CN111735954A - 梨火疫病菌快速免疫检测试纸条及其应用 - Google Patents

梨火疫病菌快速免疫检测试纸条及其应用 Download PDF

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CN111735954A
CN111735954A CN202010577165.0A CN202010577165A CN111735954A CN 111735954 A CN111735954 A CN 111735954A CN 202010577165 A CN202010577165 A CN 202010577165A CN 111735954 A CN111735954 A CN 111735954A
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王利民
田艳丽
王岩
李潇楠
姜培
郑秋菊
胡白石
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Abstract

本发明公开了一种梨火疫病菌快速免疫检测试纸条及其应用。其包括底板，底板的一面从上到下依次黏贴吸水垫、检测垫、金标垫和样品垫，相邻各垫在连接处交叠连接，检测垫以硝酸纤维素膜为基垫，硝酸纤维素膜上自上而下横向设置质控线和检测线，检测线位于质控线下方，检测线上包被有抗梨火疫病菌多克隆抗体0550，质控线上包被有羊抗小鼠IgG；所述金标垫上喷涂有纳米金标记的抗梨火疫病菌单克隆抗体。其特异性强，对亚洲梨火疫病菌、梨锈水、梨腐烂、梨炭疽等无交叉反应；试纸条灵敏度可达1×105cfu/mL，可用于田间梨火疫病菌的快速筛查。

