清华团队研发的30min核酸检测卡盒技术原理剖析，可靠不？

admin · 发表于 2020-10-14 12:12:10

                  <section data-role="paragraph" data-color="rgb(182, 228, 253)" data-custom="rgb(182, 228, 253)"><section><section><section><section powered-by="gulangu"><section><section><section><section powered-by="gulangu"><section><section><section><section powered-by="gulangu"><section><section><section><section powered-by="gulangu"><section><section><section><section><p><p><img src="image/20201014/86b79d371e9862b01189f0252a03b2cd_1.jpg" /></p></p></section><section><section><span><strong></strong></span></section><p><span>医疗器械媒体报道先锋</span></p><p><span>分享专业医疗器械知识</span></p></section><section><section><section><span>关注</span></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section></section><section data-tools="gulangu" data-id="97917"><section><section><section data-bgless="spin" data-bgopacity="30%"><br  /></section><p data-mpa-powered-by="gulangu"><span>近日清华大学微信官网上公布了一则消息“清华团队研发核酸检测卡盒，居家检测30分钟内出结果！”引起了热议。</span></p><p><br  /></p><p><span>据悉该项目由清华大学药学院白净卫、李寅青与医学院生物医学工程系刘鹏课题组在北京市科委和清华自主研发计划支持下联合研发的一体化自助式SARS-CoV-2核酸检测卡盒（BINAS），在解决以上问题上取得重大突破。该卡盒具有居家检测的便捷性，两个推杆一推一拉之间，30分钟内即可实现“样本入、结果出”！</span></p><p><span><br  /></span></p><p><p><img src="image/20201014/9c75b38d2aec3aa80711d51d936052c8_2.png" /></p></p><p><br  /></p><section data-mpa-template="t" mpa-from-tpl="t"><section data-id="94925" mpa-from-tpl="t"><section data-width="100%" mpa-from-tpl="t"><section data-autoskip="1" mpa-from-tpl="t"><section><strong><span>反应原理：</span></strong><span></span></section><section><span>第一步，移动杆带动注射器式的活塞导致裂解的样品流入第一个等温扩增腔；</span></section><section><span>第二步，反应10 分钟后，反应产物被稀释；</span></section><section><br  /></section><section><span>第三步，泵入第二个等温反应室进行带探针的进一步扩增；</span></section><section><br  /></section><section><span>最后，将反应混合物泵入装有层流试纸条的室中，以读出测试结果。这其中，卡盒含有 USB 或电池供电的简易恒温电路，可以提供扩增反应所需要的温度。</span></section><section><span><br  /></span></section></section><section mpa-from-tpl="t"><section mpa-from-tpl="t"><br  /></section></section></section></section></section><p><br  /></p><p><p><img src="image/20201014/b6ff7b34b886ca19090c11005b4e4a20_3.png" /></p></p><p><span>这个卡盒实现了从咽拭子取样、一体化卡盒反应、胶体金试读取的自助操作，在保证准确率的前提下，30分钟内即可出结果！</span></p><p><span> </span></p><p><br  /></p><p><strong><span>技术原理：</span></strong><span></span></p><section data-mpa-template="t" mpa-from-tpl="t"><section data-id="94925" mpa-from-tpl="t"><section data-width="100%" mpa-from-tpl="t"><section data-autoskip="1" mpa-from-tpl="t"><p><span>采用巢式等温扩增（ITA）技术。所谓的“巢式”，可以理解为 “嵌套”，一个环节嵌套一个环节。</span></p><p><br  /></p><p><span>最后，以上反应物在胶体金试纸上通过竞争法显色，读取方式与验孕棒类似。” </span></p><p><br  /></p><p><span>整个过程理论上22分钟即可完成，检测者个人操作的话，30分钟之内可以完成。</span></p><p><br  /></p><p><span>根据公开信息显示，该技术是基于时下较火的CRISPR技术原理开发而成。