等温扩增技术概述及其在动物病原检测中的应用
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Overview of Isothermal Amplification Technology and Its Application in Animal Pathogen Detection
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    摘要:

    等温扩增技术是一种可以在恒定温度下进行体外核酸扩增的技术,近几年其研究热度很高。本文从技术原理、优缺点及在动物病原检测应用等方面,对环介导等温扩增技术(LAMP)、重组酶等温扩增技术、依赖核酸序列扩增技术(NASBA)、交叉引物等温扩增技术(CPA)和赖解旋酶等温扩增技术(HDA)等5种常见等温扩增技术进行了综述。LAMP技术操作简单,不依赖特殊设备,但引物设计较为复杂,研发多重LAMP检测方法有很大困难。重组酶等温扩增技术不受场地条件约束,扩增时间短,但容易造成气溶胶污染,因此一般用于实验研究。NASBA技术扩增效率高,不易产生污染,但成本偏高,操作相对复杂,在目前疫病检测研究中的应用逐渐减少。CPA技术操作相对简单,灵敏度高,特异性好,但结果判断受主观因素影响易造成假阳性或假阴性。HDA技术引物设计相对简单,但反应体系较为复杂,因此不适合扩增长序列。不同的等温扩增技术需要根据实际需求和场景条件来选择。等温扩增技术作为一种新兴技术具备许多优点,但仍需要不断改进,未来与微流体芯片、纳米金、CRISPR等技术联合应用,将会实现更加快捷、准确、简便的检测,从而为动物疫病检测提供更好的服务。

    Abstract:

    Isothermal amplification technology could be used for in vitro nucleic acid amplification at a constant temperature,which has been increasing studied in recent years. In the paper,loop mediated isothermal amplification technique(LAMP),recombinase isothermal amplification technique,nucleic acid sequence dependent amplification technique(NASBA),cross primer isothermal amplification technique(CPA)and helicase dependent amplification technique(HDA)were reviewed from the aspects of technical principles,advantages and disadvantages as well as the application in animal pathogen detection. LAMP could be operated without any special equipment,but more complex primers were needed,it would be very difficult to develop multiple LAMP. Recombinase isothermal amplification technique could not be restricted by surrounding conditions with short amplification time,but aerosol pollution could be produced,which was generally used for experimental researches. NASBA was with high amplification efficiency,producing little pollution,but its cost was high with relative complex operation,so its application in disease detection and researches was reducing. CPA was relatively simple,with high sensitivity and specificity,but false positive or false negative results might be obtained due to some subjective factors. The primer design of HDA was relatively simple,but its reaction system was more complex,which was not suitable to amplify the genes with long sequence. In conclusion,isothermal amplification techniques should be selected according to actual needs and specific conditions. In spite of the above advantages,the techniques should be continuously improved to be used together with microfluidic chips,nano gold,CRISPR and other techniques in the future,which was expected to be a more rapid,accurate and simple operation,and thus provided better convenience for animal disease detection.

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禹兰平,王楷宬,潘俊慧,张富友,杨文静,周凯钰太,崔成都,孙福亮,王素春.等温扩增技术概述及其在动物病原检测中的应用[J].《中国动物检疫》编辑部,2022,39(12):96-103.

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