May 19 news, the Future Forum joint science, medicine, clinical and other fields of well-known experts in the creation of “Understanding the future” scientific lecture: virus and human health – the topic of science, the 10th live broadcast, Beijing Future Gene Diagnostics High-Tip Innovation Center Deputy Director, Peking University Professor Huang Yanyi brought us “new coronavirus laboratory testing” theme sharing.
Professor Huang introduced the technical methods, principles, results judgment and practical application limitations of detecting new coronaviruses. He said: the detection of the new coronavirus is mainly divided into nucleic acid detection and antibody detection, the two detection methods are completely different, different purposes, different sampling, different methods, the results of different interpretation, the scope of application is also different. Molecular diagnosis is an important part of disease diagnosis. The diagnosis of disease is a complex problem, especially for new infectious diseases, molecular diagnosis in the case of known pathogens has become an extremely important indicator. Molecular diagnosis in the narrow sense is generally nucleic acid detection, including qualitative and quantitative analysis of specific nucleic acid fragments, the use of nucleic acid hybrid chips, sequencing techniques and so on for sequence analysis. If you want to expand into a broad molecular diagnosis, it may also cover other molecular pathology techniques corresponding to traditional pathological diagnosis, including antibody detection. But the convention is common, in general we say molecular diagnosis is called nucleic acid testing. In infectious disease detection, nucleic acid testing refers to the test of whether the sample contains a specific sequence of DNA or RNA fragments, as evidence of the existence of pathogenic microorganisms.
At the same time, he said, the most common sample of nucleic acid testing is a nasopharyngeal swab, which requires a professional approach. More than a month ago, Huaxi Hospital raised the possibility of a new crown diagnosis with saliva. This is an easy self-sampling operation. The FDA has also approved the use of home-collected saliva samples for new corona testing. In addition, Yale University in the United States compared the effects of saliva and nasopharyngeal swabPCr nucleic acid testing. The experimental results show that the stability of saliva sampling is quite good, or even better, than the nasopharynx swab. This is a good starting point.
Speaking of antibody testing, Professor Huang Yanyi said: Antibody test samples are blood or serum, rapid detection kit can use colloidal color for test paper testing, similar to pregnancy test paper. The benefits of this approach are very simple, easy to use, inexpensive, and require no complex equipment, very fast.
The following is the full text of Huang Yanyi’s speech (with limitations):
Huang Yanyi: Hello everyone, I am Huang Yanyi. I work at Peking University and am now a researcher and deputy director of the Center for Advanced Innovation in Future Genetic Diagnostics in Beijing, and I am also a researcher at the Center for Biomedical Frontier Innovation at Peking University and a professor at Peking University’s School of Chemistry. I am mainly engaged in the research of life analysis chemistry, especially the analysis of micronucleic acid technology, focusing on gene sequencing methods, single-cell analysis and microflow control technology in several directions. At the invitation of the Future Forum, I’ll give you a chat about the detection of the new coronavirus.
This is two hours before the New Year’s bell rings on December 31, 2019, and I took a picture of it at Peking University. The lake is covered with ice, the reflection of the lake is the reflection of the lake, is the lights on the Boyata. That day I was busy, did not read the news; By the time I know the news, you may know it. I don’t use twitter, so the source is always slow. Over the next few days we slowly got a lot of news, and I began to care about the news, even though I didn’t know if the sudden situation would affect my life. About a week later, the cause of pneumonia has been identified, and that’s when I began to pay more attention to it. Why is that? Because it’s a new coronavirus, although I don’t know what a coronavirus is, I’m interested in getting the whole genome sequence of the virus, because I’m a researcher doing sequencing research. When I saw the virus from the sequence, I thought it was really remarkable. From the time I saw the news, to the fact that it was less than a week before I got the final result, it was very uncandy lyme, and it took only a few days for us to identify new pathogens.
Recall the process of identifying a pathogen, and what to do if it is a new infectious disease? It’s never easy. At first the news spread on the Internet is SARS, but also we often say SARS, come back again. It was discovered so quickly that it was not SARS, but a brand new virus, and was very close to SARS, thanks to the rapid development of high-throughput DNA sequencing technology over the past 15 years or so. Usually identify the pathogen of a disease, to comply with the so-called Koch rule. That is to say, first of all, to find the host body of pathogenic microorganisms, found that it does not exist in healthy organisms, to find ways to separate, get its pure culture, re-inoculated into the host body, again isolated to get this culture, found that the host of this culture inoculated will inevitably lead to the occurrence of disease, and so on. The Koch Law was officially published 130 years ago and has so far dominated the diagnostic criteria for the vast majority of infectious disease pathogens. But with the development of science and technology, in the new era, with the rapid development of genomics, many laws used to identify new pathogens, there are new changes.
For example, pneumonia, for example, the symptoms of which are usually poor oxygenation in the patient’s alveoli filled with fluid. What pathogen sedits pneumonia is? It can be said that there are many. In addition to the most common streptococcus pneumoniae, there are many pathogens that can cause pneumonia. For example, the various pathogens shown in the figure below, which I found online, can cause pneumonia.
