One-PCR-tube approach for in situ DNA isolation and detection.
One-PCR-tube approach for in situ DNA isolation and detection.
复制标题
用于原位 DNA 分离和检测的单 PCR 管方法。
DOI：
10.1039/c1an15116a
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Zhu,Shuifang
中科院分区：
文献类型：
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作者：
Huang,Xin;Hou,Lihua;Xu,Xiaohe;Chen,Hongjun;Ji,Haifeng;Zhu,Shuifang
传统的实时聚合酶链反应（PCR）需要纯化的DNA样品用于PCR扩增和检测。这就要求PCR检测必须在洁净的实验室中进行，并限制了其在现场检测中的应用。本工作建立了一种在一个PCR管中进行DNA纯化、扩增和检测的方法。首先用铬酸处理聚丙烯PCR管，并将肽核酸（PNA）作为DNA捕获剂固定在管的内表面上。将粗提液中的花椰菜花叶病毒35 S（CaMV-35 S）启动子与试管表面的PNA杂交，用缓冲液冲洗，可有效去除粗提液中的抑制剂、干扰剂和无关DNA。捕获靶DNA的试管可用于以下实时PCR（RT-PCR）。通过使用这种方法，可以在3小时内完成对复杂样品中少于2500个拷贝的35 S质粒的检测。对巧克力样品进行了真实的样品分析，并成功地用该方法原位鉴定了转基因巧克力样品中的35 S质粒。该方法操作简便，操作时间短，与市售试剂盒相比具有一定的竞争力。该方法可广泛应用于食品中含有修饰DNA和特定病原体的现场鉴定。
Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field.