Since the outbreak of the new coronavirus epidemic in 2020, 'nucleic acid testing' has begun to recur in our lives as an important indicator for detecting whether or not we are infected.2022 On March 10th, the State Council's Comprehensive Group on Joint Prevention and Control Mechanisms for Responding to the New Coronavirus Pneumonia Epidemic issued a notification deciding to push forward with the testing model of 'Antigen Screening, Nucleic Acid Diagnosis,' adding antigenic test as a supplement to the nucleic acid test.
What is antigen detection? How is it different from other tests? In this article, we'll talk about common rapid screening tests, the rationale for them, and their scope, using novel coronaviruses as an example.
What can we look for when we screenTo screen for a disease or a substance, the first thing we need to figure out is where to start, and then how to detect it, so that the changes in the microscopic world are reflected in front of our eyes to help us make a judgment.
Where to startWe are dealing with viruses. According to familiar knowledge from secondary school biology classes, viruses are a class of life-like organisms composed of genetic material and protein shells . If you want to detect the infection of a virus, you need to start with its components. The next part of the content, I hope you bring their secondary school biology knowledge to read.
Take the SARS-CoV-2 that is currently plaguing us. It belongs to the genus Coronavirus B in the subfamily Coronavirus, under the family Coronaviridae, and is the seventh coronavirus known to be able to infect humans. All coronaviruses are positive single-stranded RNA viruses with an envelope, meaning that their genetic material is a single RNA strand, and that this RNA strand is directly involved in translation as mRNA (messenger RNA), which directs protein synthesis.
Toxigenic species number NPRC 2020.00002, image courtesy of the National Pathogenic Microbial Resource Repository (Institute for the Prevention and Control of Viral Diseases, Chinese Center for Disease Control and Prevention).
Our goal now is to detect the presence of this virus in a specimen, either on its own or by detecting the products brought by the virus, and there are only two directions in which we can start: the protein shell (envelope), and the genetic material.
How to detectFollowing this logic, the most obvious way is to check whether it can be seen, but the virus itself is very small, the diameter of SARS-CoV-2 is 80-120 nm, and it is unrealistic to take every specimen through the electron microscope, and the manpower, material, and financial resources can not support it. A more cost-effective way is to take some measures to make the viral components, or the special substances that appear because of the virus, accumulate to a certain order of magnitude and then emit light, change color, and appear macroscopic manifestations.
Our problem then translates into choosing a way to observe a macro-scale change that correlates with a component of the virus, a substance triggered by the virus. The substances we can choose from are also on the table: the genetic material of the virus, in this case its RNA; the envelope of the virus, the protein shell; and, if you remember some basic biology, the fact that the human immune system produces antibodies to fight off the invasion of a virus after it has been infected, which is also a good choice.
Several of the tests we currently use are derived from these substances (and their related substances): nucleic acid testing for genetic material, antigen testing for the envelope, and serum antibody testing for antibodies.
Nucleic acid testingAs the genetic material of a virus, nucleic acid sequences contain the genetic features that identify a virus as a particular species, so a positive nucleic acid test means that the virus was present in the body.
The "nucleic acid test" we currently perform is actually divided into two parts. The usual "nose and throat" sampling and subsequent characterization is the first part. After the specimen is obtained, because the amount of virus is so small, the sample is amplified a certain number of times in the lab and judged positive or negative based on the fluorescence reaction.
The second part, the samples that are determined to be positive, also need to be genotyped by gene sequencing to determine the typing of the sample virus for traceability. This step is no longer part of routine screening, but it is important in epidemiologic investigations, and if you are interested in learning about it, you can see Wikipedia for a brief overview.
Nucleic acid testing, which we usually engage in as a screening tool, refers to the first part of the collection to characterization.
After infection with SARS-CoV-2, viral nucleic acids can be found in specimens such as throat swabs, sputum, lower respiratory tract secretions, and blood. Positive rates of nucleic acid detection vary among different parts of the specimen, and the detection rate of each part of the specimen changes as the disease progresses.
What we are used to calling "nasal swabs" and "pharyngeal swabs" are in fact collections of secretions and tissues from the posterior wall of the pharyngeal cavity, the former from the nasopharynx and the latter from the oropharynx. There are other standards, such as saliva, that can also be used as a test specimen, but essentially the regulations vary from region to region
Nasal (pharyngeal) swabs and (oropharyngeal) swabs are already a combination of positive rates and convenience considerations. Feces and urine can also be used for specimen collection. Moreover, according to a study of 31 patients, anal swabs were more accurate than nasopharyngeal and oropharyngeal sampling, especially late in the course of the disease, when anal swabs were positive in less than 30% of the nasal swabs in cases confirmed by anal swabs. 4 Clearly, however, operational limitations preclude its use as the preferred means of early screening.
The next step was to extract nucleic acids from the little specimen that was obtained. Since the virus in the sample was of such a small order of magnitude that it was not sufficient for analysis, it also needed to be amplified and labeled. You need to use the same knowledge that you learned in high school: polymerase chain reaction (PCR) - this step may seem troublesome, but because its principles and processes have been studied and matured, in practice, you only need to add the reagents and send the machine, the most troublesome part of the whole nucleic acid testing process is to let the person who is to be tested to get the specimen in peace (laughs).