In general, it is divided into three categories: viruses, bacteria and a few fungi. The virus is the most troublesome inside because it is particularly small. Viruses with human beings for a long time, the history of human civilization has never been interrupted, has always had its existence, difficult to deal with. Viruses have been on earth for much longer than we have, and as human civilization progresses, it will always follow us. Polio, now slowly rare due to the spread of vaccines, known as poliomyelitis, has taken more than half a century to basically eradicate it. A few months ago I saw news from the World Health Organization that polio-type III wild virus had been eliminated worldwide, a subtype that had been completely eliminated. One reason the virus is so difficult to deal with is that it is so small that its scale is very different from that of the naked eye, which is invisible to the naked eye and varies by orders of magnitude. The advent of optical microscopes helped biologists and pathologists find pathogens that helped Koch identify pathogens such as Bacillus tuberculosis and Vibrio cholerae, but he had no way of using these tools to detect the virus, which exceeded the limits of optical microscopes. It was not until 1939 to 1940 that Ruska, a German scientist, first saw virus particles under an electron microscope, including tobacco leaf virus, bovine pox virus, etc. This had to be thanked to his two-year-old brother, who was the inventor of the electron microscope and later won the Nobel Prize in his later years. This time we’re determined for the new coronavirus, thanks to these new technologies.
Let’s go back to January this year. In late January, several scientific papers written by Chinese scientists were published, giving us a comprehensive understanding of this new pathogen. News reports are news, but read scientific papers and you can feel the importance of detail. First, in the New England Journal of Medicine on January 24th, we saw the entire genome sequence of the virus. From the sequence, biologists can clearly determine that this is a coronavirus, and from the sequence analysis can determine that this is a new virus, in human history has not been found before the new virus, belong to the Beta genus, is infected with the seventh member of the coronavirus family infected with humans.
Above, the first scientific paper showing a photo of the new coronavirus’s transmission electron microscope, which shows its small head – by scale, is only about 100nm in size, but looks very similar to other coronaviruses.
On the same day, The Lancet published the results of the study of the clinical characteristics of new crown patients by the team of Professor Wang Jianwei of the Chinese Academy of Medical Sciences and Director Cao Bin of the Sino-Japanese Friendship Hospital. The first mention here is that a technique, the RT-PCR method, is used for nucleic acid testing when diagnosing a patient. There are indications that this is a new disease that presents us with new challenges, not only for science, for medicine, but also for patients.
The third important paper, published in our own Chinese journal, the Chinese Journal of Medicine, systematically describes the characteristics of the new coronavirus. In the subsequent news in the China Science Daily, we can see that the pathogen lock-in process is very rapid and efficient. Several teams moved in parallel to begin sequencing, greatly increasing the reliability of the results. In the serological study, it is also revealed that the antigen antibody response between the virus and the serum during the patient’s recovery period is a very complete scientific discussion.
The fourth is the other team in the three parallel tests, Wuhan Virus Institute team. Their results, like those of other teams before them, were given a full sequence of several viruses.
Another important result is that these colored optical microscopes on the right show (above) that the human ACE2 protein is an important way for viruses to invade human cells, which provides important clues for the study and treatment of viral pathogenic mechanisms and drug development.
The fifth paper was published at the same time as the previous one, the Shanghai team, from an early Wuhan a single patient to take samples, through high-throughput sequencing methods, to obtain the full sequence of the virus, with it and SARS virus sequence comparison. It was thanks to the rapid response and high-quality work of Chinese scientists in the first week of January that the virus’s reference genome was soon obtained. By referring to the genome, we have a coordinate system.
First, it is understood what each sequence of the virus genome is. For example, the above image marks the road, is a gene, used to encode a piece of functional protein, exercise specific functions, some allow the virus can be copied, some let the virus is conducive to invasion of the human body, some are to build the virus’s structural framework. Second, with this reference coordinate, the virus mutates again later, and we know what is going on, which is very important for our deeper understanding of the basic functions of virus biology, as well as its natural history, and the occurrence and development of disease.
The image above is the main protein encoded by the virus genome, showing the predicted protein structure, the result of a team of Professor Zhang Yang of the University of Michigan. From here, there is a very important protein called Spike protein ( S protein), and other such as M protein, N protein, and so on, these different proteins are very specific expression of the virus protein. These results can be built out, now can often be seen in the news model of the new coronavirus, the virus appearance of these proteins are generally referred to by me these proteins, these proteins give the virus specific function, not only give it a structural support, but also for us to test can also become an important goal for us. We can determine the presence of these viruses by detecting specific components of these viruses, such as target proteins or the genes that encode them.
It is because of our interpretation of the virus sequence that we immediately know that it is a close relative of its arch-enemy SARS coronavirus 17 years ago. There’s no need to tell everyone how to see this diagram, you can see that they are quite like. It’s not just like SARS, it’s also about MERS 10 years ago. This also shows that we can’t look down on the virus, which can be devastating. Due to the similarity with SARS, the International Committee on Virus Naming, which originally named it the 2019 new coronavirus, is SEVERE Acute Respiratory Syndrome coronavirus 2, or SARS-CoV-2. SARS-CoV-2 indicates that it is still relatively close to the original SARS virus. A recent paper by German scientists also showed that the new SARS coronavirus and the original SARS virus did use a very similar strategy when attacking cells, by attacking the same protein to invade the cells. It is also found that antibodies of the previous SARS virus also have a certain neutrality effect on the new virus, revealing the high correlation between the two viruses.
Entering the era of biomoleculars, the understanding of infectious diseases begins to change, starting with the understanding of the molecular level. Knowing the coronavirus is not like it was in 2003, when we weren’t sure about the pathogen, called it atypical pneumonia. The International Health Organization now names this new disease as “COVID-19”, a disease caused by coronavirus infection, rather than simply a symptom. Our national health care committee is named “new coronavirus pneumonia”. So most of the time we are in China, or the new coronary pneumonia to call COVID-19.
