Contagion in this country, we pay close attention to everyone all the time dynamics of the epidemic, especially in the case of diagnosis of substance supply and new coronavirus. Among them, as the main force to detect new viruses crown, nucleic acid detection kit often appear in our field of vision, then it is how to detect suspected patients carrying the novel coronavirus?
Know thyself
Before identifying novel coronavirus we first look at it:
This class of enveloped virus is a genome is a linear single-stranded positive strand RNA virus, circular or elliptical particle diameter of about 60-140nm.
It should be noted that the above picture is virus amplification using electron microscopy to nano-scale shooting, that is, by normal means of detection, hardly notice it's there. After the virus infected humans, the symptoms will be infected from the common cold to severe pulmonary, and a long incubation period, virus carriers will inadvertently infect more people. Therefore, early detection of its presence to reduce the risk of HIV infection to others, so that the new coronavirus patients receive timely professional treatment will become a priority.
According to the National Health health committee issued a "novel coronavirus infection pneumonia treatment program (Trial Fourth Edition)" confirmed cases need to have the following etiological evidence:
1. respiratory samples or blood samples of real-time RT-PCR, fluorescent novel coronavirus nucleic acid positive;
2. respiratory specimens or blood samples and other virus gene sequencing, known novel coronavirus highly homologous.
Currently, most of the kit for nucleic acid using the first method, real-time RT-PCR fluorescence to identify novel coronavirus, probably this technology process :
- Obtaining a sample suspected patients, the nucleic acid sequence of samples extracted.
- These nucleic acid sequences (which may contain viral nucleic acid sequences) are mixed with the starting material
- Amplification reaction (this reaction can only expand the number of viral nucleic acid sequences) in a reaction vessel, to facilitate subsequent detection of
- Nucleic acid sequence is amplified with a fluorescent, a fluorescence signal is detected if the virus signature curves meet, then the patient is suspected that the positive.
- In the actual process of a patient will be detected several times
Desired material : nucleic kit, QPCR instrument, computer, samples, etc.
Here, we learn more about this technology is how to ferret out new coronavirus.
如何放大病毒信号
我们都知道作为核酸序列的DNA可以复制自身，这个过程如下：
- DNA先打开双链，暴露出模板
- 引物（左右两个DNA片段）会特异性的结合到模板上
- DNA聚合酶（黄色的几何体）就可以利用碱基（彩色的小圆点）从引物开始复制，这个过程也叫扩增
- 只要这个反应有足够原料，新合成的DNA链又会作为模板链开始新的合成
- 最终，开始的序列会被以呈指数级的增加。利用这个特性，可以放大样品中微弱的病毒序列信号
实际上，早在上个世纪，一位叫穆利斯（Kary Banks Mullis）的牛人在体外实现了DNA复制过程。该技术被命名为聚合酶链式反应（PCR），因这个伟大的发明他在1993年获得了诺贝尔化学奖。
但是，需要注意的是2019新型冠状病毒是一种RNA病毒，PCR是针对DNA复制来设计的。
因此，我们需要将RNA逆转录为cDNA，然后进行DNA复制的过程，这个技术也叫RT-PCR（逆转录-PCR）。
如何只放大新型冠状病毒的信号
接下来的问题是怎么去识别2019新型冠状病毒，避免检测到其他的序列信号？
大家会注意到在PCR过程中，只有引物会特异性的结合到模板上，也就是说，设计好的引物可以识别新冠病毒的核酸序列。引物的设计工作就成为病毒检测成功与否的关键。
科学家根据已发布的2019-nCoV全基因组序列，通过序列比对寻找具有 2019新冠状病毒高度特异性的核酸序列，该序列可以特异鉴别2019新冠状病毒。
根据这个特点，在此区域设计特异引物用于2019新型冠状病毒鉴定。
如何进行信号检测
为了能够实时检测到核酸序列的变化情况，在PCR的反应体系中往往需要加入探针或染料用于荧光信号捕获。同时需要QPCR仪，这种仪器可以将荧光信号采集为计算机可以识别的数字信号，用于后续诊断依据。这里涉及的两种产生荧光的物质有着不同的发光原理：
- 荧光染料：一般这种游离的染料几乎没有荧光信号，一旦结合双链DNA后，其荧光信号可呈数百倍的增加。
- 荧光探针：依据目的基因设计合成一个能够与之特异性杂交的探针，该探针的核酸片段两端分别带有荧光基团和淬灭基团。
- 一般情况下，两个基团的空间距离很近，荧光基因因淬灭而不能发出荧光。
- PCR扩增时，探针的结合位置位于上下游引物之间。当扩增延伸到探针结合的位置时，一种切割酶会将荧光基团切下来，从而使其发出荧光。
- 检测到的荧光分子数与PCR产物的数量成正比，因此，根据PCR反应体系中的荧光强度即可计算出初始DNA模板的数量。
为了提高每次检测的人次，检测人员会将样本放置在孔板上，每个孔即为一个样本。同时，会加入空白对照，阳性对照，阴性对照来提高准确率。
接下来，使用计算机软件分析，会有几种结果：
- 空白对照，不会扩增
-
内参样本：会加入人源性内参基因的检测，满足临床对样本质量控制的需要，降低假阴性率。如果，该样本没有扩增曲线，需要检查质控情况
-
阴性对照：这里阴性对照，不会扩增
-
阳性对照：同样，阳性对照样本是存在已知新冠病毒的核酸（也就是说，已知该样本肯定要扩增），为了降低假阳性率。
病原检测人员会根据以上的数据来判断该患者是否为新冠病毒阳性。
最后，看看检测人员平时的工作状态：
新冠病毒对我们来说只是个新闻字眼，但是对于他们来说只隔着一层防护服。而且几乎所有奋战在一线的科学家，医护工作者，志愿者们都是身披这身战甲，在前线日夜奋战。他们无数人洒下泪水与汗水，更有人奉献出自己的生命，但是更多的人不是悲伤和恐惧，而是继续奔赴前线。也许艾青在《我爱这土地》中，早已给出他们奋不顾身的原因：
If I were a bird,
I should hoarse throat singing:
This is the land against the storm,
it is always our grief raging rivers,
which no stop to the sweeping winds angered,
and that from the forest very gentle dawn ......
- then I die,
even feathers putrefied in the land.
Why is my eye often tears?
Because I this land dearly ......
