新型冠状病毒检测试剂盒的“庐山真面”?
p style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "抗击新型冠状病毒肺炎疫情,这场发生在属相轮回之交的“战役”愈演愈烈。“战役”之初,因试剂盒短缺而导致被病毒感染的人无法及时确诊,甚至有人因次错过有效的针对性治疗而丧失生命。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "在1月21日病毒病预防控制所发布新型冠状病毒核酸检测引物和探针序列后,多家企业开始研发出病毒检测试剂。1月25日,国家药监局为7家新型冠状病毒检测试剂盒生产商开通快速审批通道,同一天,湖北省实行新型冠状病毒检测试剂盒先试用后申报的办法。span style="text-indent: 2em "而今,已有数十家企业在夜以继日的生产着这关乎性命的试剂盒,并且已有4家企业的检测产品相继获得国家药品监督管理局注册证。/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "那么,这个在此次抗击新型冠状病毒肺炎疫情战役中进入公众视线的病毒试剂盒究竟是什么?/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "检测试剂盒,顾名思义,是一个装有检测某样目标物质所需试剂的小盒子。只需要按照厂商随盒附上的说明书进行加样操作,便能进行检测。试剂盒所提供的方案,是经过多次实验所产出的最优解,包括试剂浓度、加样量等,以保证结果的可靠性和准确性。除却检测试剂盒,另有核酸提取试剂盒、ELISA试剂盒、特定蛋白检测试剂盒以匹配不同操作平台和实验目的,我们先按下不表。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "目前,用于检测新型冠状病毒的试剂盒多采用荧光PCR方法,在获取病毒样本当天即可出具病毒核酸检测结果。短短一月间,从发现新型冠状病毒,到研发出病毒检测试剂盒,这一切,离不开科研人员、国家等多方的迅速反应。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "实际上,开发一个核酸诊断试剂盒到它真正上市投入使用,短则2-3年,长则4-5年。这里,我们将结合此次2019-nCoV病毒检测来大致介绍核酸检测试剂盒的开发流程。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "科普时间:/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "核酸检测试剂盒开发流程/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "引物与探针设计/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "引物:分为正向和反向引物,通过对GC含量(50%-60%)的控制来避免引物二聚体形成以及 控制退火温度。一般会把此次病毒病预防控制所发布的两对引物,GC含量都在50%左右且退火温度均维持在50-60℃之间。/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "探针:使用探针以增加扩增子的特异性,减少假阳结果。探针序列必须有别于引物序列,且C含量多于G含量,过高的G含量会降低反应效率。探针的长度控制在18-30bp最佳,过长会导致淬灭效果不佳。图1展示的两个探针长度分别为28和20bp。/span/pp style="text-align: center"img style="max-width: 100% max-height: 100% width: 513px height: 345px " src="https://img1.17img.cn/17img/images/202001/uepic/f1fa5b86-2452-4524-a20d-f029ff4eb792.jpg" title="试剂盒序列.jpg" alt="试剂盒序列.jpg" width="513" height="345"//pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 0em text-align: center "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "图1:病毒病预防控制所公布的新型冠状病毒核酸检测引物和探针序列/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "扩增子:即目的病毒核酸扩增片段,同理,选择含有特定GC量的片段来避免形成二级结构,长度在60-120bp。/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "我们在这里只是粗略地介绍了引探设计中需要考虑的因素,即便从理论出发所设计的引物与探针看似一切无虞,但在实际操作中仍会出现各式各样的问题,在后文我们会提及体系优化以达到最佳检测效果。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="color: rgb(79, 129, 189) "strong模板制备/strong/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "首先需要明确模板类型,此次需要检验的2019-nCoV病毒便是单股正链RNA病毒。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "现下的新冠肺炎是呼吸道疾病,且可由飞沫进行传播,咽部是病毒高度聚集的部位,因此样本采集途径是收集就诊者咽拭子标本或者是痰液;所有的样本模板制备过程中都进行纯化和质控管理。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="color: rgb(79, 129, 189) "strong体系优化/strong/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "聚合酶在这个实际体系优化中作为最重要的关键原料,这对检测试剂盒的灵敏度,特异性,重复性,扩增效率等性能指标至关重要。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "催化酶的优化在最初期,厂家会选择多家供应商的产品进行测试,目的是选择最适合该反应体系的催化酶。而原料供应商提供的聚合酶配套的buffer也往往非工业生产过程中的最优配方,需要不断调试离子浓度,pH值等以达到最佳反应体系。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "探针浓度的优化主要依靠背景信号强度、探针与靶的结合效率、PCR扩增的效率、引物的浓度等进行优化。目的是既使背景荧光最小化,又没有降低实验的敏感性,从而得到最佳实验结果。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "引物也需要浓度优化,并且在设计过程中,会准备多对引物与探针匹配,选择能够给出最低Ct值,到达但是却能给出最高反应终点荧光值的引物。(Ct值:即荧光量到达阈值所需的循环数)/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "对于引物退火温度,理论值与实际温度会有一定出入,通常使用梯度PCR来确定最佳效率的退火温度。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "由于2019-nCoV与SARS CoV高度同源,因此还需制备阳性对照来避免假阳性的可能。span style="font-family: 楷体, 楷体_GB2312, SimKai color: rgb(79, 129, 189) "(假阳性:一般来说,阳性(+)是表示疾病或体内生理的变化有一定的结果。相反,化验单或报告单上的阴性(-),则多数基本上否定或排除某种病变的可能性。然而,由于环境因素、操作因素、实验方法或者患者自身因素等等可能把不具备阳性症状的人检测出阳性的结果)/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="color: rgb(79, 129, 189) "strong性能检测/strong/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "当一切准备就绪后,就需要进行性能测试,而这也是最耗费时间的。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "大致为:/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "灵敏度测试、重复性测试、稳定性测试、最低检测浓度、特异性及其他干扰物质的影响、精密度分析等。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "以上就是大致的荧光PCR核酸检测试剂盒研发流程,每一个环节都需要多次确认才得以成就最后产品。