Local CDCs or testing centers purchase appropriate nucleic acid extraction kits and nucleic acid detection kits. Extraction kits are responsible for extracting RNA from mixed samples (cellular debris, secretions, dust, and other contaminants), and are commonly available as magnetic beads, centrifugal columns, and releasing agents, which may have a slight impact on the accuracy of the assay at a later stage. After that, the purified RNA is handed over to the test kit (or some kits combine the two) for subsequent processing.
The process that the kit carries the sample through the machine is the main reaction in the assay: RT-qPCR (real-time quantitative reverse transcription-polymerase chain reaction).
The next step requires you to pick up your knowledge of high school biology. A typical PCR reaction has the following steps:
Heating: allows the double-stranded DNA to deconvolute and become two single strands; Annealing: allows the mixed single-stranded DNA to bind to primers designed for the fragments that need to be replicated; Extension: adjusts the temperature so that the DNA polymerase starts working down the primers, replicating the new strand and forming a new double strand.In the detection of viruses, we have to do the same thing as above, but with two additional things:
before the first reaction, we use reverse transcriptase (RNA-dependent DNA polymerase) to synthesize the complementary strand of the viral single-stranded RNA, which is assembled into the cDNA ; in the annealing and extension stages, we use the reverse transcriptase to synthesize the complementary strand of the viral single-stranded RNA. At the annealing and extension stage, the TaqMan probe is needed in addition to the primers and enzymes.You can think of a TaqMan probe as follows: its main part is an oligonucleotide chain designed to pair up in a double-stranded fashion with a small portion of the gene fragment to be replicated; it has a fluorescent molecule attached to one end and a switch (quenching group) attached to the other end, so that when the two are connected to the probe the fluorescence will be suppressed by the quenching group and will not be detected. When annealed, this probe binds to the single-stranded fragment to be replicated along with the primer. During elongation, DNA polymerase cuts up any obstacles in its way, including this probe, and the quenching group and the fluorescent molecule are thus separated, and the fluorescence manifests itself.
As the number of cycles increases, so does the number of amplified DNA fragments and fluorescence. Comparing the fluorescence brightness of each cycle to the baseline brightness of the previous cycles, we can derive the current amount of DNA fragments, or we can directly use the number of cycles and fluorescence brightness to make a qualitative judgment.
So which part is being replicated? Since we are detecting viruses, we pick the most representative nucleic acid fragment. In the current standard, the ORF and N genes are the most common loci.
The test kit is responsible for putting in the extracted RNA (the sample), setting the PCR temperature and duration according to the instructions on the kit, controlling the amplification process by the machine, and collecting the fluorescent signal at a fixed point to record the number of cycles (Ct value). The criterion for determining whether a negative-positive / still infectious is the current cycle count when the fluorescent signal reaches the threshold value. According to the current "Novel Coronavirus Pneumonia Diagnostic and Treatment Program (Trial Version 9)," the criteria for release from quarantine management is a Ct value ≥35. The time between discharge and release from isolation is much shorter than the previously accepted criterion of ≥40.
ImmunoassaysNucleic acid testing via RT-PCR has been the gold standard for diagnosis until now because it is (theoretically) 100% accurate from a methodological standpoint. However, nucleic acid testing is time-consuming, environmentally and operatorally demanding, and when environmental conditions are substandard and supplies and instrumentation are not available, large-volume nucleic acid testing can be very labor-intensive and costly.
In this outbreak, the immunoassay we used included a rapid antigen test and an antibody test, which uses the antigen-antibody reaction as the basic principle, aiming to detect the presence or absence of the substance to be tested in the sample with less time and cost through the rapid neutralization effect of antigen and antibody. Both belong to the category of immunochromatography.
Self-administered antigen tests, which come in a boxed format, are a great supplement to self-assessment when supplies are low.
Antigen testingNucleic acid testing examines the (signature) genetic material of the virus, the "inside" of the virus. A (rapid) antigen test examines the "outside" of the virus, directly examining the intact virus particles. Currently approved antigen test kits include three types: colloidal gold, latex, and fluorescent immunochromatography. All three have the same intrinsic principle. However, the fluorescent immunochromatography kit still requires a special detector or ultraviolet flashlight, which is not suitable for home self-testing; colloidal gold and latex methods are converted into visible bands, the difference is that the substances used to mark the color are different.
Of course, the antigen test naturally has its disadvantages in that its false negative rate (positive but negative) is higher, which may lead to missed tests and wrong tests. But given the time advantage of getting results in a cup of tea, the accuracy gap can give way in certain circumstances.
Image source: How the SARS-CoV-2 EUA Antigen Tests Work | ASM.org
Compared with nucleic acid testing, antigen testing reduces the difficulty of self-testing for individuals by adding a 'nasal swab' as a sampling route. The sample on the swab is eluted in buffer, and when a drop of liquid is added to the spiking well, the liquid passes through an area preloaded with antibodies (the conjugate pad) due to capillary action, carrying the underlying antigen with it.