Description

梨火疫病菌快速免疫检测试纸条及其应用
技术领域
本发明属生物检测领域，具体涉及一种梨火疫病菌快速免疫检测试纸条及其应用。
背景技术
由梨火疫病菌(解淀粉欧文氏菌，Erwinia amylovora)引起的火疫病(Fireblight)是梨和苹果等仁果类果树上的毁灭性细菌病害。该病害最早发生在美国，然后通过各种途径传播和扩散，迄今，病害已经扩散到中欧、东南欧、地中海及澳大利亚等40余个国家。根据有关专家风险评估结果，我国大部分梨和苹果产区为火疫病菌的适生区，且我国种植的本地及引进的梨和苹果品种中，大部分为高度感病品种。因此，火疫病菌一旦传入，除了直接危害造成的损失外，由于其检疫重要性，还会失去大多数的国际市场，我国的梨和苹果生产因此将会受到巨大打击。为此，我国政府十分关注该病害，将其列入禁止进境的检疫性有害生物。
除梨火疫病外，在邻国韩国和日本局部地区沙梨系列的梨树上分别发生了与梨火疫病极为相似的亚洲梨火疫病(Erwinia pyrifoliae)和梨细菌性枝枯病(Erwiniaamylovora pv.pyri)(最新研究表明两者病原菌均为亚洲梨火疫病菌)，自然情况下，病菌主要为害梨树，特别对沙梨系统的梨品种威胁很大。鉴于亚洲梨火疫病菌对我国梨产业的潜在威胁性，2007年农业部和国家质量监督检验检疫总局也将其列为禁止进境的检疫性有害生物。
对梨火疫病菌的准确、快速检测是对梨火疫病执行检疫措施的重要前提。目前，对该病原菌的检测主要是在基因分子水平对病原菌进行检测，包括PCR等。基因分子水平检测技术具有准确、灵敏的优势，但需要较为严格的实验条件、措施及操作，不能完全满足现存快速检验检疫的需求。因此，建立对梨火疫病菌现场快速、准确的轻简型检测方法对严格执行检疫措施具有极大的现实意义。
胶体金免疫层析试纸条由于其快速便捷、操作简单、成本低、性能稳定等特点而备受关注。以胶体金颗粒为标记物，利用抗原-抗体的特异性反应，通过裸眼可见的颜色反应来检测目标分析物。因此，胶体金免疫层析试纸条对病原物的现场快速检测具有重要意义。
在该发明中，制备出能特异性识别梨火疫病菌的的单克隆抗体，与高效识别梨火疫病菌的多克隆抗体配对制备出梨火疫病菌的胶体金免疫层析试纸条。该试纸条具有特异性强、灵敏度高、操作简便、反应快速等特点，对梨火疫病菌的检测限为1×105CFU/mL，与亚洲梨火疫病菌无交叉反应，10分钟内可完成检测。
发明内容
本发明所要解决的问题是提供一种梨火疫病菌快速免疫检测试纸条及其应用。其可用于梨、苹果等作物上梨火疫病菌的园间快速筛查。
本发明通过以下技术方案来实现：
1、梨火疫病菌快速免疫检测试纸条，其特征在于：包括底板，底板的一面从上到下依次黏贴吸水垫、检测垫、金标垫和样品垫，相邻各垫在连接处交叠连接，所述检测垫以硝酸纤维素膜为基垫，硝酸纤维素膜上自上而下横向设置质控线和检测线，所述检测线位于质控线下方，检测线上包被有抗梨火疫病菌多克隆抗体0550，所述质控线上包被有羊抗小鼠IgG；所述金标垫上喷涂有纳米金标记的抗梨火疫病菌单克隆抗体；所述的抗梨火疫病菌单克隆抗体由杂交瘤细胞株EA2019分泌产生。
2、所述的梨火疫病菌快速免疫检测试纸条，其特征在于：所述的吸水垫长18-20mm，宽4mm；检测垫长25mm，宽4mm；金标垫长5mm，宽4mm；样品垫长20-22mm，宽4mm，相邻各垫的交叠长度为3-5mm；所述的吸水垫为吸水板；所述的底板为纸板。
3、所述的梨火疫病菌快速免疫检测试纸条，其特征在于：所述检测垫上质控线于检测线的间距为5-6mm，检测线与硝酸纤维素膜上沿的间距为15-16mm。
4、所述的梨火疫病菌快速免疫检测试纸条，其特征在于：所述金标垫中所用的纳米金粒径为20-40nm；所述金标垫上每厘米喷涂长度所需的纳米金标记的抗梨火疫病菌单克隆抗体的用量为100-200ng；检测垫检测线上每厘米喷涂长度所需的抗梨火疫病菌多克隆抗体的用量为50-100ng。
5、所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：应用方法如下：剪取适量样品于研磨袋中，将单面研磨袋的磨砂面沿中间对折，样品置于对折曲面中充分研磨，加入样品缓冲液2mL，混匀得到待测样品溶液；塑料滴管吸取待测液，滴入检测条加样端，平放，反应5-10分钟，根据检测线和质控线颜色判定结果。
6、所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：所述的适量样品，是指叶片或果肉。当样品为叶片时，在病健交接处剪取约10mm*10mm大小的样品；当样品为果肉时，可在病部取约1立方厘米大小的果肉。
7、所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：所述的待测样品溶液的用量为100-200μL，检测时间为5-10分钟。
8、所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：试纸条检测特异性强，对亚洲梨火疫病菌、梨锈水、梨腐烂、梨炭疽等无交叉反应；试纸条灵敏度可达1×105cfu/mL。
有益效果
(1)本发明中提供的梨火疫病菌快速免疫检测试纸条，其廉价易得，极大的降低了田间细菌性果斑病的快速筛查成本；同时该试纸条检测时间短，5-10分钟可完成检测，大大提高了梨火疫病菌的快速筛查效率；其使用的单克隆抗体EA2019的亚型为IgG2b，轻链均为κ链，大大提高了产品批次间的稳定性，降低了生产成本。
(2)本发明中提供的梨火疫病菌快速免疫检测试纸条，灵敏度高，试纸条对梨火疫病菌检测限为1×105cfu/mL；稳定性好，试纸条在室温下可保存18个月；田间检测对采样量要求低，单次检测只需10mm*10mm大小的叶片，降低了田间采样量。
(3)本发明中提供的梨火疫病菌快速免疫检测试纸条，特异性强，对亚洲梨火疫病菌、梨锈水病菌、梨腐烂病菌、梨炭疽病菌等无交叉反应。
附图说明
图1为本发明的梨火疫病菌快速免疫检测试纸条灵敏度测试，图中自右往左依次为：108，107，106，105，104，0cfu/mL。
具体实施方式：
实施例1：梨火疫病菌快速免疫试纸条灵敏度测试
取梨火疫病菌，在LB平板培养基上划线后，置于28℃恒温培养箱培养。待培养1-3天长出菌落后，挑取单菌落接种于NA培养基中，置于摇床(28℃，200rpm)培养。待菌体生长至OD为0.3时，取培养好的菌液4℃、6000rpm离心5min，用试纸条层析液离心洗涤两次后悬起，配制成108cfu/mL的菌悬液，待用。
将上述待测液梯度稀释为：108、107、106、105、104CFU/mL的待测梯度菌液，取150μl待测梯度菌液滴入梨火疫病菌快速免疫检测试纸条的样品垫，待层析5-10分钟后，根据检测线颜色有无判断梨火疫病菌快速免疫检测试纸条的灵敏度。