在这场举国防控防疫攻坚战中,检测试剂盒的出现为广大一线医务工作者提供了有利工具,再结合临床检查,加快病情排查。普通流感与新冠肺炎症状类似,对于前往发热门诊的病患来说,试剂盒的存在也让他们吃了一颗定心丸,尽快确诊病情便可着手进行治疗。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "span style="color: rgb(79, 129, 189) "strong从“一盒难求”到“先试后报”/strong/span/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "为何现在试剂盒仍旧供不应求?首先,正值春节,工厂产能有限、物流人手有限;其次,2019-nCoV具有高传染性,因此对于检验实验室和检验人员都有一定要求,并非每一家医院都有配备负压条件的实验室。但是我们同时也非常欣喜地看到,截止目前,共有7家企业研发的新型冠状病毒检测试剂盒进入了国家药监局的快速审批通道,湖北省更是提出采取先试用后申报的办法,允许在医疗机构先试用,疫情解除后再按程序审核。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "小彩蛋:br//pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "默克作为体外诊断试剂的原料供应商,疫情发生以来,就收到来自全国各地的检测试剂盒原料供应需求,默克员工春节不休,全球调货以满足众多检测试剂盒生产厂家的需求。/pp style="margin-top: 10px margin-bottom: 10px line-height: 1.5em text-indent: 2em "默克核酸检测试剂盒原料解决方案:/ptable width="677" style="margin-top: 0px margin-right: 0px margin-left: 0px padding: 0px max-width: 100% box-sizing: border-box color: rgb(51, 51, 51) width: 648px " helvetica="" pingfang="" hiragino="" sans="" microsoft="" yahei="" font-size:="" letter-spacing:="" text-align:="" white-space:="" background-color:="" overflow-wrap:="" break-word="" width:=""tbody style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box 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break-word !important "项目/strong/p/section/tdtd colspan="1" rowspan="1" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:0.td@@1" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px "p style="max-width: 100% box-sizing: border-box clear: both min-height: 1em overflow-wrap: break-word !important "核酸提取/p/section/tdtd colspan="1" rowspan="1" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:0.td@@2" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px "p style="max-width: 100% box-sizing: border-box clear: both min-height: 1em overflow-wrap: break-word !important "引物探针合成/p/section/tdtd colspan="1" rowspan="1" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:0.td@@3" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px "p style="max-width: 100% box-sizing: border-box clear: both min-height: 1em overflow-wrap: break-word !important "PCR/qPCR/p/section/td/trtr opera-tn-ra-comp="_$.pages:0.layers:0.comps:38.classicTable1:1" powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important "td colspan="1" rowspan="7" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:1.td@@0" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px background-color: rgb(77, 195, 207) border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px color: rgb(245, 245, 245) "p style="max-width: 100% box-sizing: border-box clear: both min-height: 1em overflow-wrap: break-word !important "strong style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important "原料/strong/p/section/tdtd colspan="1" rowspan="1" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:1.td@@1" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px line-height: 1.8 letter-spacing: 0px "p style="max-width: 100% box-sizing: border-box clear: both min-height: 1em overflow-wrap: break-word !important "液体蛋白酶k/p/section/tdtd colspan="1" rowspan="1" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:1.td@@2" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px "p style="max-width: 100% box-sizing: border-box 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style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px letter-spacing: 0px "p style="max-width: 100% box-sizing: border-box clear: both min-height: 1em overflow-wrap: break-word !important "固体蛋白酶k/p/section/tdtd colspan="1" rowspan="1" opera-tn-ra-cell="_$.pages:0.layers:0.comps:38.classicTable1:2.td@@1" width="25.0000%" style="margin: 0px word-break: break-all max-width: 100% box-sizing: border-box border-radius: 0px border: 1px solid rgb(0, 0, 0) padding: 5px overflow-wrap: break-word !important "section powered-by="xiumi.us" style="margin: 0px padding: 0px max-width: 100% box-sizing: border-box overflow-wrap: break-word !important text-align: center font-size: 14px "p style="max-width: 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