CN103088153A - 一种用于检测甲肝病毒的分子马达生物传感器试剂盒 - Google Patents
一种用于检测甲肝病毒的分子马达生物传感器试剂盒 Download PDFInfo
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Abstract
一种用于检测甲肝病毒的分子马达生物传感器试剂盒，属于食源性病毒快速检测领域，主要解决传统检测方法培养增殖周期长、增殖能力低，不形成细胞病变，难以满足检测要求。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亚基上连接甲肝病毒各型的共有序列特异性探针，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10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。本方法具有反应时间短，抗原抗体结合充分，操作使用方便等特点，缩短了反应时间，提高了检测灵敏度。本试剂盒可对待测食品样本中的甲肝病毒进行快速、灵敏、高通量的检测。
Description
技术领域
本发明是关于食源性病毒快速检测技术的一种。
背景技术
传统甲肝病毒(HAV)的检测方法是通过细胞培养，但甲肝病毒不仅增殖时间长、增殖能力低，且不形成细胞病变，难以满足检测的要求。20世纪80年代建立的RT-PCR和ICC/PCR(integrated cell culture，PCR)等检测技术提高了检测的灵敏度和特异性，在甲肝病原监测中有很好的应用，但其检测时间仍需5-6h，且容易受PCR扩增产物污染而产生假阳性的可能，这类技术仍然需要依据放射性物质标记的RNA探针或通过凝胶电泳中的特定RNA条带来判断检测结果，且整个过程需要扩增、电泳等步骤，操作繁琐。不仅费时费力，给使用亦带来很大不便。因此急需可靠有效的快速检测方法，缩短窗口期，避免因食源性甲肝病毒感染而引起的甲型肝炎的发生和流行。
发明内容
本发明核心技术是利用载色体Chromatophore上的F0F1-ATPase分子马达生物传感器，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1h，且检测灵敏度能达到0.01ng/mL，满足现有的食源性病毒检测范围。
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为甲肝病毒特异性探针，5为甲肝RNA链。
附图2为chro HAV对不同种病毒的检测结果，纵坐标表示荧光值，横坐标表示检测的病毒种类。
附图3为chro HAV对13个添加甲肝病毒食品样品和2个不含有甲肝病毒食品样品的检测结果，纵坐标表示荧光值，横坐标表示检测的样本，1～15为食品检测样本编号。
具体实施方式
根据甲肝病毒各型设计具有各型共有的特异性的核酸探针5’-AGCGGCGGATATTGGTGAGTTGTTAAGAC-3’，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，利用荧光探针DHPE标记的载色体Chromatophore上的F0F1-ATPase分子马达生物传感器即可对待测样本的RNA进行检测。当样品为甲肝病毒RNA时，检测时与生物传感器结合的同时启动ATP合成，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、快速对甲肝病毒进行检测。
试剂盒组成：
| 编号 | 组分名称 | 数量 | 保存条件 |
| 1 | ChroHAV | 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 eppendorf(EP)管，加入待测样本10μL。
2.取2μL chro HAV，用合成buffer稀释到一定的倍数。取稀释后的chro HAV 10μL加入上述EP管。
3.另取1个EP(eppendorf)管加入10μL无菌水，再加入10μL合成buffer作为本底对照，短暂振荡使反应体系混匀。
4.再向2个EP管中分别加入30μL启动buffer(启动buffer由ADP(1.6moL/L)和合成buffer按体积比1∶3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。
5.将上述反应体系置于中温育10min。
6.将EP管从摇床中取出，分别加入200μL PBS缓冲液，振荡使体系混匀。
7.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。
8.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 HAV具有良好的特异性，用chro HAV对水、甲肝病毒、轮状病毒、诺如病毒进行检测，甲肝病毒的荧光值明显高于水和其他对照病毒的荧光值，具体数据见下表，结果见附图2(*表示样本荧光值与H2O对照相比较差异显著(p＜0.05)。
| 样品(0.9ng/mL) | 荧光值 |
| 水 | 95460 |
| 甲肝病毒 | 120463 |
| 轮状病毒 | 94842 |
| 诺如病毒 | 95508 |
用chroHAV对15株食品样品进行盲样检测，对添加甲肝病毒的食品样品均能检出，其中2号和9号样品不含甲肝病毒。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 | 60622 | 8 | 72537 |
| 1 | 71691 | 9 | 60292 |
| 2 | 61135 | 10 | 72402 |
| 3 | 71652 | 11 | 72730 |
| 4 | 75910 | 12 | 73525 |
| 5 | 74326 | 13 | 68529 |
| 6 | 72014 | 14 | 72444 |
| 7 | 73125 | 15 | 74157 |
实验结果表明，该试剂盒对甲肝病毒RNA的检测时间为1h，检出限为0.01ng/mL。对15株食品检测样本菌株的检出结果与甲肝病毒传统方法检测的结果一致。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)
- 本发明要求保护的内容有：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、生物素标记的甲肝病毒特异性探针。
- 2.试剂盒检测体系的反应条件：室温温育10min。
- 3.合成buffer、启动buffer、PBS的配方。合成buffer：甘油20％，氯化镁5mM，Tricine50mM，磷酸氢二钾5mM；启动buffer：ADP(1.6mM)：上述合成buffer＝1∶3(v/v)；PBS：137mM氯化钠，2.7mM氯化钾，10mM磷酸氢二钠，2mM磷酸二氢钾。
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| 《食品工业科技》 20111230 张捷等 "分子马达传感器对沙门氏菌快速检测方法的初步研究" 第33卷, 第12期 * |
| 张捷等: ""分子马达传感器对沙门氏菌快速检测方法的初步研究"", 《食品工业科技》 * |
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