The use of biological magnetic beads for nucleic acid extraction is still a relatively novel nucleic acid extraction method in China. Compared with the traditional chloroform isoamyl alcohol extraction method and spin column kit method, this method is still understood by many people. There are also some misunderstandings in the process of using magnetic beads to purify nucleic acids.

Misunderstanding 1: The more magnetic beads used, the better the extraction effect

Many teachers like to increase the amount of magnetic beads when the extraction effect is not good. They think that adding a little more magnetic beads can attract more nucleic acids. I have to say that this idea is not advisable.

The main feature of magnetic beads is that they can be dispersed in a liquid or separated from a liquid phase in a solid state under the action of an external magnetic field. For any reagent system, the ratio of magnetic beads to liquid should have a certain threshold, exceeding a certain ratio Excessive magnetic beads will lose their dispersion characteristics because they cannot be uniformly dispersed in the liquid, and the efficiency of contact between the nucleic acid magnetic beads and the liquid cannot be fully increased during the washing process. Excessive magnetic beads will also adsorb more impurities, which has a great influence on the effect of impurity removal. Even sometimes, too many magnetic beads will adsorb protease, lysozyme and other functional components that play a major role in the liquid system, resulting in low efficiency of the entire kit. In many cases, when the extraction effect is not good, reducing the amount of magnetic beads used is the best way to improve the extraction effect.

Normally, the amount of reference magnetic beads given by the magnetic bead method kit is slightly excessive. Therefore, it is not often necessary to increase the amount of magnetic beads to improve the adsorption efficiency. However, if it is determined that the extraction effect is not caused by insufficient amount of magnetic beads Well, it is possible to improve the extraction effect by increasing the amount of magnetic beads within a certain range.

Take GNT-02 series magnetic beads as an example. When extracting macro samples (plant tissue, whole blood, etc.), the usual dosage is 10ul/time; when extracting trace samples (such as serum free DNA, buccal swabs, etc.), magnetic beads The dosage is 15~20ul/time. If you need to exceed this usage, you need to communicate with a technical engineer.

Misunderstanding 2: The more reagents used, the better the extraction effect

The cracking effect is not good? Add more lysis buffer. The washing effect is not good? Add more detergent. This is the inertial thinking of many customers in the use of kits.

However, for the magnetic bead method, each increase in the volume of a part of the liquid reduces the probability of more magnetic bead collisions, and reducing the probability of magnetic bead collisions will result in a significant drop in the adsorption rate. Therefore, in many cases, although adding lysis solution and washing solution can indeed enhance lysis and enhance washing, the core of magnetic bead extraction is the efficiency of magnetic bead adsorption of nucleic acids. The efficiency of magnetic bead collision cannot be guaranteed, but the efficiency of nucleic acid extraction cannot be guaranteed. Yes, so simply increasing the amount of reagents used to improve the extraction effect may not be completely effective.

For the GNT-B02 whole blood genomic DNA kit, the general lysis solution should not exceed 400ul/time, and the washing solution should not exceed 500ul/time. If the system really needs to be amplified, the amount of magnetic beads and samples should also be increased accordingly. This amplification is not necessarily in equal proportions.

Misunderstanding 3: The more washing times, the better the extraction effect

When there are too many impurities in the extracted nucleic acid, the user will consider washing several times to obtain a purer nucleic acid. Increasing the number of washes is indeed conducive to the purification of nucleic acids, but considering that each wash will lose a certain amount of nucleic acid and increase the possibility of nucleic acid fragmentation and hydrolysis, it is generally appropriate to control the number of washes at 2 to 4 times.

For the GNT series of kits, the washing times of a single purification kit is 2 times, the washing times of plant and animal kits are 3 times, and the washing times of blood kits are 3~4 times.

Misunderstanding 4: The more samples are used, the better the extraction effect

When the sample is not fresh enough or the nucleic acid content itself is low, the nucleic acid extraction effect is often not good, and many teachers will use multiple samples to increase the amount of nucleic acid extraction.

However, simply increasing the sample sampling volume may sometimes introduce too many impurities, exceeding the lysing capacity of the lysate, and also reduce the extraction efficiency. Therefore, it is not recommended to simply increase the sample sampling volume to achieve the purpose of increasing the extraction volume.

If the extraction volume is indeed too low due to insufficient sample volume, it is recommended to go through the enrichment or concentration step before starting the extraction. Or increasing the completeness of lysis and exposing more nucleic acids is also a solution.

Misunderstanding 5: If a certain kind of magnetic bead is good, it should be effective in all tests

There are many types of magnetic beads, different particle size, different dispersion, different magnetic response time, different coating base matrix, different outer modified functional groups, different coating densities, and different functional group arm lengths, which will lead to magnetic beads Features vary greatly.

