3) The sample pad, a nitrocellulose membrane Step 1) Preparation of gold labeling pad, Step 2) Preparation of an absorbent pad attached to the PVC plate sequentially, is cut out to prepare a test strip card width 3-5mm; final the test strip card into the card housing made detection kit.
 对比例试纸卡的制备：采用25mM甘氨酸缓冲液预处理金标垫，其他试剂及实验方法均同实施例1。  Preparation of test paper card ratio of: using 25mM glycine buffer pretreatment gold labeling pad, other reagents and experimental methods were the same as in Example 1.
 1.取实施例1制备的本发明试剂盒和实施例2的对比试剂盒，将试剂盒放置在水平台面上。 The kit of the present invention and Comparative Example Kit  1. Take 1 Preparation Example 2 of the embodiment, the kit will be placed on a horizontal surface.
 2.待测样品的制备：按表1的四类实验对象，用专用取样棒随机从检测者粪便的不同部位取样，取样量以沾满专用取样棒前端的小圆环为准；准备一个样品处理管，竖直放于处理管支架上，并在样品管里加入0. 6ml蒸馏水；将专用取样棒上取好的样品插入样品管中的蒸馏水充分搅拌均匀作为待测样品。 Preparation  2. The test samples: four subjects in Table 1, with a special random sampling from different parts of the sample bar stool test subject, sample volume to rod distal end covered with a small specific sampling prevail ring; a sample preparation process tube, the process tube vertically placed holder, and distilled water was added 0. 6ml sample tube in; and taking a good sample into the tube of distilled water, stir on a dedicated test sample as the sample rod.
 由上表可知，本发明的检测试剂盒对大肠杆菌及痢疾杆菌均无交叉反应，特异性良好。  The above table shows that the detection kit of the present invention, Escherichia coli and Shigella no cross reaction good specificity.
 综上所述，本发明所提供的检测试剂盒具有良好的抗干扰性和特异性，有效克服了现有技术中的种种缺点而具高度产业利用价值。  In summary, the present invention provides a test kit having good resistance specificity and effectively overcome the drawbacks of the prior art and the use of highly industrial value.
 上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。  The above-described embodiments are only illustrative of the principles and effect of the present invention, the present invention is not intended to be limiting. 任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。 Any person skilled in this art can be made at without departing from the spirit and scope of the present invention, the above-described embodiments can be modified or changed. 因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。 Thus, one skilled in the art that whenever all having ordinary knowledge in the technical ideas and spirit of the present invention is disclosed without departing from the completed equivalent modified or altered, yet the claims shall be encompassed by the present invention.
1. 一种霍乱弧菌0139胶体金检测试剂盒，包括试纸卡，试纸卡包括底板、及位于底板表面的从加样端开始依次排列的样品垫、金标垫、硝酸纤维素膜和吸水垫，所述金标垫上包含抗霍乱弧菌0139型单克隆抗体，所述硝酸纤维素膜上包被有检测线和质控线，所述金标垫上的抗霍乱弧菌0139型单克隆抗体采用胶体金标记。 A V. cholerae 0139 colloidal gold detection kit, comprising a paper card, paper card includes a bottom plate, the bottom plate surface of the sample pad and starting from the loading end arranged sequentially, the gold conjugate pad, a nitrocellulose membrane, and an absorbent pad the pad comprises gold-labeled anti-Vibrio cholerae 0139 monoclonal antibody, the nitrocellulose membrane coated with test and control lines, the pad 0139 gold-labeled monoclonal antibody against V. cholerae using colloidal gold labeled.
2. 如权利要求1所述的胶体金检测试剂盒，其特征在于，所述硝酸纤维素膜上，检测线位于离加样端较近一侧，质控线位于离加样端较远一侧。 2. The colloidal gold assay kit according to claim 1, characterized in that, the nitrocellulose membrane, the detection line is located close to the side away from the loading end, the control line is located farther away from a side loading side.
3. 如权利要求1所述的胶体金检测试剂盒，其特征在于，所述检测线上包被有抗霍乱弧菌0139型单克隆抗体。 Colloidal gold assay kit according to claim 1, characterized in that the detection line 0139 is coated with monoclonal antibodies against Vibrio cholerae.
4. 如权利要求1所述的胶体金检测试剂盒，其特征在于，质控线上包被羊抗鼠抗体。 The colloidal gold assay kit as claimed in claim 1, wherein the line quality control package is goat anti-mouse antibody.
