In general, we here climbed the Philippines and looked for cunning bacteria. Then we went to Iceland with chemists, and they told us about the expeditions of biologists to the mountains for thermophilic bacteria (who live near hot springs) - and it turned out that the whole story was needed for the polymerase chain reaction. It became wildly interesting to me how the laboratory has to do with blood tests to a geothermal source, and now I will tell you this exciting story.
It means that yesterday Rospotrebnadzor and the Far Eastern Federal University opened a PCR training center in Vladivostok. I drove there with the journalists and found normal mad scientists who explained everything on their fingers. Because that is exactly what they will do there - to teach the Vietnamese and the Chinese. Basically - learn to deal with biohazard.
So what is PCR?
This is a story about how to make several million of exactly the same from a single DNA molecule.
Basically, this is a three-step reaction: unwinding the DNA into separate helices, then searching and marking the beginning and end of the desired sections, then completing the marked molecules with polymerase to complete. That is, this is such a simulator of DNA replication in our body, only done in vitro. And extremely fast.
That is, we cut all the DNA into halves, find only the halves of the pathogen, mark them, then the polymerase builds up to the marked pairs. Repeat. Repeat.
The current reaction doubles the amount of DNA in the flask every 40 seconds. More precisely, the efficiency of the cycle is slightly less - from 78% to 99%, because not everything and not always meshes in the real world.
Why do you need it?
If any pathogen floats in your blood, then you can build a reaction just for it, which will amplify (increase the amount) of its DNA. At some point in the brew they will be so much that you can notice it. And here the robot will shout: “Aha, got caught!”, And the result will get the label “positively”.
As a starter, we need only 50 molecules per milliliter in the general case. For comparison, the flu virus is not even in the blood, but in the patient's saliva with the first symptoms - about 10 thousand pieces per milliliter.
Why is it still needed?
You can literally make a couple of DNA from the mummy of the Pharaoh, the mammoth or the skin of a tiger and sequence its genome. That is, it is realistic to completely count, because from an insignificant amount of material for research, there will be a lot of DNA molecules. Hence, the use in forensic examination - it is possible to establish which of the dead kings to whom he was the son. Or a couple of molecules of saliva at the crime scene to find the perpetrator (right Gattaka). Or even cooler - you can replicate molecules with changes, and you get controlled mutagenesis. But the latter is difficult.
And PCR is the same reaction that changes the social behavior of society. Because with the help of it, paternity is determined - it is possible to isolate only different DNA segments and evaluate where and what is different (and how much), that is, to look for close relatives. Well, or just to sequence and evaluate the differences.
But then we will talk about the diagnosis of viral and bacterial infections - this is the widest application. And they teach him here in Vladivostok.
How is PCR in general?
We take the material, such as blood or something else. Carefully prepare the drug (usually 1/3 of the material is taken so that in case of errors in the analysis two more doses remain in the archive until the end of the study). The drug is mixed with the test system. In the closed flask we place this “soup” into the instrument for carrying out the reaction.
Then the cycles of DNA unraveling, primers hitch to it, and alignment with the polymerase of full molecules begin. Modern real-time PCR techniques suggest that labels are accumulated and glowing. This is cool because you do not need to open the tube to react to the selection of the desired DNA sequences. When the luminescence can be registered, it is decided that the strains in question were in the materials.
A few more cycles allow us to estimate the degree of signal change and build a hypothesis about how many DNA molecules were originally in the material, that is, to determine the concentration of the pathogen with sufficient accuracy.
How does the primer know what to cling to?
A primer is a crap that “sticks” to that which matches its nucleotide code. If you need a rabies virus, you need to sequence its genome (more precisely, we are interested in the beginning and end of DNA) and record the places on both sides of the molecule where the nucleotide chains are unique. And make them primers. Primers stick to DNA halves, and polymerase will start working from above.
Then the DNA of blood cells clings to nothing, only to rabies.
Rabies primers will only cling to rabies. To diagnose your cow for rabies and flu, you need two doses of blood and two different reactions.
Now the most fun. We do not need the exact beginning and end of the DNA molecule for diagnosis. We just need a lot of recognizable areas. That is, on the full genome, for example, bird flu, it is necessary to distinguish two unique fragments - the beginning of copying and the end of copying. So that between them there is something that can be unambiguously identified as bird flu without false positive and false negatives.
Further mathematics comes into play - it is necessary to select unique places for the genome, and so that they are very short, ideally from 15 to 30 nucleotides. And so that between them was necessary. Two primers give something like a hash from a DNA molecule, that is, they allow it to be described in 30-60 nucleotides.
Primers are attached to these minimum sufficient sites for recognition. If they cling to something else - it was a bad targeting, and the accuracy drops.
So what have the hot springs?
