新冠病毒各类检测方法异同

发布日期：2017-09-04 16:13
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新冠病毒各类检测方法异同

2020-12-07

         新型冠状病毒的检测方法主要包含核酸检测、抗体检测、抗原检测几种方法。由于抗原检测检出率较低，目前新冠检测主要集中在抗体和核酸检测。核酸检测目前是新型冠状病毒检测的“金标准”，具有早期诊断、灵敏度和特异性高等特点;但抗体检测操作便捷、检测迅速，可作为核酸诊断的补充手段。
         The detection methods of novel coronavirus mainly include nucleic acid detection, antibody detection and antigen detection. Due to the low detection rate of antigen detection, the new crown detection mainly focuses on antibody and nucleic acid detection. Nucleic acid detection novel coronavirus detection is currently the "gold standard", which has the characteristics of early diagnosis, high sensitivity and specificity. But the detection of antibody is convenient and rapid detection, which can be used as a complementary means of nucleic acid diagnosis.

核酸检测

     检测原理：

     所有生物除朊病毒外都含有核酸，核酸包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。新型冠状病毒(2019-nCoV)属于Beta冠状病毒属(Betacoronavirus)，该病毒是蛋白包裹的单链正链RNA病毒，寄生和感染高等动物(包括人)。病毒中特异性RNA序列是区分该病毒与其它病原体的标志物，新型冠状病毒出现后，我国科学家已经成功发现了新型冠状病毒中的特异性核酸序列。如疑似患者样本中能检测到新型冠状病毒的特异性核酸序列，则认为该患者可能被新型冠状病毒感染。
     新型冠状病毒常用的核酸诊断方法有两种：病毒核酸特异基因检测和病毒基因组测序。最常见的检测新型冠状病毒特异性核酸序列的方法是荧光定量PCR(聚合酶链式反应)。由于新型冠状病毒是RNA病毒，试剂盒检测基本都采用反转录加实时聚合酶链式反应法(RT-PCR)，扩增病原体的核酸 (RNA) ，同时通过荧光探针实时检测扩增产物。在PCR反应体系中，包含一对特异性引物以及一个Taqman探针，该探针为一段特异性寡核苷酸序列，两端分别标记了报告荧光基团和淬灭荧光基团。探针完整时，报告基团发射的荧光信号被淬灭基团吸收;如反应体系存在靶序列，PCR反应时探针与模板结合，DNA聚合酶沿模板利用酶的外切酶活性将探针酶切降解，报告基团与淬灭基团分离，发出荧光。每扩增一条DNA链，就有一个荧光分子产生。荧光定量PCR仪能够监测出荧光到达预先设定阈值的循环数(Ct值)与病毒核酸浓度有关，病毒核酸浓度越高，Ct值越小。不同生产企业的产品会依据自身产品的性能确定本产品的阳性判断值。
     Detection principle:
     All organisms except prion contain nucleic acid, which includes DNA and RNA. The novel coronavirus (2019-nCoV) belongs to the Beta coronavirus (Betacoronavirus), which is a single strand positive strand RNA virus wrapped by protein, which infect and infect higher animal (including human). The novel coronavirus is a novel coronavirus that has been identified by RNA. The new DNA sequence has been successfully identified by scientists in China. If the novel coronavirus novel coronavirus sequence can be detected in suspected patients, it is believed that the patient may be infected by a new coronavirus.
Novel coronavirus is commonly used in nucleic acid diagnosis. There are two kinds of methods: virus nucleic acid specific gene detection and viral genome sequencing. A novel coronavirus specific nucleic acid sequence is detected by fluorescence quantitative PCR (polymerase chain reaction). Since the novel coronavirus is RNA, the detection kit is based on real-time reverse transcription and real-time polymerase chain reaction (RT-PCR), amplifying the nucleic acid (RNA) of the pathogen, and real-time detection of the amplified product by fluorescent probe. In the PCR reaction system, a pair of specific primers and a TaqMan probe were included. The probe was a specific oligonucleotide sequence, and the two ends were labeled with reporter fluorescent group and quenched fluorescent group respectively. When the probe is complete, the fluorescence signal emitted by the reporter group is absorbed by the quenching group; if there is a target sequence in the reaction system, the probe is combined with the template in PCR reaction, the DNA polymerase digests the probe along the template by using the exonuclease activity of the enzyme, and the reporter group is separated from the quenching group to emit fluorescence. Every time a DNA strand is amplified, a fluorescent molecule is produced. The fluorescence quantitative PCR instrument can monitor that the number of cycles (CT value) of fluorescence reaching the preset threshold is related to the concentration of viral nucleic acid. The higher the concentration of viral nucleic acid is, the smaller the CT value is. The products of different manufacturers will determine the positive judgment value of the product according to the performance of their own products.

