The minimum detection limit of PCR is only 0.5 template? do you believe it?

First of all, we must correct a concept. When we develop nucleic acid detection technology, a key performance indicator is the minimum number or concentration of the analyte that this detection method can reliably and stably detect. Usually, this technology The detection sensitivity of the method is called sensitivity in English. However, in the field of in vitro diagnosis, a detection method generally requires the cooperation of detection equipment and detection reagents, and the detection equipment uses optics, electricity, electromagnetics, etc. to identify the corresponding signals sent by the detection reagents when biochemical reactions occur. Therefore, This leads to confusion in performance indicators. A "sensitivity" indicator alone is not enough to distinguish the detection performance of hardware devices for detecting the corresponding physical signals from the detection performance of the overall method for detecting the quantity or concentration of the analyte. That said, the word "sensitivity" should be used with extreme caution.

To measure the "sensitivity" index as the saying goes, the word "limit of detection" is used, which is called LoD in English, limit of detection.

The detection limit generally refers to the minimum number or concentration of the analyte that can be detected under the condition that the confidence interval (false positive rate and false negative rate does not exceed 5%) are met.

In fact, what I mainly want to discuss today is the issue of "copy/ml", the unit of measurement usually used for detection limits.

Most of them use real-time fluorescent PCR technology. Some can reach 100 copies/ml, and some need 1000 copies/ml. The difference between them is ten times, which is an order of magnitude. It is true that the technical performance of different manufacturers is so different. ?

Since the covid-19 epidemic, the continuous changes of nucleic acid detection kits are a bit interesting. We know that the nucleic acid detection process generally has to go through the steps of sampling, sample separation, nucleic acid extraction, and amplification detection. Before the covid-19, the detection limit of general nucleic acid detection kits is to use the concentration of the DNA or RNA template added to the amplification detection reagent system as the detection limit index. The covid-19 epidemic has been three years, and many new nucleic acid detection technologies have been born. , For example, new methods such as extraction-free, integrated, fully automatic, etc., emerged as the times require. If only the template concentration is used as the detection limit index of the detection method, it will bring a lot of confusion and incomparability, so it seems The current detection limit index is directly defined as the copy number concentration of the new coronavirus in the sample.

Let's take the most classic experimental method as a chestnut. After taking the sample with the swab, break the swab in the preservation tube with the sample preservation solution (just say the single tube for a single person). Now there must be at least 1 ml of preservation solution in the single tube for a single person. It was then transported to the testing laboratory, first shaken to release the virus collected on the swab as much as possible, and then sucked out 140 microliters (taking Qiagen's nucleic acid extraction kit method as an example) for nucleic acid extraction. The first step is to elute nucleic acids from magnetic beads or chromatography columns, generally 60 microliters are used for elution, that is to say, assuming that there is no loss in the previous steps, according to the performance requirements of our country’s covid-19 nucleic acid detection kit, the detection limit is ≤500 copies/ml, then the concentration of the nucleic acid template should be about 1.17 copies/μl, which is equivalent to concentrating the original sample concentration to about 2.3 times.

500 copies/ml × 140 μl ÷ 60 μl

= 1.17 copies/μl

= 2.3x (500 copies/ml)

The next step is to do the amplification reaction. Add the nucleic acid template to the amplification reagent. Generally, the amplification system is as small as 20 microliters and up to 50 microliters. The amount of template added is generally 5 microliters to 20 microliters. According to the above calculations, there are at least 5 copies in this amplification reaction, and there is no problem with PCR amplification. Well, the indicator of "detection limit ≤ 500 copies/ml" set by the Chinese state is quite scientific.

However, let's turn around and look at the detection limit indicators of various manufacturers. At the same time, combining the detection limit and the number of the sample addition system, it is still based on the ideal condition that there is no loss in the extraction process. The lower detection limit × 0.14 ÷ 60 × sample addition System, look at what kind of numbers each product will get, most of the kits based on PCR technology, this number is around 5~10, a few can be as low as 2 points, and some "special" innovative technologies Possibly hundreds. Of course, there are also one less than 0.5.

What does this number represent? In fact, it is the minimum number of target nucleic acid molecules (copy number) that meet the detection limit specification in an amplification detection reaction, not the concentration. We can imagine the PCR reaction tube as a dark room, in which we play the game of blind man touching people. One person is responsible for arresting people (amplification reagents), and one or more people are responsible for being arrested (nucleic acid templates), and the molecules cannot see anything. , you can only bump around in the room, so the speed of catching people directly depends on how many people are caught in the room. In theory, as long as there is a template in the room, you can catch them sooner or later, but theory is theory after all. When the fate is not good, these two people just can't get together. This is called the single-copy amplification efficiency. Some people have studied this probability problem many years ago, and it seems that the highest probability is 80~85%. So this is also the so-called absolute quantification of single-copy amplification that digital PCR claims, but the theoretical basis for fitting the calculation with Poisson distribution is also the reason why I have long opposed calling digital PCR as absolute quantification and single-copy detection sensitivity.

