CN103088111A - 一种用于检测霍乱弧菌的F0F1-ATPase旋转分子马达传感器试剂盒 - Google Patents

一种用于检测霍乱弧菌的F0F1-ATPase旋转分子马达传感器试剂盒 Download PDF

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CN103088111A
CN103088111A CN2011103360397A CN201110336039A CN103088111A CN 103088111 A CN103088111 A CN 103088111A CN 2011103360397 A CN2011103360397 A CN 2011103360397A CN 201110336039 A CN201110336039 A CN 201110336039A CN 103088111 A CN103088111 A CN 103088111A
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张捷
张惠媛
张昕
卢晓宇
汪琦
陆琳
张雷
刘岩
顾德周
陈广全
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Beijing Entry Exit Inspection and Quarantine Bureau of Peoples Republic of China
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Abstract

一种用于检测霍乱弧菌的F0F1-ATPase旋转分子马达传感器试剂盒属于食品微生物快速检测领域，主要解决传统检测方法时间过长(约5天)。本发明的核心技术是利用载色体Chromatophore上的F0F1-ATPase分子马达生物传感器，F0F1-ATP合酶由于能快速地旋转，通过旋转它建立了催化位点与质子转运之间的偶联。首先在ATP合酶的ε亚基上连接ompW探针，将待测样品和阴性对照分别与生物传感器结合后，比较其催化ATP合成20分钟后的ATP产生量，ATP合成的多寡可以通过环境中H+的量进行测量，而H+的多寡通过H-DHPE所体现的荧光强度大小来获得。本试剂盒可对待测样本中的霍乱弧菌进行快速、灵敏、高通量的检测。