                                                           

       由于每个RT-PCR反应需要2个小时左右才出结果，因此，试剂盒在开发时一般都针对病毒序列中高度保守的2-3个序列比如编码replicase(复制酶)或者necleocapsid(核蛋白衣，核鞘)的核酸序列设计引物，但是各个方法用的序列都不完全相同。一些方法使用multiplex，也就是把多个目标序列在同一反应管中扩增;另一些采用分步，先用一个目标序列筛查，阳性后再用另一个序列确认，这样可以有效缩短整个流程时间，加快检测速度。另外RNA病毒变异很快，用多个保守目标序列可以防止病毒变异造成的假阴性。
       Because each RT-PCR reaction takes about 2 hours to produce results, the kits are generally designed for the highly conserved 2-3 sequences in the virus sequence, such as the nucleic acid sequences encoding replicase or nuclease capsid, but the sequences used in each method are not exactly the same. Some methods use multiplex, that is, amplifying multiple target sequences in the same reaction tube; others use step-by-step, screening with one target sequence first, and then confirming with another sequence after positive, which can effectively shorten the whole process time and speed up the detection speed. In addition, RNA virus mutates rapidly, and multiple conserved target sequences can prevent the false negative caused by virus mutation.
       目前批准产品均基于新型冠状病毒基因组中开放读码框1a/b(open reading frame 1ab，ORF1ab)、包膜蛋白(Envelope protein，E)和核衣壳蛋白(nucleocapsid protein，N)进行选择。不同产品的检测原理基本一致，但是其引物、探针设计存在不同，有单靶区段(ORF1ab)、双靶区段(ORF1ab、N蛋白)、三靶区段(ORF1ab、N蛋白和E蛋白)的检测和判读差别。一般检测位于病毒ORF1ab和N基因上的两个靶标，同一份标本需满足双靶标阳性或重复检测为单靶标阳性或两种标本同时满足单靶标才能确认SARS-CoV-2病毒核酸阳性。
       
       Because each RT-PCR reaction takes about 2 hours to produce results, the kits are generally designed for the highly conserved 2-3 sequences in the virus sequence, such as the nucleic acid sequences encoding replicase or nuclease capsid, but the sequences used in each method are not exactly the same. Some methods use multiplex, that is, amplifying multiple target sequences in the same reaction tube; others use step-by-step, screening with one target sequence first, and then confirming with another sequence after positive, which can effectively shorten the whole process time and speed up the detection speed. In addition, RNA virus mutates rapidly, and multiple conserved target sequences can prevent the false negative caused by virus mutation.
       Currently approved products are novel coronavirus genome based on open reading frame 1a/b (open reading frame 1ab, ORF1ab), envelope protein (Envelope protein, E) and nucleocapsid protein (nucleocapsid protein, protein) to choose. The detection principles of different products are basically the same, but the design of primers and probes are different. There are differences in the detection and interpretation of single target region (orf1ab), double target region (orf1ab, N protein) and three target region (orf1ab, N protein and E protein). Generally, two targets located in orf1ab and N genes of SARS CoV are detected. The same sample needs to meet the requirements of double target positive or repeated detection for single target positive, or two samples meet the requirements of single target at the same time to confirm the SARS cov-2 virus nucleic acid positive.
                                                                  
                                                           



     检测原理：
     检测程序需要经过五个步骤，取样、留样、保存、核酸提取、上机检测。首先根据试剂盒说明书进行样本采集，样本类型包括咽拭子、鼻拭子、痰液、支气管灌洗液、肺泡灌洗液等。由于RNA易降解，因此，采集样本时使用无RNA酶的拭子和无RNA酶的储存管。获得患者样本后，需尽快进行检测，如无法立即检测需要进行低温封装，并送到专门的检测机构进行检测。检测机构收到样本后，对样本进行核酸提取，核酸提取试剂应使用批准产品说明书中指定的核酸提取试剂盒。最后是荧光PCR核酸检测，也就是上机器检测，将提取物进行荧光PCR扩增反应，需要70—80分钟。
     Detection principle:
     The detection procedure includes five steps: sampling, sample retention, preservation, nucleic acid extraction and computer detection. Firstly, samples were collected according to the instructions of the kit, including throat swab, nasal swab, sputum, bronchoalveolar lavage fluid, bronchoalveolar lavage fluid, etc. Because RNA is easy to degrade, samples are collected using RNase free swabs and RNase free storage tubes. After obtaining the patients' samples, it is necessary to carry out the test as soon as possible. If the test cannot be carried out immediately, it needs to be packaged at low temperature and sent to a special testing agency for testing. After receiving the sample, the testing institution shall extract the nucleic acid from the sample. The nucleic acid extraction reagent shall use the nucleic acid extraction kit specified in the approved product manual. Finally, fluorescent PCR nucleic acid detection, that is, on the machine detection, the extract for fluorescent PCR amplification reaction, takes 70-80 minutes.

                                                   
抗体检测
      病毒侵入人体后，人体会产生相应的特异性抗体进行防御。免疫球蛋白是机体在抗原物质刺激下由浆细胞产生的一类能与抗原特异性结合的血清活性成分，根据分子结构和抗原特异性的不同，将免疫球蛋白分为五类：IgG、IgA、IgM、IgD、IgE。
    After the virus invades the human body, the human body will produce the corresponding specific antibody for defense. Immunoglobulin is a kind of serum active component produced by plasma cells under the stimulation of antigen. According to the different molecular structure and antigen specificity, immunoglobulin can be divided into five categories: IgG, IgA, IgM, IgD and IgE.