Back to the issue of detection limits. I just did a series of calculations at the elementary school level, and they were all under ideal preconditions. If we take into account factors such as virus release, nucleic acid extraction and recovery efficiency, nucleic acid amplification efficiency, etc., the "lower limit of detection" in the formula just now "×sample system" can reach 1000, which is basically the theoretical limit. I personally think that such a detection limit index is more meaningful, that is, it is described by copies/tests, which can better reflect technical capabilities. the level of. Departing from volume only emphasizes concentration, which is inaccurate.

As I mentioned just now, some "innovative technologies" have relatively high values of "lower detection limit × sample addition system". Is the performance of this technology poor? In fact, it cannot be generalized. The covid-19 epidemic has spawned many new detection technologies. The effect of many new technologies is to reduce the manual operation and difficulty of the entire nucleic acid detection process as much as possible, and improve the efficiency of detection work. Whether it is extraction-free, automatic or isothermal amplification, it is all in the The traditional detection process can be reduced as much as possible by biochemical or automated methods to reduce labor burden and save time. At the technical level, there are advantages and disadvantages. It improves the overall detection time efficiency, may reduce the virus release efficiency, may reduce the nucleic acid extraction efficiency, and may reduce the detection limit of the amplification detection reaction, which requires such a new technology solution, It is necessary to comprehensively optimize the overall technical process in order to achieve the purpose of comprehensive detection performance to achieve the expected indicators. Among them, the most representative are the many so-called nucleic acid POCT technical solutions, because such technologies usually use the flag of "sample in, result out", which not only ensures the airtightness and safety of the overall detection process, but also guarantees performance indicators To meet the requirements, we need to think hard about sample processing and nucleic acid extraction, and introduce as many copies as possible into the detection system.

The state has stipulated a rigid indicator that the detection limit is ≤500 copies/ml. This is the main policy. The formula of "lower detection limit × sample addition system" is mentioned again. Technology determines the number after multiplication, so there is only one way to reduce the lower detection limit. Yes, increase the sampling system. This is also the reason why the so-called rapid testing equipment and POCT equipment require a relatively large sample size. The most exaggerated method I have seen, it seems that the volume is adjusted to 2 ml, and then the detection limit index reaches 20 copies/ml.

Taking the new crown nucleic acid detection kit as an example, in order to maximize the needs of on-site detection, we designed the swab after sampling to not go through the preservation solution preservation tube link (because there is a preservation tube process, it will inevitably face the problem of secondary opening, press The regulations are to be operated in the P2 laboratory), broken directly in the sample cavity of the microfluidic chip, and it is difficult to describe this detection limit. The result of our test is that 50 copies of the virus can be detected on a swab, but the detection limit of this 50 copies/reaction or swab is not recognized by the industry. One method is not to swab Come on, add virus preservation solution, our chip can only hold 50 microliters of samples at that time, that is 1000 copies/ml, and the indicator is not in compliance. Another method is that a swab should be placed in 1 ml of preservation solution anyway, and that 50 copies of a swab can be converted into a detection limit of 50 copies/ml.

Now the nucleic acid detection technology is more and more, faster and faster, from the traditional two to three hours, to the fully automatic one hour, to the isothermal amplification and extraction-free rapid detection of 30 minutes, to the ultra-fast 5-minute PCR .

One of the main factors affecting the speed of PCR is the speed of heating and cooling, but it is definitely not the only factor. Now there are a lot of crossovers engaged in electricity and light to crush life science instrument technology, which is a good thing, but then again, no matter how different the principles of biological detection technology are, in the end, it still comes down to biology itself. The temperature rise and fall is fast, can the temperature be effectively and uniformly conducted into the reagent system, whether the biological enzyme molecules and the reaction substrate have sufficient time to form the corresponding structure and react, whether the reaction is sufficient, life science and even Biochemistry is about a dynamic balance. It is undeniable that the progress of technology is continuous and endless, but no matter how advanced it is, it is not advisable not to follow the laws of science.

When sequencing was done in the past, the industry was not big, and each company seemed to be able to create a market of 100 billion. Now doing digital PCR, after thinking about it, I can't figure out what differentiated applications this thing has at this stage. I really didn't expect that it has become a trend in the past few years. Digital PCR can kill the revolutionary technology of traditional PCR in one fell swoop. The real pandemic of nucleic acid testing has benefited from the pandemic of the covid-19. It can be said that the covid-19 epidemic has created a rocket-like rise in the nucleic acid testing market. A business that could compete with technological advancement and innovation was abruptly turned into a quagmire of cost and manpower. Nearly three years after the covid-19, innovative technologies that can reach the application level have appeared abroad, such as electro-infiltration microfluidics, infrared optical temperature control, RTF-EXPAR technology without reverse transcription, and so on. In China, nucleic acid detection reagents have been raised from 200 yuan to 3 yuan in three years.