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遗传  2017, Vol. 39 Issue (6): 525-534    DOI: 10.16288/j.yczz.17-031
生物学   技术与方法 |遗传学 本期目录 | 过刊浏览 |
一种载样简单的多重可视化PCR微芯片
陈建伟1(),邵宁1,张雨晨2,朱元首1,杨立桃2(),陶生策1(),卢大儒
1. 上海交通大学系统生物医学研究院,系统生物医学教育部重点实验室,上海200240
2. 上海交通大学生命科学与技术学院,上海200240
A visual multiplex PCR microchip with easy sample loading
Chen Jianwei1(),Shao Ning1,Zhang Yuchen2,Zhu Yuanshou1,Yang Litao2(),Tao Shengce1(),
1. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
2. School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
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摘要: 

在核酸检测领域急需一种能够经济、快速、操作简便且同时检测多个靶标的新技术。常规多重PCR能够同时扩增多个靶标,但由于在一个试管中引物间的相互作用和竞争,重数往往受到限制。本研究设计了一种操作简单的多重PCR芯片,可以同时进行54个靶标的扩增。芯片结构简单,一条微通道将正下方平行排列的多个微孔连接在一起,微通道只留加样口和出样口。同时整个微通道呈疏水状态,微孔呈亲水状态。将不同的引物对和低溶点琼脂糖提前固定于不同的微孔中,通过一次加入PCR mix,并加入矿物油推动PCR mix进入到每个微腔室中,同时微孔也被矿物油相互隔离避免交叉反应。加样后将芯片放置于平板PCR仪上进行扩增,在整个反应过程中低溶点琼脂糖呈液态,反应结束后凝固成固体,便可引入核酸染料进行染色,最后利用自制小型的紫外照射仪上进行可视化检测和拍照记录。利用此平台成功实现了对7种非常重要和常用的转基因作物靶标的并行检测,结果显示此平台具有较高的灵活性和特异性。此技术经过进一步优化可应用于包括转基因检测在内的多重核酸检测领域。

关键词 多重PCR微芯片亲疏水低溶点琼脂糖转基因作物检测    
Abstract

There is an urgent demand for affordable, rapid and easy-to-use technology to simultaneously detect many different DNA targets within one reaction. Conventional multiplex PCR is an effective methodology to simultaneously amplify different DNA targets. However, its multiplicity is limited due to the intrinsic interference and competition among primer pairs within one tube. Here, we present an easy multiplex PCR microchip system, which can simultaneously detect 54 targets. The design of the microchip is quite simple. There is a microchannel connected with multiple underlying parallel microwells. And every microchannel has an inlet/outlet for loading PCRmix. The surface of the microchannel is hydrophobic and the inner surface of the microwell is hydrophilic, which enables us to load and separate the PCRmix into different microwells simultaneously. Different primer pairs and low melting agarose are pre-fixed in different microwells, and the microchip is assembled with top glass. The PCRmix is loaded into inlets and then mineral oil is sequentially pipetted into channels to push the PCRmix into all microwells and subsequently mineral oil fills the channels to avoid cross contaminations. After the PCRmix is loaded, it would be placed on a plat thermal cycler for PCR. During PCR, the low melting gel in the well is liquid and after PCR it would be solidified due to temperature changes. When PCR is completed, a nucleic acid dye is introduced into channels and then results are visualized by a home-made, potable UV detector. In our platform we successfully detected seven frequently used targets of genetically modified (GM) organisms. The results demonstrate that our platform has high flexibility and specificity. Due to the excellent performance of this technology, we believe that it can be applied to multiple nucleic acid detection fields including GM organisms.

Key wordsmultiplex PCR    microchip    hydrophobic and hydrophilic    low melting agarose    genetically modified organisms
收稿日期: 2017-02-24      出版日期: 2017-05-03
基金资助:“十三五”国家重点研发计划(2016YFA0500601);国家自然科学基金(31670831, 31370813)
第一作者简介: 陈建伟,硕士,专业方向:生物芯片。E-mail: naughtygene@sjtu.edu.cn|杨立桃,博士,副研究员,研究方向:转基因作物检测。E-mail: yylltt@sjtu.edu.cn|陶生策,博士,研究员,研究方向:生物芯片。E-mail: taosc@sjtu.edu.cn
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引用本文:

陈建伟,邵宁,张雨晨,朱元首,杨立桃,陶生策,卢大儒. 一种载样简单的多重可视化PCR微芯片[J]. 遗传, 2017, 39(6): 525-534.
Chen Jianwei,Shao Ning,Zhang Yuchen,Zhu Yuanshou,Yang Litao,Tao Shengce, . A visual multiplex PCR microchip with easy sample loading. Hereditas(Beijing), 2017, 39(6): 525-534.

链接本文:

http://www.chinagene.cn/CN/10.16288/j.yczz.17-031      或      http://www.chinagene.cn/CN/Y2017/V39/I6/525

表1  7个转基因靶标特异性引物
图1  芯片和检测器结构图 A:芯片组成图,由下往上依次为底层玻片、不锈钢板、FR4板和顶层玻片;B:芯片封装和引物点制图。底部3层贴合完成并疏水处理后进行引物点制;C:已制作完成待用芯片;D:自制芯片检测器结构图,包括开关、稳压器、电池组、UV灯管、ZWB2滤光片和CB565滤光片。芯片置于ZWB2滤光片和CB565之间的空格进行检测。
图2  加样过程图 A:样本加载示意图。先加入PCRmix,再用矿物油推动PCRmix进入各个微孔,最后矿物油完全充满通道覆盖反应微孔;B:样本加载真实图片,PCRmix中添加溴酚蓝便于显色观察。
图3  芯片和试管PCR扩增效率比较 A:10 pg/μL含T-E9序列质粒;B:0.1 pg/μL含T-E9序列质粒;C:1 ng/μL含T-E9序列转基因棉花基因组MON88913;D:0.1 ng/μL含T-E9序列转基因棉花基因组MON88913。M:DNA Marker;1~3:3个芯片PCR重复;4~6:3个试管PCR重复。
图4  芯片交叉污染测试 A:测试不同行之间的交叉污染。两行都点制T-E9靶标引物,第一行加入含DNA模板的PCRmix,第二行加入不含DNA模板的PCRmix对照;B:测试相邻反应孔间交叉污染测试。隔孔点制引物,1表示点制引物,0表示没有点制引物。
图5  5个含有不同模板组合的样本进行芯片PCR扩增 A:芯片引物预点制由左到右依次为:0、1.P-CaMV35s、2.P-CmoVb、3.T-nos、4.T-E9、5.nptII、6.zSSIIb、7.Hmg1/y、0,0为无引物点制。5个样本a~e中含有不同的模板组合。B:5个样本在芯片上的扩增结果。a~e分别为加入A图中5个样本的芯片结果图。每个结果平行重复2次。
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