The fact is that the reaction takes place at a high temperature, otherwise the DNA does not denature. Polymerase at this temperature usually breaks. Usually, because specifically with thermophilic bacteria, she calmly experiences everything. Therefore, in the last century, there were expeditions to Kamchatka, where geologists were looking for means for molecular medicine, shooting back from bears. And in Europe they drove to Iceland.
The funny thing is that in 1976 and in 1980 there were publications about polymerase (our and American), but it seemed to everyone a funny fact, and they did not find practical application.
Now further. It turns out that Taq-polymerase from Thremus aquaticus works on the UDP type protocol, that is, without error correction. At all. Collects what happens - and then intercourse there yourself. But quickly.
Pfu- and Pwo-polymerases were isolated from archaea - they work with error correction, that is, they do not give mutations in the replication process (well, they don’t give much), but they act much slower.
Previously, the stocks of this thirst were scooped from sources, now polymerase is diluted in laboratories. And they make an artful mixture of Pfu, Pwo and Taq - it turns out at a lightning speed quickly and relatively correctly.
What it looks like in the real world
In the real world, you have a box of test systems with the intriguing inscription "syphilis", for example. Or "flu." Inside one test system - all components for PCR, except for the DNA matrix, that is, the patient's blood. Or another piece of it where the pathogen is searched.
Then it is necessary to mix the blood and the test system, heat and cool many times under pressure, and the catalyst, primers, polymerase and auxiliary components will do everything themselves. At the exit - a test tube with 10 in the twelfth DNA molecules per milliliter.
Can I do it in the kitchen?
In theory. The device itself is very simple, all the scientific capacity in the test system - and a bit of software that records the change in the light signal of the label. Still need the accuracy of the temperature of 0.1 degrees and the program for its change. Plus photosensor. That is, iron is not very complicated. Therefore, there are so many of these laboratories - they are really cheap to deploy.
But in the kitchen it will not work, because the strongest story is the contamination of the sample (pollution). Purity protocols are what are taught more than the reaction itself. If one of the resultant pathogen DNA tubes is broken in the laboratory, everything will be covered with an even layer of false positive result. That is, all the current analyzes are done at once by the forest, then a long cleaning, checking, then taking out archived blood and again starting the line.
Well, and I remind you that the pathogen DNA and the pathogen itself are different things, like the source code and the compiled code. And the danger from the fact that you broke a test tube with 10 in the twelfth DNA of the tuberculosis pathogen is about the same as scattering on a floor a pack of sheets of the source software of a nuclear warhead launch.
4-7% of the results may be false-positive due to inaccurate work and features of the real world. Therefore, a thorough validation of the result is performed, in particular, using mathematical methods.
Along with the usual PCR materials, a test tube is subjected to water. If there something begins to amplify, the lab technician will tear off his hands to the tonsils.
This is not a safe; it is a gateway for transferring material to the laboratory. Personnel are transferred to the laboratory in a clean area after a shower, changing clothes and a couple of degrading procedures.How can you understand that there is something wrong with the reaction?
By the nature of its development. The usual reaction begins sluggishly, then the phase of exponential growth, then in the area of the last cycles - the exit to the plateau. The amount of DNA does not increase with new cycles for dozens of reasons, from the depletion of primers to the completion of one of the resources, pyrophosphates accumulate. Plus, halves of DNA, which have not reacted earlier, are swimming alongside in the soup, and they crawl to hug everyone. As a result, artifacts and an atypical course of the reaction can be determined - this is already done by software for small data mining. Domestic, free, regularly updated, but not open source.
But why do we need to increase the amount of DNA in the preparation, if all we need to improve the methods of detection?
There is such a path of development. Physico-chemical methods are improving, but not enough. Last experience - they took blood plasma, centrifuged, "heavy" viruses fell to the bottom of the tube. Then they were fried with a mass spectrograph laser. Nifiga It works with colonies of bacteria (they have been doing it in Moscow laboratories on a stream for a long time), but there are no viruses. And the colony still need to grow. Therefore, for now (and it looks like the next ten years), PCR will be the fastest thing for analysis.
By the way, a full analysis is done in about 45 minutes (with preparation of the material), that is, half a day is
cito or 4 days if the laboratory is in the city and you are in the village.
For some infectious diseases, the concentration of the virus is extremely low. For example, there was a case of encephalitis - the clinician saw focal symptoms (damage to the central nervous system), blood was taken — even by PCR, there is still nothing, and the antibodies are already deployed. That is, PCR would give the result after the analysis of antibodies, and mass spectrography - even later, it is orders of magnitude less accurate than PCR.
That is, only one specific genome is amplified?
In the usual case, yes. The most modern strain of the disease that is relevant for the region is located, and the primers target its DNA. But this is a little utopian situation, because at the same time it is necessary to look for immediately six pieces of different strains. To do this, different primers are added to the kit, plus a number of changes are being made. Naturally, the test system is more expensive. Or it is necessary to do several reactions for different pathogens.
Is it possible to multiply RNA?