Above, scientists at the University of Hong Kong saw the coronavirus in January, which has been replicated and released from cells.
Above, a large head image of the coronavirus taken by American scientists in February, shows the surface of the “flower crown”, which is a pseudo-color photo, a good indication of the existence of the “flower crown.”
Not long ago, the U.S. Centers for Disease Control and Prevention released a new collective photo of the coronavirus (pictured above), blue marked with virus particles, the virus particles in the small black spot is cut on the section of the nucleic acid, is the coronavirus contains parts of the genome. This genome is of great importance to our testing and is the material basis for our testing.
Molecular diagnosis is an important component of disease diagnosis, which is a complex problem, especially when new diseases arise, and molecular diagnosis in the case of a known pathogen is an extremely important indicator for an infectious disease. Its main purpose is to: First, it determines clinical symptoms, such as whether fever is caused by a new coronavirus, to prevent it from being confused with other diseases. Second, characterize the course of the disease (e.g. positive) or quantitatively described, such as the amount of the virus. Third, from a more nuanced point of view, certain molecular diagnoses can also provide a treatment plan for the disease, helping to determine its treatment plan and providing evidence support. Fourth, from the point of view of viral diseases, it is also very important to track the mutation, research and predict the virus in the transmission process of infection and the evolution of disease-causing capacity, such as my colleague Lu Jian teacher’s article, he pointed out that there are some particularly closely related mutation sites, and the disease is likely to have a very close indication of the link, so it is likely to be the treatment of future diseases, and understand how the virus host and co-existence is very important.
What is molecular diagnostics? Molecular diagnosis in the narrow sense is generally nucleic acid detection, including qualitative and quantitative analysis of specific nucleic acid fragments, the use of nucleic acid hybrid chips, sequencing techniques and so on for sequence analysis. If you want to expand into a broad molecular diagnosis, it may also cover other molecular pathology techniques corresponding to traditional pathological diagnosis, including antibody detection. But the convention is common, in general we say molecular diagnosis is called nucleic acid testing. Nucleic acid testing is the test of whether a specific sequence of DNA or RNA fragments in the middle of a sample is present as evidence of the presence of pathogenic microorganisms. In addition to the molecular diagnosis of pathogenic microorganisms, this method has been used before the outbreak has been very common, such as a large number of other infectious diseases diagnosis, genetic disease diagnosis, tumor classification, chemotherapy medication guidance and many other medical practices are used in nucleic acid, but at that time did not mention so much.
In addition, we do antibody testing is antibody testing, antibody testing is not the virus itself, but to see whether there are antibody molecules in the middle of the blood or body fluids.
Another test is antigen testing, the target is the virus body of substances, such as the blue mark we see in the figure above, both are virus-specific proteins, protrusion S protein and n-shell N protein, to see if they exist in the sample, is antigen testing. It’s all based on the material basis of the time we’re trying to detect.
Detection, in the final analysis, is a measurement of molecules, measuring the number of molecules; The Israeli and American scientists listed some key data on the coronavirus. From the test, in addition to nucleic acid molecules (the green arrow spout in the image above), it is a molecule in each virus, there are many protein molecules (a few red arrows marked), in the number of viruses. But the number of these molecules is distributed differently, with S proteins, probably hundreds per virus, and N proteins and M proteins, thousands of per virus. Understanding these quantities will greatly help us to develop specific detection techniques in the future to determine the presence of viruses.
As can be seen from the seventh version of the new coronavirus pneumonia, the diagnostic indicators are very clear, the evidence on the pathogen has two, one is fluorescent RT-PCR, and the other is gene sequencing. The nucleic acid sequence characteristics of the virus are very different from those of other species, such as animals and humans, and these two methods are less error-prone to identify sequences. With the development of the epidemic and our understanding of the natural history of the virus deepened, the diagnosis and treatment program adjusted to the later time, the serological testing also added, we will talk about later.
Regarding testing, the CIRC’s notification and FDA approval have always been around nucleic acids and antibodies. The two detection methods are completely different, different purposes, different sample sources, different detection methods, the results of different interpretation, the scope of application is also different. Here’s a little introduction to these two methods.
Nucleic acid detection, first of all sampling.
Now to accept nucleic acid testing more people, we see more news, see the picture above will not feel too fresh. In fact, I haven’t experienced swallowing swabs before last year. More people have only seen such a laboratory environment in pictures, and have not really experienced it. The real process is more complicated than the picture might be. Sampling, generally in the laboratory outside the relatively empty place, the sampler must be professionaltraining, but also pay attention to protection. Because this is a risky operation, not only in the face of people with the potential to be infected, but also in the sampling process itself increases the risk of exposure. For example, a sneeze or cough in the sample increases the risk of exposure.
There are two keys to ensuring successful sampling, one is that the material used, the swab, is fine, not just a cotton swab, and the preservation environment after swabs is sampled is also exquisite. In addition, the operation of sampling itself is exquisite, the method is also important, can not be taken in the past. The selection and operation of sampling sites are the basis for obtaining high-quality samples, and only if the sample quality is good can reliable detection be made. For example, the most common sample is a nasopharyngeal swab, note that the sampled swabs should be extended all the way to the last side of the nasopharynx, above the mouth and throat. This is not an easy operation, very pay attention to experience and methods, do it is also difficult, generally do not recommend their own so sampling. There is a throat swab, open mouth, but also to go long swabs deep into the back of the mouth, in the throat, tonsil edge. This sampling is not easy to do, usually to press down the tongue. Why is sampling so elaborate? Because the harvest did not pick, did not pick good, directly determine son you do testing in the end can see reliable results.