The antibodies in this area are monoclonal antibodies against the target antigen (SARS-CoV-2), and each antibody molecule is conjugated to a specific marker, which reacts with the antigen in the sample to form an antigen-antibody complex, which travels by capillary action to the next band.
Immediately after this is the test line, to which the same monoclonal antibody against the target antigen is attached, which you can take to mean that it is the same thing as on the binding pad, but without the marker. At this point, if the subject has been infected with SARS-CoV-2, the antigen-antibody complex formed by the antigen he left in the sample will bind again to the antibody on the line. Here, these labeled complexes are deposited and eventually show a dark or light band. The source of the color of the band is the markers attached to the antibody molecules on the previous binding pad, which are gold particles in a colloidal state in the colloidal gold method, colored latex droplets in the latex method, and fluorescent molecules in the fluorescence method. So you'll notice when you use this type of paper that just after you finish spiking the sample and the liquid just starts to diffuse, there's a little bit of very light color that keeps pushing at the very tip of the diffusion, and that's the color of the markers that haven't been fixed and deposited yet.
Next, the liquid continues to spread and passes through the quality control line (C line, control line), where another antibody is attached - the 'anti-'monoclonal antibody against the target antigen'' (monoclonal antibody against the target antigen). monoclonal antibody, or secondary antibody for short. The new antibody is made by reacting the previous antibody in the immune system of another animal. For example, if the antibody that binds to the pads comes from a rabbit, then the antibody in this case comes from a sheep, and it is a monoclonal antibody of a sheep against a rabbit. In other words, the antigen of the secondary antibody is the antibody that was previously on the binding pad. This line is there to detect if the liquid is spreading properly, if the antibody on the binding pad is not failing, and so on. At this point, the large amount of antibody remaining in the liquid from the binding pad acts as an antigen and reacts with the secondary antibody on the QC line, forming a complex that shows a distinct band.
Because of the abundance of antibody on the binding pad, this QC line band appears very quickly and is very colorful, whereas the detection line varies in speed due to the amount of antigen (virus), but is generally sufficient to determine the result within 15 minutes. So don't look at the C line as obvious and the T line as faint and think, "It's okay," because the T line, no matter how dark or light it is, is positive.
For more information on how to do this, please refer to the instructional video published by the Medical Affairs Bureau. The state is also gradually promoting antigen self-testing kits, which to a certain extent can reduce the pressure on medical care and streets in the future.
Serum antibody testingIn addition to the first two tests, there is a less commonly used but equally important test, the serum antibody test, which is also an immunoassay.
Antibody testing uses a kit that is very similar to antigen testing, but the specimen is much more restrictive - since the test is for antibodies, the specimen must be blood (or plasma, or serum) in which antibodies are clearly present. Moreover, the body does not produce antibodies in the first place after the initial infection with the virus. Antibodies can definitively reach the order of magnitude to be detected, typically one to two weeks after the initial infection (or vaccination). These conditions limit the ability of antibody testing to be used as a confirmatory test. Currently, serum antibody testing is used only as a means of checking whether the vaccine is working in certain circumstances, or to verify that the subject has had a recent infection with a new coronavirus.
There are five antibodies in the human body, IgA, IgD, IgE, IgG, and IgM. IgA is responsible for mucosal immunity, IgD is associated with the activation of the immune response, and IgE protects against parasites and is also involved in allergic reactions. IgE protects against parasites and is also involved in allergic reactions. The remaining IgG and IgM are the mainstays of the immune system in the fight against pathogens.
SARS-CoV-2, as a pathogen, is stimulated to secrete IgG and IgM. Existing antibody test kits also target IgG and IgM produced by the body in response to the N (nucleocapsid protein) or S (spiny protein) proteins of SARS-CoV-2.
The appearance of the antibody test device is similar to that of an antigen test. The difference between the two is the material attached to the binding pad, detection line, and quality control line as mentioned above.
In this case, the sample dropped in the spiked well may have antibodies to SARS-CoV-2. Therefore, the binding pad should have the tagged antigen on it - it is not possible to put live virus on it. Typically the antigenic protein designed to be detected is used here, such as the aforementioned N or S proteins, or a recombinant virus, either of which must contain a receptor-binding domain (RBD) as a target for antibody binding. On the detection line, it is the anti-IgM or anti-IgG antibody that is attached to capture the antibody protein that has bound the antigen. Finally, an antibody specific for the antigen is attached to the quality control line to capture the remaining free antigen.
SummaryOverall, the three types of assays address different needs and complement each other. Nucleic acid testing, as the gold standard, directly checks the RNA of the virus and is responsible for determining whether a person has the virus or not; antigen testing, as a rapid test, checks the protein of the virus, but is less accurate than nucleic acid testing and is more effective for highly contagious infections; and antibody testing checks whether the vaccine has taken effect and whether a person has been infected with the virus in the recent past.
With the recent resurgence of the new virus, the Omicron variant has a shorter incubation period, faster replication, and increased infectiousness compared to the previously prevalent Delta variant, although the mortality and severe disease rates are significantly lower. I hope everyone stays healthy in this environment.