Claims (8)

1.梨火疫病菌快速免疫检测试纸条，其特征在于：包括底板，底板的一面从上到下依次黏贴吸水垫、检测垫、金标垫和样品垫，相邻各垫在连接处交叠连接，所述检测垫以硝酸纤维素膜为基垫，硝酸纤维素膜上自上而下横向设置质控线和检测线，所述检测线位于质控线下方，检测线上包被有抗梨火疫病菌多克隆抗体0550，所述质控线上包被有羊抗小鼠IgG；所述金标垫上喷涂有纳米金标记的抗梨火疫病菌单克隆抗体；所述的抗梨火疫病菌单克隆抗体由杂交瘤细胞株EA2019分泌产生。
2.根据权力要求1所述的梨火疫病菌快速免疫检测试纸条，其特征在于：所述的吸水垫长18-20mm，宽4mm；检测垫长25mm，宽4mm；金标垫长5mm，宽4mm；样品垫长20-22mm，宽4mm，相邻各垫的交叠长度为3-5mm；所述的吸水垫为吸水板；所述的底板为纸板。
3.根据权力要求1所述的梨火疫病菌快速免疫检测试纸条，其特征在于：所述检测垫上质控线于检测线的间距为5-6mm，检测线与硝酸纤维素膜上沿的间距为15-16mm。
4.根据权力要求1所述的梨火疫病菌快速免疫检测试纸条，其特征在于：所述金标垫中所用的纳米金粒径为20-40nm；所述金标垫上每厘米喷涂长度所需的纳米金标记的抗梨火疫病菌单克隆抗体的用量为100-200ng；检测垫检测线上每厘米喷涂长度所需的抗梨火疫病菌多克隆抗体的用量为50-100ng。
5.权力要求1所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：应用方法如下：剪取适量样品于研磨袋中，将单面研磨袋的磨砂面沿中间对折，样品置于对折曲面中充分研磨，加入样品缓冲液2mL，混匀得到待测样品溶液；塑料滴管吸取待测液，滴入检测条加样端，平放，反应5-10分钟，根据检测线和质控线颜色判定结果。
6.权力要求5所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：所述的适量样品，是指叶片或果肉。当样品为叶片时，在病健交接处剪取约10mm*10mm大小的样品；当样品为果肉时，可在病部取约1立方厘米大小的果肉。
7.权力要求6所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：所述的待测样品溶液的用量为100-200μL，检测时间为5-10分钟。
8.权力要求6所述的梨火疫病菌快速免疫检测试纸条的应用，其特征在于：试纸条检测特异性强，对亚洲梨火疫病菌、梨锈水、梨腐烂、梨炭疽等无交叉反应；试纸条灵敏度可达1×106cfu/mL。
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