The differences in sampling sites are reflected in the test results are also different, and Chinese scientists have long summarized some of the rules.
For example, from the figure above a, it can be seen that as the symptoms progress, slowly as the signal goes down. As time increases (after symptoms), fewer nucleic acids are sampled. As a result, the amount of nucleic acid taken from the nasopharyngeal swab can be seen on the c-graph will be higher and the stability will be slightly better than the larynx swab. Early experience is of great significance to the process of guiding sampling later, namely, how to take and where to take the sample.
The characteristics of the disease lead to the virus infection site has characteristics, both preferences, but also by the time of onset of the effect. In fact, as a test, you can use a variety of samples, not just two swabs. The seventh edition of the diagnosis and treatment of the treatment of the nasopharyngeal swab, sputum, other lower respiratory secretions, blood, feces samples can be detected in the virus nucleic acid. There are also research studies that show that so many papers, it seems that in many samples such as tears, feces may detect nucleic acid virus, but the sputum or lower respiratory tract secretions inside is the most difficult to leak, that is, the detection rate is the highest, followed by nasopharyngeal swabs. So can find really do high-quality measurement research, the source of the sample is very sophisticated, the requirement stoush must ensure the quality of sample sampling, only to get a good sample can get good results. The World Health Organization in January has guidance documents, the collection of different samples, preservation, use have done a good combing, with such guidelines, researchers and medical personnel around the world can follow a relatively formal and reliable process to ensure the reliability of results.
Sample collection after testing, the first step is usually nucleic acid extraction, that is, the nucleic acid part of the sample left, other parts of impurities, human shedding cells, other molecules such as protein molecules are removed. The usual nucleic acid extraction, the use of nucleic acid molecules and its affinity strong materials, such as silicone, first adsorption, wait until the other substances are separated, in the adsorption of nucleic acid molecules washed off, to achieve extraction, but also the steps of purification and enrichment. It’s possible here, in the lab, there are two methods. Both methods are now widely used in nucleic acid detection of new coronaviruses. Either the silicone film is used to adsorption, or the surface-modified magnetic particles are used to achieve adsorption. This step may seem simple, but the most important thing here is to take biosecurity into account. Interested viewers can watch the Chinese Academy of Medical Sciences Beijing Concord Hospital training and teaching video, this video completely demonstrated how to do nucleic acid extraction, but also highlighted the operator’s biosecurity issues. In order to ensure this, it is noted that all samples should be placed in a 56-degree water bath tank for 30 minutes before operation, so that the virus is incapacitated. The New Coronary Virus Laboratory Biosecurity Guidelines issued by the National Health and Reform Commission also say that the operation of uncultivated infectious materials needs to be done and should be maintained in a biosafety secondary standard laboratory, with the use of biosafety level III laboratory personal protection.
Above, a cartoon of the CDC in the United States. Biosecurity level three, simply put in the enclosed space in the use of negative pressure, all air and other substances will not be without treatment directly discharged to the outside world, such a biological experimental environment. The people inside and the heavily armed white soldiers we often see on television are not very different, are the same outfits, will only be more stringent, the need for no exposed body parts. Bio-secondary requirements are less stringent, air and the outside world are exchanged, and sometimes in individual areas can increase the level of biosecurity, to do more demanding experiments. This time Wu Chen led the Wuhan square cabin testing national team, is in a mobile biosecurity three-level laboratory work.
Above, a photo of Professor Wu Chen working at the scene in Wuhan. The working environment on the left is biosecurity level II, the car on the right is a mobile biosecurity level III laboratory, Wu Chen teacher’s dress is the standard biosecurity level three dress, if not have a name written there, you do not know who she is. The experiment was conducted with full force for biosecurity reasons.
Nucleic acid extraction is followed by amplification steps, which are achieved through several tubes of reagents provided by the manufacturer. On the left side of the image above is the reagent of Huada gene, which contains several small test tubes, the reagents distributed by the U.S. Centers for Disease Control and Prevention on the right, and several small test tubes. The detection process, called RT-PCR, is a reverse transcription-polymerase chain reaction. This is an in vitro replication operation of DNA molecules using the naturally present nucleic acid replication mechanism, which has been invented for nearly four decades, is the basic technology of molecular diagnosis and an important foundation technology for modern biology and medical progress. In each test tube, in addition to the nucleic acid extract in the sample to be tested, DNA polymerases, reverse transcriptases, and primers and probes are added. The new coronavirus is an RNA virus, the virus encased this genomic RNA, through reverse transcriptase to turn this RNA into complementary DNA, and then copied again, into double-stranded DNA, and then through the heat cycle over and over to untie the pairing of the double strands, respectively. 1 change 2, 2 change 4, 4 change 8, after the N-wheel cycle, theoretically can get the geometry of the growth of specific pieces of DNA. In the process of replication, a clever design allows each replication to produce a specific fluorescent molecule, and the more fluorescence the copied molecules produce, the more we can detect this signal, so that a round of replication goes on, and we watch the fluorescence grow. If there is fluorescence growth is the substance to be tested, that is, the so-called positive; The faster fluorescence grows, the more molecules, the slower the growth, the fewer molecules, and probably this process.
This process takes not too long, can give such a graph to come (see above), draw a line to cut past, and the fluorescent intensity curve cross the value if it is low proof that you may be positive, this value is too high or does not appear (up) to prove negative.