Yes, you just need to make DNA from it. The reaction is more complicated, but interesting because it allows to separate dead cells from living cells. DNA is incredibly durable, so it can be broken off even from a mammoth. If the virus is already interrupted, the DNA of its dead samples will "glow" for another 3 weeks. And RNA crumble much faster. Therefore, NASBA methods (search for RNA viruses, for example) are much more accurate in some cases. But much more expensive.
A couple more beautiful pieces
The whole history of the implementation of PCR after the invention of the theory is directly tricky physical and chemical tasks. For example, it is possible to amplify unknown regions of DNA. To do this, the molecule is cut at a known site, and then trimmed together. Some turn over, and molecules with a known code at the beginning and end are obtained. Primers are hooked - and on those where there is a beginning and an end, and not one marker, amplification is carried out.
A very funny story with how to avoid unnecessary reaction steps when heating the tube. The fact is that the first part of the reaction takes place at a high temperature, but as long as the test tube heats up, you can accidentally start from the wrong tact - this will dramatically increase the noise and the probability of error. Therefore, options with the fact that one of the reagents of the system is placed in a wax capsule (it melts only after passing through the desired point and releases the component in time) came to the option when a substance is added to the solution that blocks the reaction but collapses at high temperature.
There is protection against contamination - molecules on amplification can be labeled in a certain way, and then at the beginning of the reaction, destroy all labeled alike. That is, if something from one tube crawled into another, it will be automatically filled up before the main reactions.
Fluorescence is also cool: a substance is put in the reagents that is destroyed as a result of reactions with DNA. That is, the more DNA is formed, the more molecules of this substance are broken. Whole it does not shine, and in chunks it begins. And the more DNA molecules react during replication, the more the test tube glows.
Given that the complexity of the test systems is growing, life hacking and elegant solutions to the sea.
Why do I need to do PCR training centers at universities?
Because this is still the main story about the diagnosis of diseases, and not necessarily in humans. Here, in the Far East, for example, the focus may be on fitoviruses. There is a binary virus for rice (more precisely, two well-combined viruses) in the bins of the Motherland, which can take and put the entire crop in Asia when it is distributed. Naturally, such a thing is not only with us - and not necessarily it will be released maliciously, it can develop according to idiocy or simply in a natural hearth. Like Japanese encephalitis in Primorye, there are constantly seasonal foci in mosquitoes. And the death rate from it - 60 percent.
The alleged enemy also has viruses in animals and humans.
So, PCR specialists represent the country's biosafety. They monitor infections, catch all the passengers of the plane, where someone has coughed up the wrong way and so on. Mikhail, for example, flew on ebola, on both bird flu and many more. He also sequenced the complete genome of the only living copy of the bear rabies virus in the world.
Mikhail Schelkanov, Professor of the Far Eastern Federal University, Head of the Virology Laboratory of the Federal Science Center of Biodiversity of the Far Eastern Branch of the Russian Academy of Sciences.With the bear in general is an awesome story. The bear lifted 5 dogs and then attacked the grandmother. He twisted her lungs and took off his scalp, plus he doused it all and covered it with blood. About the beast, hunting experts decided that it was some kind of wrong bear. And gave it the remnants of scientists on the experiments.
Here are more details.
Michael is also a fan of fur seals because he loves islands with rookeries. They are extremely quickly transmitted to the entire population of viruses and parasites. They found a cool louse with no less steep bacteria in their nostrils. Seals when diving, he slammed his nostrils. The louse inside feels warm and seductive.
In addition to biosafety, the second story is the training of foreign specialists. There are two important things here: first, they learn from our test systems (and then they will be more likely to buy them), and this is important for export - we are leaders in the CIS (approximately 25 million reactions per year), but not in the world . Secondly, the uniformity of experience makes it possible to monitor equally according to general protocols, that is, then change databases and experiences. Bases, of course, more important. This means a much more consistent response to possible epidemics.
In Vladivostok, the official opening did not coincide with the technical one - a group of Vietnamese doctors are finishing off the last lessons. Two people know Russian, the others learn through them. There is a fully English course. The same center works in Novosibirsk for Russian doctors and doctors from the CIS countries, and specialists have been graduating there for a year.We also do everything in Russia for our test systems, and in practical molecular biology are among the strongest in the world (in theory, no, but we quickly and engineering implement everything). Therefore, education will help expand this market for us. In total, 3881 specialists have been trained in such centers (small groups, because there is a lot of practice), and now the task is to go widely to study foreign specialists.
Fully, this is called the International Training Center of Rospotrebnadzor at the School of Biomedicine of the Far Eastern Federal University. It was opened, in fact, by Mikhail Shchelkanov from the photo above, and German Shipulin (FBUN TsNII Rospotrebnadzor in Moscow). Proofs in the media will be tomorrow, apparently. Well, I am not a microbiologist, so if you make a mistake somewhere higher, please send me a message.
UPD: here is the news on
RG , in which there is still a point of view on this story.