                                                                     

      
         抗原初次进入机体后，经过一定的潜伏期产生浆细胞并合成分泌抗体。最早出现的是IgM，但该抗体维持时间短、浓度低、亲和力较低，在血中持续数日至数周，是急性期感染的诊断指标;在IgM接近消失时，IgG的含量达到高峰，IgG产生晚，但浓度高、维持时间长、亲和力高，血清IgG阳性提示感染中后期或既往感染，当数月乃至数年再次接触相同抗原时，最初原抗体量稍有下降，可能是由于一部分原有抗体被再次进入的抗原所结合，从而暂时降低了抗体含量，但短期内抗体含量迅速增加，可能比原来抗体含量高数倍至数十倍，以IgG为主，在体内持续时间也长，IgM很少增加。
         After the antigen enters the body for the first time, it produces plasma cells and secretes antibodies after a certain incubation period. IgM was the first to appear, but it had a short duration, low concentration, low affinity, and lasted for several days to several weeks in the blood, which was a diagnostic index of acute infection; When IgM was close to disappear, the content of IgG reached the peak, IgG was produced late, but the concentration was high, the maintenance time was long, and the affinity was high. The positive serum IgG indicated the middle and late stage of infection or previous infection. When the same antigen was contacted again for several months or even years, the amount of the original antibody decreased slightly, which may be due to a part of the original antibody was combined by the re entered antigen, thus temporarily reducing the anti-virus activity However, in a short period of time, the antibody content increased rapidly, which may be several to dozens of times higher than the original antibody content. IgG was the main body, which lasted for a long time in the body, and IgM rarely increased.
                                                                  

      本次疫情中，通过研究者对COVID-19患者进行研究发现，病毒侵入人体后，IgM抗体大约需要5-7天产生，IgG抗体在10-15天时产生。2020年3月4日，国家卫生健康委员会发布了最新版本的《新型冠状病毒肺炎诊疗方案(试行第七版)》在原有诊疗方案的基础上增加了血清学检测，作为确诊病例和疑似病例的诊断依据之一。血清学检测便是通过检测血液样本中的特异性抗体IgM和IgG的存在及含量，来间接判断体内有无病毒及病毒感染情况。目前临床中常用的抗体血清学检测方法有3种：酶联免疫吸附试验法、化学发光免疫分析法、胶体金免疫层析法。
       In this epidemic, the researchers found that it takes about 5-7 days to produce IgM antibody and 10-15 days to produce IgG antibody after the virus invades the human body. The novel coronavirus pneumonia treatment plan (trial version 7) was released by the national health and Health Committee in March 4, 2020. Serological detection was added to the original diagnosis and treatment plan as one of the diagnostic bases for confirmed cases and suspected cases. Serological detection is to indirectly determine whether there is virus and virus infection in the body by detecting the presence and content of specific antibodies IgM and IgG in blood samples. At present, there are three kinds of antibody serological detection methods commonly used in clinic: enzyme-linked immunosorbent assay, chemiluminescence immunoassay, colloidal gold immunochromatography.

酶联免疫吸附法（ELISA）
Enzyme linked immunosorbent assay (ELISA)

     ELISA是一种结合抗原、抗体特异性反应和酶对底物高效催化作用的高敏感性免疫学实验技术。测定时，受检的标本与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体，也通过反应结合在固相载体上。固相上的酶量与标本中的受检物质的量成比例。加入酶反应底物后，底物被催化成有色产物，产物的量与标本中受检物质的量直接相关。所以，可根据呈色的深浅进行半定量或定性分析。该检测方法灵敏度较高，载体标准化难度较低，但检测速度慢、易污染、步骤较为繁琐。在实际应用中，需要根据抗原抗体的特性设计不同的检测法，目前有双抗体夹心法、免疫抑制法、竞争法、直接法&间接法等类型。 
     ELISA is a highly sensitive immunologic experimental technique which combines antigen, antibody specific reaction and enzyme catalysis. During the determination, the tested sample reacts with the antigen or antibody on the surface of the solid carrier. The antigen antibody complex formed on the solid carrier is separated from other substances in the liquid by washing. Then the enzyme labeled antigen or antibody was added to the solid carrier by reaction. The amount of enzyme on the solid phase is proportional to the amount of tested substance in the sample. After adding enzyme reaction substrate, the substrate is catalysed into colored products, and the amount of products is directly related to the amount of tested substances in the sample. Therefore, semi quantitative or qualitative analysis can be carried out according to the shade of color. The detection method has high sensitivity and low difficulty of carrier standardization, but the detection speed is slow, easy to pollute and the steps are complicated. In practical application, we need to design different detection methods according to the characteristics of antigen and antibody. At present, there are double antibody sandwich method, immunosuppressive method, competitive method, direct method & indirect method and so on.
 
                                           





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