This judgment in fact still have to pay attention to, do in the process must be qualitative control, Yin nature control and yang nature control should have. Generally negative is pure water, inside should not be tested nucleic acids, should not signal; Positive, generally is a low concentration of samples, if we do not measure the signal, prove that the experiment is not sensitive enough, perhaps where there is a problem, need to check and redo.
In addition, the choice of probe sequence is the primer sequence is very exquisite, in about 30,000 base about the length of the sequence, to detect which paragraph still needs a lot of effort. A group of South Korean scientists, who recently published a comparative results, made a relatively objective comparison of the effectiveness of the nucleic acid testsites used separately in January. There is a difference between the sites identified by scientists from different countries for diagnosis. Here, I marked the location of these detected nucleic acid fragments in the genome, such as two sites in China, one orF1ab and the other N;
The image above is a sequence of n genes, which are color-coded, indicating the location of the primer used at the sites detected by each country. As can be seen, there are small overlaps that scientists in each country have determined in a small number of locations, but in general, they are not precise together. The results are very interesting, the Korean scientists’ comparative experiments put forward the location selected by China and the United States are the most sensitive of these tests, which also reflects from the side when our scientists select the sites very accurately, which requires experience, capability, and scientific judgment.
Can nucleic acid detection be improved? First look at the speed, a standard nucleic acid testing process, sampling is very fast, and then stored it in the transport medium, the sample saved to a certain number of times, concentrated inactivation, 56 degrees, 30 minutes, and then nucleic acid extraction; Can this process be accelerated? If it is a single sample, sample collection after direct lying, you can skip many steps, such as directly into the lyse, and then do rapid nucleic acid extraction, or even do not do extraction to do a quick PCR, so that may be in about 30 minutes to get the results. As can be seen, PCR testing itself can be done quickly, especially when the sample volume is small, but when the sample is more, it is best to operate in batches more efficiently.
I’ll talk about some testing methods later, let’s take a look at a review of the American scientists of the last few days and summarize the various nucleic acid testing methods that have been reported recently.
As you can see above, most of the time it takes to test the various methods can be done in an hour or two, with prices ranging from a few dollars to a dozen dollars. There are many methods here, some are no longer the so-called RT-PCR method, but still fully borrow the power of nature, the use of natural genetic material replication mechanism, to amplification of specific DNA fragments, and ultimately to achieve strong enough signal acquisition. The amplification reaction itself can be achieved in a variety of clever ways. In addition to RT-PCR, which has been repeatedly validated and optimized for more than 20 years and has proven to be an extremely reliable clinical test, many other amplification methods have been invented and are being rigorously tested in the face of the detection of new coronaviruses. Based on the pursuit of detection speed, even through the RT-PCR method, in order to reduce technical barriers and errors in operation, so-called POCTt equipment, that is, can be used in the field of small instrument devices.
The two above are the first two instruments approved by the Fda administration for emergency use in late March, allowing the entire sample-to-results testing process to be completed in 30 or 45 minutes, respectively. The use of such equipment during an infectious disease epidemic has at least two important benefits, one is to decentralize the processing of patient testing needs, avoid ingsampling of samples and difficulties in transportation. The second is the use of almost completely enclosed laboratory design, reducing the operator’s biosecurity risks.
The instrument above is probably the most popular of its kind, the u.S. super-sale of Trump’s own small instrument, unfortunately he did not put it right, the picture is not the correct placement method. The machine can give a positive result in 5 minutes, a negative result for ten minutes, and the overall time it takes to actually use it will actually take a little longer. To be clear, this is not a new invention, not a new coronavirus suddenly emerged such a new invention. The device has been on the market for a long time, and before it was bought by its new owner, Abbott, it was a portable device on the market for detecting flu virus microbes.
Above, for example, a 2014 paper by researchers at Futian Hospital in Shenzhen evaluated the device’s performance in detecting influenza. The basic technique used in this instrument is a relatively cold amplification method called NEAR (incision enzyme amplification reaction), the natural world there are many strange enzymes, can do a lot of things. Incision enzyme is the formation of a small incision on the surface of the DNA, and then let the polymerase find that place, can start to make replication, the continuous formation of incision, continuous replication. This is a very clever method that does not require PCR reaction and heat cycle, which belongs to the same temperature amplification. The meaning of sopeer amplification is that there is no need to require a heat cycle, and that the amplification process is carried out at a constant temperature, with the benefit that the process becomes very simple; In addition to NEAR, there are many isowarming amplification reactions that can be used in nucleic acid detection. For example, a relatively well-known LAMP ring-mediated temperature amplification, which was invented by Japanese scientist Notomi et al. around 2000, this method is also used four or six primer sequences added to the substance to be tested, its temperature slightly raised a little, usually at about 60 degrees temperature to do the same temperature amplification reaction, do not need a very complex instrument can be done quickly. One advantage of this method is that the device can do very simple, can be easy to operate, the naked eye can make a difference. The China Drug Administration has also approved in vitro diagnostic reagents based on such reactions, such as the new coronavirus diagnostic device based on LAMP amplification by Chengdu Boao Core.
There are other methods, one of which is RPA, which is relatively easy. It uses a combination of two enzymes, recombinant enzymes and polymerases, to amplify nucleic acid molecules through a very complex cycle, which is complex but very efficient and very fast. Based on RPA, Zhang Feng, a well-known young Chinese scientist in Boston, USA, combined it with gene editing to graft it to achieve RNA testing, they also named SHERLOCK (Sherlock Holmes’s name) also used the name to register the enterprise to do the development of this reagent. In San Francisco, California, Jennifer Donna, along with her collaborators, has developed a new method called DETECTR, a company called Mammoth Biosciences, based on the LAMP method. In comparison, both methods can be detected for 30 to 50 minutes, relatively fast. These two methods can be used in the form of test notes to read the results, but also easy to understand, do not need complex instruments.