</span></p></section><section mpa-from-tpl="t"><section mpa-from-tpl="t"><br  /></section></section></section></section></section><p><p><img src="image/20201014/996edcc4540a74871b8e6ce65abad22a_4.png" /></p></p><p><br  /></p><p><span>对比实验数据显示：</span></p><p><br  /></p><section data-mpa-template="t" mpa-from-tpl="t"><section data-id="94925" mpa-from-tpl="t"><section data-width="100%" mpa-from-tpl="t"><section data-autoskip="1" mpa-from-tpl="t"><p><br  /></p><p><span>新研发的技术对19 例 QPCR（传统的实时荧光定量PCR方法）阳性患者样本进行了复合，结果全部可以检出。</span></p><p><span> </span></p><p><span>“结果证明了本项目的灵敏度接近QPCR的灵敏程度，QPCR是300 拷贝/毫升（copies/ml），我们的灵敏度小于400拷贝/毫升。</span></p><p><span><br  /></span></p><p><br  /></p><p><span>此外，与近期于国际顶级期刊 Nature Biotechnology 报道的美国加州基于CRISPR原理开发的高灵敏度技术相比，本项目独创的带标签的巢式等温扩增技术检测时间更短灵敏度更高。</span></p></section></section></section></section><section data-mpa-template="t" mpa-from-tpl="t"><section data-mid="" mpa-from-tpl="t"><section data-mid="" mpa-from-tpl="t"><section data-mid="" mpa-from-tpl="t"><br  /></section><section data-mid="" mpa-from-tpl="t"><span><strong><span>关于CRISPR</span></strong></span></section><section data-mid="" mpa-from-tpl="t"><br  /></section></section></section></section><p><span><strong><span>1、CRISPR技术的定义</span></strong></span></p><p><span><br  /></span></p><p><span>CRISPR的全称是Clustered Regularly Interspaced Short Palindromic Repeats（成簇的规律间隔的短回文重复序列），Cas的全称则是CRISPR associated（CRISPR关联），合在一起简称为CRISPR/Cas系统。</span></p><p><span><br  /></span></p><p><br  /></p><p><span>Cas9（CRISPRassociated nuclease）是CRISPR相关核酸酶，CCRISPR/Cas9是最一种由RNA指导，利用Cas9核酸酶对靶向基因进行编辑的技术。该技术是被人们发现并加以利用，而非为人类开发合成的。</span></p><p><span><br  /></span></p><p><br  /></p><p><span>CRISPR/Cas9成功与否的关键在于gRNA的设计，gRNA和非目标区域过多的非特异性结果，脱靶效率较高，特别是靠近PAM的8-10bp不能和非目的区有高同源性。目前为了提高基因组编辑的准确性，对Cas9核酸酶进行了突变，突变后的Cas9只能切割DNA双链中的一条链，通过两个gRNA引导Cas9对DNA切割，可以显著提高基因组编辑的特异性，降低脱靶效应。</span></p><p><br  /></p><p><span><strong><span>2、CRISPR技术原理</span></strong></span></p><p><br  /></p><p><p><img src="image/20201014/63eda13f1ae8e50320de079ce6eb2d5e_5.png" /></p></p><p><br  /></p><p><span>CRISPR/Cas9主要由两部分组成：gRNA和Cas9蛋白。gRNA由CRISPR RNA（crRNA）与反式激活crRNA（trans-activating crRNA,tracrRNA）组成，crRNA的一部分序列能与tracrRNA配对，形成双链RNA结构；一部分序列与靶目标互补区域，以此识别靶序列。</span></p><p><span><br  /></span></p><p><br  /></p><p><span>Cas9蛋白先与gRNA形成复合体（Cas9-sgRNA），使Cas9蛋白构象变化，产生一条通道，使其更容易与DNA结合。该复合体随机碰撞DNA，若未识别到PAM序列，则会快速离开DNA，发生下一次碰撞；若识别PAM序列，Cas9蛋白的Arg1333和Arg1335会与二核苷酸鸟嘌呤结合，此时，Ser1109和Lsy1107形成磷酸锁结构，与PAM序列旁的+1位核苷酸发生相互作用，促使双链DNA解旋，利用RNA-DNA碱基互补配对原则，核对序列与crRNA的互补情况，若在种子序列出现较多错配，则序列识别过程终止，从而确保Cas9在正确的目标处发挥作用。</span></p><p><br  /></p><p><br  /></p><p><span>从原理上来说，CRISPR分子诊断方法实际上是将扩增方法与Cas蛋白的检测相结合。CRISPR检测在提高灵敏度的同时，也提高了特异性，而且降低了对仪器依赖。另外，经过方法优化，CRISPR检测也做到了多靶标、一步法定量检测、DNA和RNA相同步骤检测和适合野外偏远地区的快速分子检测。<br  /></span></p><p><br  /></p><p><span>参考资料：清华大学、Sam的行研笔记、IVD从业者等</span></p><section powered-by="gulangu"><section><section><br  /></section></section></section></section></section></section><section data-role="outer" label="Powered by gulangu"><section data-role="paragraph" 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