Description

一种用于检测霍乱弧菌的F0F1-ATPase旋转分子马达传感器试剂盒
技术领域
本发明是关于食品微生物快速检测技术的一种。
背景技术
传统的霍乱弧菌检测方法要求对每个检验项目进行非选择性增菌、选择性增菌、分离、筛选和鉴定等步骤，一般需要4天才能出具检测报告，严重影响货物的品质和货架寿命。一些新的技术如PCR技术、免疫学检测技术、生物芯片技术等检测方法仍依赖于传统的检测步骤，即需要进行增菌，耗时耗力，完成一次检测通常需要几天的时间。这样不仅增加了实验室的工作量，而且也不利于食品工业中生产流程的监测、终产品的质量控制以及政府部门对食品安全的管理和控制。
发明内容
以受控分子马达技术为核心，集成核酸探针识别、荧光探针标记与检测等技术，建立了一个全新概念的快速检测技术体系。利用ATP酶作为载体对目标物进行检测具有灵敏度高、特异性强、快速的特点。在F0F1-ATPase分子马达连接上特异的核酸探针，可以实现对特定食品微生物的快速、特异性、高通量的检测。本发明可将检测时间缩短至2天，且检测灵敏度能达到102CFU/ml，满足现有的食品微生物检测范围。
附图说明
附图1为分子马达生物传感器模式图(其中a、b、c、α、β、δ、γ、ε均为ATP合酶亚基)，1为ε亚基抗体，2为链霉亲和素(Strptavidin)，3为N-biotin，4为ompW探针，5为霍乱弧菌单链DNA。
附图2为chro ompW对不同种菌株的检测结果，纵坐标表示荧光值，横坐标表示检测的样本，其中1为水，2为霍乱弧菌，3为副溶血性弧菌，4为沙门氏菌，5为单增李斯特氏菌，6为大肠杆菌。
附图3为chro ompW对30株霍乱弧菌样本的检测结果，纵坐标表示荧光值，横坐标表示检测的样本，1～30为食品检测样本编号。
具体实施方式
根据霍乱弧菌ompW基因设计特异性核酸探针5’-caccaagaaggtgactttattgtg，其中探针的5’用生物素进行标记。将该探针通过生物素抗体连接在分子马达上面，利用分子马达传感器即可对待测样本的DNA进行检测，当样品为霍乱弧菌DNA时，检测体系的荧光值会发生明显的变化，通过这一变化即可判断样本为阳性。为此，我们设计了一个试剂盒，可以方便、快速对食品中的霍乱弧菌进行检测。
试剂盒组成：
  编号   组分名称   数量   保存条件
  1   chro ompW   20μl   -20℃
  2   合成buffer   10ml   室温
  3   ADP(1.6mol/l)   1ml   -20℃
  4   1×PBS   25ml   室温
  5   荧光素酶/荧光素   100个单位   -20℃
  6   荧光素酶/荧光素重组缓冲液   12ml   -20℃
  7   无菌水   5ml   室温
操作步骤如下：
1.取1.5ml EP管，加入待测样本10μl。
2.将上述EP管放入沸水中3min，然后立即转移到冰上至完全冷却。
3.取2μl chro ompW，用合成buffer稀释到一定的倍数。取稀释后的chro 10μl加入上述EP管。
4.另取1个EP管加入10μl无菌水，沸水中3min，然后立即转移到冰上至完全冷却，再加入10μl合成buffer作为本底对照，短暂振荡使反应体系混匀。
5.再向2个EP管中分别加入30μl启动buffer(启动buffer由ADP(2.2mol/l)和合成buffer按体积比1∶6配制，每次实验按需要取一定的量配制，现用现配)，振荡使反应体系混匀，然后立即短暂离心以除去管盖内壁上的水珠。
6.将上述反应体系放入37℃恒温摇床中温育20min。
7.将EP管从摇床中取出，分别加入450μl PBS缓冲液，振荡使体系混匀。
8.取一块干净的96孔板，将2管中的最终反应体系加入其中，每个体系加3孔，每孔加样50μl，然后对每个加样孔分别加入30μl已配置好的荧光素酶溶液(将荧光素酶/荧光素重组缓冲液加入装有荧光素酶/荧光素的棕色玻璃瓶中，盖上瓶塞，反复颠倒几次混匀，不可振荡。使用前应将混合液在室温放置1h)，用枪反复吹打几次使体系混匀。
9.将96孔板上机检测，处理数据，对各组数据取平均值，然后用样本数值减去本底数值即为样本的实际荧光值。
通过实验，分子马达生物传感器chro ompW具有良好的特异性，用chro ompW对水、霍乱弧菌、副溶血性弧菌、沙门氏菌、单增李斯特氏菌、大肠杆菌进行检测，霍乱弧菌的荧光值明显低于水和其他对照菌的荧光值，具体数据见下表，结果见附图2。
  样品(10ng/mL)   荧光值
  水   639037
  霍乱弧菌   600750
  副溶血性弧菌   644647
  沙门氏菌   650209
  单增李斯特氏菌   660825
  大肠杆菌   661486
用chro ompW对30株食品样本进行检测，均能检出，具体数据见下表，结果见附图3。
  样本编号   荧光值   样本编号   荧光值
  水   639037   16   378975
  1   401080   17   390871
  2   437383   18   418190
  3   396535   19   437624
  4   394478   20   431979
  5   404590   21   489020
  6   402205   22   483579
  7   442514   23   512370
  8   462857   24   502238
  9   457680   25   501172
  10   451325   26   465526
  11   399178   27   466292
  12   386717   28   456464
  13   401744   29   488729
  14   395816   30   492164
  15   385561   ——   ——
实验结果表明，该试剂盒对霍乱弧菌DNA的检测时间为1h，检出限为10ng/ml。对30株食品检测样本菌株的检出结果与PCR检测的结果一致。通过大量的实验验证该分子马达生物传感器具有良好的特异性和较高的灵敏度，并可通过96孔化学发光板高通量的对样本进行检测。

Claims (3)

1.特异性核酸探针与F0F1-ATP合酶连接的方式：在F0F1-ATP合酶的ε亚基上依次连接ε亚基抗体、生物素、链霉亲和素、生物素标记的ompW探针。
2.试剂盒检测体系的反应条件：37℃恒温摇床中温育20min。
3.合成buffer、启动buffer、PBS的配方。合成buffer：甘油20％，氯化镁5mM，Tricine 50mM，磷酸氢二钾5mM；启动buffer：ADP(2.2mM)∶上述合成buffer＝1∶6(v/v)；PBS：137mM氯化钠，2.7mM氯化钾，10mM磷酸氢二钠，2mM磷酸二氢钾。 
CN201110336039.7A 2011-10-31 2011-10-31 一种用于检测霍乱弧菌的F0F1-ATPase旋转分子马达传感器试剂盒 Expired - Fee Related CN103088111B (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108018365A (zh) * 2016-11-01 2018-05-11 北京出入境检验检疫局检验检疫技术中心 一种绝对定量检测总霍乱弧菌与致病性霍乱弧菌的试剂盒及检测方法

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