In addition to shortening the time, for large-scale testing, improve the detection flux is very important, the kit can take a box can do a good job of multi-test, but the sample to one test. There are nucleic acid extraction so originally particularly physical and requires experience, skills, training work, it is best to give to the tireless, not easy to make mistakes machine to operate. So we see a large number of nucleic acid extraction automation instruments appear in the detection site, such instruments are located in various fixed-point hospitals, at all levels of the CDC.
The two instruments on the right of the image above are in the University Laboratory of the United States, which is now heavily added to the queue for large-scale testing to eliminate some of the anxiety of delays in detection. If it is a centralized processing of large-scale detection structure, it is necessary to use more complete automation of the instrument.
Above, Roche’s Big Mac instrument, Cobas8800, a machine that can complete about 3,000 nucleic acid tests without stopping a day, and is almost fully automatic, and starts doing it after putting the sample in. What’s the benefit? In addition to fast, there is an important advantage in ensuring that the results achieved are basically reliable and stable in different parts of the world. In this way, in the foundation is relatively weak, the accumulation is not strong enough, the same can achieve high-quality large-scale testing. In Kenya, Canada, the United States, China, the results of the test will not be due to operational problems and large deviations.
As the outbreak progresses, more and more testing needs emerge. As home isolation becomes the norm, can self-detection and self-sampling be feasible? The first home self-sampling kit product in the U.S. to get FDA approval was LabCorp’s Pixel, which uses a cotton swab in the nasal cavity and sends it back to the testing center for a large-scale test, eliminating the need for complex sampling. At present, no large-scale evaluation data have been seen. But after all, taking cotton swabs to the nose, is not easy to do a good stability, so more than a month ago, Huaxi Hospital researchers raised the possibility of using saliva for a new crown diagnosis. This is an easy self-sampling operation. Less than a month ago, scientists at Yale University published a paper comparing the effects of saliva and nasopharyngeal swabPC nucleic acid testing. The experimental results show that the stability of saliva sampling is quite good, or even better, than the nasopharynx swab. What’s more interesting is that the vast majority of patients see the results, saliva detected more viruses, the results may be more accurate, reduce the appearance of false negatives. It’s a good starting point, so a few days ago Rutgers University in New Jersey and their testing agency received approval from the U.S. Drug Enforcement Administration to start using saliva-collected samples for testing for the new coronavirus. They used a streamlined, automated machine for large-scale processing that could detect 10,000 samples a day. Saliva sampling detection is a very encouraging new approach, and if replicated it is expected to be a good solution to the problems we have faced in our past sampling.
As the first peak of the outbreak passes and the phase of resumption of work approaches, antibody testing becomes a new buzzword. How is antibody testing done? I’ve found that the first nine sessions have been all about these issues, and you’ve got a better idea of what antibodies are than I am. So I’m technically talking about how antibody testing is done.
First of all, the sampleised very differently, all with blood or serum testing. Antibody detection methods are varied, the most common is enzyme-linked immunosorption and immunochemical luminescence. The principles of these two methods are very similar and are the most common testing methods in laboratory research. Take the principle of enzyme-linked immunosorption detection as an example, in the detection of the substrate we first spread a layer of artificially prepared antigen, and then add the serum sample, incubating for a period of time, specific binding to the antibody; Changes in the color of the solution. Eventually we derive the concentration of the antibody you want to detect using the concentration of the substance that produces the color rendering reaction. This method, the principle is simple, specific through antibody recognition to ensure that no particularly complex operation, can be automated or semi-automated, experimental flux is not low. From these holes, the color is deep and shallow, you can calculate the number of antibodies. So, this is a quantitative approach, and it also has the capacity for mass screening. The chemical luminescence method used in the hospital is similar to this principle, except that the detected signals are not quite the same.
Another method is the use of colloidal gold color test paper test method, test whether pregnant test paper is a similar method. The benefits of this approach are very simple, easy to use, inexpensive, and require no complex equipment, very fast.
The image above shows the structure of the test paper: the sample pad, the colloidal gold binding pad, the cellulose acetate film and the absorption pad. If you want to detect the sample includes the antibody you want to detect, such as the antibody IgM or IgG of the new coronavirus, the sample into the test paper, this time in the colloidal gold binding pad has gold-label antigen, gold-label single resistance, that is, colloidgold joint antigen and monoantigen, together to the direction of the quality control line flow, gold standard single anti-resistance will be combined to the gold standard single antibody, as a result of the quality control. If there are these two antibodies to be tested in this sample, it will be added to the two detection lines, and because it can adsorption of gold-label antigens, so it will color. If the color rendering is, you will know if there are antibodies to be tested in the sample. If the IgG line is very deep, we know that there is IgG, and IgM this line is very light color, you can think that perhaps there is no antibody, this result can be very simple to understand.
Why do colloidal gold color testing is because the experiment can be applied to large-scale screening. Many places have to investigate the infection rate, if the infection rate is high, most people have antibodies, then the virus is not easy to spread. If the infection rate is very low and most people do not have antibodies, the virus can easily spread, in order to better prevent the next epidemic.
So do you get the results you want to know if you do the test? This requires very careful consideration, because no method is perfect. Molecular detection has never been perfect, and there is no perfect test ingress in the world. Understand the advantages and disadvantages of each detection method to better select and judge.