5. 如权利要求1所述的胶体金检测试剂盒，其特征在于，所述样品垫采用缓冲液处理， 所述缓冲液选自PBS缓冲液、Tris-HCl缓冲液、甘氨酸缓冲液、硼酸盐缓冲液和柠檬酸-磷酸盐缓冲液中的一种或多种的组合，缓冲液的浓度为50~lOOmM。 5. colloidal gold assay kit according to claim 1, wherein said buffer treated sample pads using the selected buffer PBS buffer, Tris-HCl buffer, glycine buffer, borate citric acid salt buffer, and - a combination of one or more of the phosphate buffer, the buffer concentration of 50 ~ lOOmM.
6. 如权利要求1所述的胶体金检测试剂盒，其特征在于，所述金标垫采用缓冲液处理， 所述缓冲液选自甘氨酸缓冲液、Tris-HCl缓冲液、硼酸盐缓冲液的一种或多种的组合，缓冲液的浓度为10~40mM。 As claimed colloidal gold assay kit of claim 1 Tris-HCl buffer, borate buffer, characterized in that, using the gold labeling pad buffer treated, the buffer is selected from glycine buffer, one or more thereof, the concentration of the buffer is 10 ~ 40mM.
7. 根据权利要求6所述的胶体金检测试剂盒，其特征在于，所述缓冲液中还包括反应增强剂，所述反应增强剂选自PEG4000、PEG6000、PEG8000和PEG20000中的任意一种，所述反应增强剂的浓度为20~40g/L。 The colloidal gold detection kit according to claim 6, wherein said buffer further comprises a reaction enhancer, said enhancer is selected from any reaction PEG4000, PEG6000, PEG8000 and PEG20000 of one, the reaction enhancer concentration is 20 ~ 40g / L.
8. 如权利要求1所述的胶体金检测试剂盒，其特征在于，还包括卡壳，所述卡壳包括背卡和上盖，所述背卡设有试纸卡卡槽，所述试纸卡嵌于所述试纸卡卡槽内，所述上盖设有测试窗和加样孔，所述测试窗的位置与所述检测线和质控线的位置相配合，所述加样孔的位置与所述样品垫的位置相配合。 8. colloidal gold assay kit according to claim 1, characterized in that, further comprising a card housing, the card housing includes a back cover and a card, the card is provided with a back strip card slot, the card is embedded in the paper the paper card slot, the cover is provided with a window and processing the test wells, and the position detecting lines and the control line of the test window cooperates, the loading position of the aperture said sample pad engaged position.
9. 如权利要求1所述的胶体金检测试剂盒，其特征在于，所述检测试剂盒用于检测霍乱弧菌0139。 9. The colloidal gold assay kit according to claim 1, characterized in that the test kit for the detection of Vibrio cholerae 0139.
10. 根据权利要求1~9任一权利要求所述的胶体金检测试剂盒的制备方法，具体包括如下步骤： 1) 用胶体金标记的抗霍乱弧菌0139型单克隆抗体溶液喷涂经预处理的金标垫，制得包含抗霍乱弧菌0139型单克隆抗体的金标垫； 2) 在硝酸纤维素膜的检测线和质控线上分别喷涂抗霍乱弧菌0139型单克隆抗体及羊抗鼠抗体，制得包被后的硝酸纤维素膜； 3) 将样品垫、步骤1)制备金标垫、步骤2)制备的硝酸纤维素膜、吸水垫依次粘贴在底板上，切裁制得检测试纸卡；最后将检测试纸卡装入卡壳制得检测试剂盒。 Preparation of colloidal gold 10. A test kit according to any one of claims 1 to 9, claim, comprises the following steps: 1) Vibrio monoclonal antibody solution was sprayed 0139 pretreated with a colloidal gold labeled anti-cholera the gold standard pad, made against cholera comprising V. 0139 of gold-labeled monoclonal antibody pad; 2) in the detection line nitrocellulose membrane was sprayed and quality control lines 0139 of Vibrio cholerae monoclonal antibodies anti goat and anti-mouse antibody, to prepare the package after being nitrocellulose; 3) the sample pad, step 1) preparation of gold labeling pad, step 2) preparation of a nitrocellulose membrane, and an absorbent pad sequentially attached on the base plate, cut tailoring to give test strip card; the last test strip card into the card housing made detection kit.
MXPA03006044A (en) 2005-02-14 Biosensors having improved sample application.

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