First, the point in time for detection is very important.
The image above is a diagram published in the Journal of the American Medical Association a few days ago. The relationship between different subjects and disease progression varies greatly. The virus is easily isolated a few days before the onset of the disease, when the nucleic acid load is not much. Can nucleic acid detection be detected? The watering fluid for swallowing swabs or alveoli is easier than fecal testing, but feces may be longer and easier to detect later. Antibodies are only out in the late stages of the disease, IgM antibody decline is relatively fast, IgG antibody decline is slow, stay longer. So what kind of detection method to choose, with the time node has an extremely important correlation, each method has the limit. In our analysis, we often use the detection limit to indicate the sensitivity of the detection method, the detection limit is the minimum concentration or minimum amount of the component to be measured from the sample under the given confidence conditions. For example, the gene editing method detection technology is about one magnitude higher than the traditional RT-PCR method. In fact, the detection limit of RT-PCR, which is now heavily used, is very low, that is, the sensitivity of the detection is very high, probably in one reaction can detect the presence of several nucleic acid fragments. Most of the other detection methods, fast or slow, not necessarily more sensitive.
Also, consider whether the specificity of a detection method is really good. The specificity of nucleic acid detection depends on the location of the primer selection, the sequence of primers and the matching of the target nucleic acid, and the examination of whether it can really be detected. The study, published in Clinical Chemistry by Hong Kong scientists, shows that two nucleic acid detection sites are generally easier to detect than the other, specifically that the N protein gene is easier to detect than ORF1b, although at first the design looks like the performance of both sites is similar, but the actual use of the time is different.
Although the method is not perfect, but it also has certain merit, as long as the proper use, find the applicable scene of the number. For example, in the crowd of 35 healthy people, 35 infected people, I now do a test, test positive results 35, people think this is a very good result, right? But that is not the case. The 35 patients detected were not really infected. Results can be divided into four pieces: the real infected person tested out, is true positive, healthy people are counted as infected, is false positive, healthy people test out health, is true negative, infected people have not been detected, is false negative. So true positive, false positive, true negative, false negative together constitute the total number of test samples.
What do you think? The first evaluation indicator: sensitivity. How many positives were tested in all the positive samples? The molecule is true positive, the denominator is true positive and false negative. Results 25 were true positive and 10 false negatives, so it was 71% sensitive.
Another indicator: specificity, i.e. how many true negatives are in the negative sample. True negative and false positive sit as denominator together, while the molecule is true negative. The result was 25 true negatives, 10 false positives and 71.4 percent. Accuracy, which is the number of true and true negatives, adds up to the total, so that it counts 71.4%. On many occasions we are concerned that the positive result is really positive. This is called a positive prediction (PPV), which means that when you see the test results, both positive and false positive, but how many of them are true positive? In this example, 25 were true positive, 10 were false positives, and PPV was 71.4%. That means about 7 positive sons for 10 people and three false positives.
I have just given a very strange example, and we can see that many of the values are the same, one reason is that the false positives and false negatives in the examples are the same. In the middle of a real detection method, the non-specificity and sensitivity must be the same. For example, there is such a method, is also the same population test, we found that all negative samples were negative, but 5 of the positive samples were negative. At this time it was found that its specificity is 100%, that is, no false positive. Its sensitivity is 86%, accuracy is 93%, and the PPV positive prediction is 100%. Although this test has false negative existence, but it does not have false positive existence, so the positive test must be positive, the positive prediction is 100%. Is there any method and how close is it? Yes, it is. For example, we use the PCR method, if done, it can make the positive prediction high. This is a Geneva-based non-profit organization FIND, on the market some of the kits made an objective evaluation, you can see different local production of kits, detection of different gene sites, although the detection limit has high and low, but overall do very good, specific this indicator is very high, many are 100%. With this method, you know that when you get the test results, at least the positive prediction is still close to 100%.
The examples just mentioned are examples where both positive and negative samples account for half. This example is a bit extreme and less applicable in the current situation of neo-coronavirus disease. Why? Because the positive prediction is related to the infection rate of infectious diseases. For example, in a group of 500 people, if the sensitivity of the test method is 95%, the specificity is 95%, and this group infection rate of 5%, what is the result? There should be 25 real infected persons and 475 uninfected persons. But the results of the test out of 452 negative, of which 1 false negative, this result is good, indicating that if you test the results are negative, you are really negative probability is 99.8%, which is still quite good. However, the positive results are not encouraging. There were only 25 positive samples, and 48 positive results were detected, 24 of which were real and 24 were false. So the probability of true positive 50%, that is, your results are positive, there is also half of the possibility that you do not have antibodies in your body, you can not go out to hang around, or honeststay at home is better.
What is the case just said about the infection rate of 5%, if the infection rate reaches 25%, what will be the result? There should be 125 infected people out of about 500 people, and the results were 6 false negatives and 19 false positives. If the result is negative, about 98.3% of the probability is true negative, and good. If it is a positive result, the true positive probability of 86%, indicating that you still have no small possibility, there are no antibodies in the body, you also want to go out to hang around?
The example just given is based on the antibody testre performance is very good hypothesis, that is, the hypothesis reagent detection sensitivity of 95%, 95% specific, and this is not so good to do.
Let’s imagine if these two indicators weren’t that high. Assuming there is a large city with an infection rate of 1%, we sit on an antibody test for 1,000 people. The detection reagents used are 90% sensitive and 80% specific, which is in line with the true indicators of many reagents now. What will happen? 1000 people, because the infection rate is 1%, so the real antibody should have 10 people, no antibodies people have 990. Among these 10 people, because the sensitivity of the test reagent is 90%, so 9 test sit positive, 1 is false negative. In people without antibodies, 792 of the 990 were tested negative because the specificity of the test was 80%, but 198 tested positive. So, after the test results came out like this, 1000 people were tested, the results were 207 positive, 793 negative. This shows that if a person tests positive results, in fact, there is only a 4% chance of positive, there is a 96% probability is not positive. How do you change this state? The fundamental solution is to make the reagents better and better, for example, by adding some specificity. Assuming that specificity increases to 90%, the result changes, with the false positive severing reduced to 99 people. However, the positive prediction rate increased from 4% to 8%, and more than 90% of the positive results were false positives. Assuming that specificity increases to 99%, the positive prediction rate can increase to about 47%. 99% specificity is a challenge for antibody testing because it is already a very high indicator. This is why the testing capacity is not ideal antibody testing in the case of low infection rate to do large-scale screening is very much a problem and risk, we must keep the basic concept of statistics firmly in mind.
A few days ago, a group of scientists in California teamed up to organize mutual cooperatives, they cross-phase compared different antibody detection methods, a total of 12 methods, 10 kinds of test paper, 2 are enzyme adsorption. Many of the test notes are Chinese manufacturers’ products, but also The products of American manufacturers, the results look similar, and no particular one. So it’s not that the production capacity varies greatly, but that the challenges of this approach are universal in themselves.
The image above shows sensitivity, which is not particularly good in the early stages, probably because the antibody itself concentration is low at the time of detection, so the detection rate is low. Slowly sensitivity increases, about 80% or 90% later. Specificity is very different, some are quite good, some of the indicators are very good, some of the comprehensive consideration can also be. Most manufacturers in 80%, 90% or so, small sample comparison, some methods reached 100%. So antibody testing, especially colloidal gold testing, in general, false positive rate is a big problem.
How to do specific tests is to use new coronanucleic acid negative samples. Because there is no way from the current large-scale epidemic when the sample, to find a real positive negative sample to do QC, had to use the new coronary pneumonia before, before July 2018 before the sample test cross-test, finally see if it will be positive in the end. They made a very strict comparison, and the results were very interesting.
It can be found that these methods are quite good, their method stability is relatively good, but so far no method really has as stable and excellent specificity as nucleic acid testing, can not fully believe its results, so the false positive problem will always bother us.
Finally, take a moment to talk about whether there are other detection methods that go beyond nucleic acid detection, such as sequencing. Sequencing is a test that has only been on the agenda in the past few years. In the past few months, we happen to have a method basically formed, in January, When I, Tsinghua Wang Jianbin and our center Professor Xie Xiaoliang published a paper, by Dilling and Fu Lisi two students mainly completed. The paper reports on a new, simple rna sequencing treatment, named SHERRY. After putting the sample in, the three-step method was formed to sequence, greatly reducing the difficulty of these experimental operations, which required very high experimental skills, and making the experimental results more stable, reliable and fast to obtain. Hospitals are overstretched and hospital researchers don’t have much time to think about complex operating processes, so the simpler the operation process, the more conducive it is to serve clinical new corona sequencing. In the past few months, although we have not been able to fully resume work, but we have the honor to work with the collaborators of Beijing Ditan Hospital, based on SHERRY to develop a new method called MINERVA, the starting point of this method is to really start from the practical point of view, how to serve the clinical. This method does not pick samples, pharynx swabs, sputum, feces can be, after extraction to establish the first step of the SHERRY library, and then after enrichment and other steps will have a new library to do virus whole genome sequencing.
Through this sequencing we have studied the nucleic acid sequences of more than 80 samples, from which we can see that there is no way to obtain information with simple nucleic acid PCR detection methods. We see interesting phenomena, such as the many samples may have different mutations at a particular site. This is a brand new virus, the more deeply we know about it, the better we can prevent and control the outbreak, and prepare for possible future threats. In addition, we don’t know how the mutation of the virus occurs. What does mutation have to do with disease development? Will it affect the research of molecular detection reagents in the future? Its accuracy cannot be guaranteed? Does the mutation affect vaccine development? Does it affect drug experiments? We don’t know anything about it, so we need more tools to study it.
Finally, I would like to thank Professor Xie Xiaoliang of Peking University for his guidance, and many of the details of the testing experiments were asked by Ren Lili, a researcher at the Chinese Academy of Medical Sciences, and Professor Wu Chen of the Chinese Academy of Medical Sciences, who works on the front line, and long-time collaborator Professor Wang Jianbin of Tsinghua University. Through this outbreak, we worked with the team of Chen Chen researchers at Ditan Hospital to obtain a large amount of genomic data, laying the foundation for future research. And thanks to the students and staff who worked during the outbreak, who worked hard to get this happened. My work is mainly done by the Beijing Future Genetic Diagnostic sorgin Innovation Center in Beijing, supported by the Beijing Municipal Education Commission, and the Center for Biomedical Frontier Innovation at Peking University, both of which are multidisciplinary research bases led by Professor Xie Xiaoliang. We are focusing on the analysis and sequencing of micronucleic acid samples, hoping to bring more results to everyone in the near future. Thank you.
(Note: PpT images are provided by speakers and may not be used without permission)
Special thanks For finishing the manuscript:
Guo Lijie, Ph.D. student, Institute of Biophysics, Chinese Academy of Sciences
Min Li, Ph.D. student, Institute of Biophysics, Chinese Academy of Sciences