Therefore, the experiments and systems that different magnetic beads adapt to are also different. Just like the reagents for nucleic acid extraction, the formulas are not exactly the same, and the properties of the magnetic beads that are also used for nucleic acid extraction are not exactly the same.

Some magnetic beads show higher adsorption efficiency in the extraction of constant nucleic acid, and some magnetic beads are more suitable for the extraction of trace nucleic acid. Some magnetic beads are suitable for more acidic reagent systems, and some magnetic beads are suitable for more alkaline reagent systems. Some magnetic beads have good magnetic responsiveness but fast settling speed, which is more suitable for magnetic rod type automatic extractor; some magnetic beads have slow settling speed but long magnetic response time, and are more suitable for pipette type automatic extractor.

There is rarely a kind of magnetic beads that can be applied to all experimental situations. Except for the fixed kit, in most cases, the magnetic beads and the reagent system need to be adjusted for a certain period of time.

Misunderstanding 6: Compared with a certain kit, the effect is not good, that is, the magnetic beads are not good

In the process of screening magnetic beads, many customers simply replace the magnetic beads with the same amount under the mature reagent system to compare the effects of magnetic beads.

In this way, it is easy to conclude that certain magnetic beads are not effective, but in fact, because different magnetic beads are suitable for different systems and dosages, they often need to be adjusted to obtain better extraction results.

In-vitro diagnosis refers to products and services that obtain clinical diagnostic information by testing human samples (blood, body fluids, tissues, etc.) outside the human body to determine diseases or body functions.

One of the key steps in the detection of human samples is the purification of nucleic acids (DNA and RNA) in the sample. The method of nucleic acid purification is an important factor affecting the quality of the extracted nucleic acid, and only high-quality nucleic acid can meet various downstream applications.

Nucleic acid is the basis of molecular biology, and nucleic acid extraction is a threshold for nucleic acid detection, and even the entire molecular industry cannot bypass the threshold. In many cases, the quality of nucleic acid extraction from a sample directly determines the validity of the test results.

Basic knowledge of nucleic acid

Nucleic acid is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), among which RNA can be divided into ribosomal RNA (r RNA), messenger RNA (m RNA) and transfer RNA (t RNA) according to different functions.

DNA is mainly concentrated in the nucleus, mitochondria and chloroplasts, while RNA is mainly distributed in the cytoplasm.

Why is nucleic acid extraction needed

Nucleic acid extraction provides answers to a large number of extensive research and applications, and the obtained nucleic acid can be used in a variety of ways. The exact research purpose determines the type of nucleic acid to be extracted; the application of nucleic acid often affects the choice of extraction method. In order to determine the best research method, it is necessary to have a clear understanding of the downstream applications of nucleic acids and any potential limitations related to the type of sample. Although the method of cell lysis is different depending on the sample type, the core principle of the overall nucleic acid extraction remains the same: cell or tissue samples are lysed to remove non-nucleic acid contaminants.

Principles and requirements of nucleic acid extraction and purification

1. Ensure the integrity of the primary structure of nucleic acid;

2. Eliminate pollution from other molecules (such as eliminating RNA interference when extracting DNA);

3. There are no organic solvents and excessively high concentrations of metal ions that can inhibit enzymes in nucleic acid samples;

4. The pollution of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized;

5. Eliminate the contamination of other nucleic acid molecules, such as removing RNA when extracting DNA molecules, and vice versa.

Common nucleic acid extraction and purification methods

1. Phenol and chloroform extraction method

Phenol and chloroform are used to extract DNA by using phenol as a protein denaturant. Repeated extraction will denature the protein. SDS (sodium dodecyl sulfonate) will lyse the cell membrane and digest the protein or polypeptide or small peptide in the presence of proteinase K and EDTA. Molecules, denature and degrade the nuclear protein, freeing DNA from the nuclear protein. While DNA is easily soluble in water, but insoluble in organic solvents. The surface of protein molecules has hydrophilic groups, which are also easy for hydration, and a hydration layer is formed on the surface, so that the protein molecules can smoothly enter the aqueous solution to form a stable colloidal solution.

When the organic solution exists, the colloidal stability of the protein is destroyed, denatured and precipitated. After centrifugation, the organic solvent is in the bottom of the test tube (organic phase), the DNA is in the upper aqueous phase, and the protein is precipitated between the two phases. Using the extraction principle, according to the protein nucleic acid dissolved in different reagent layers, the pipette tip is extended into the different liquid layers to extract the required components, and purified nucleic acid is obtained after multiple washings.

The disadvantage is that due to the use of phenol, chloroform and other reagents, the toxicity is relatively high, long-term operation has a greater impact on personnel health, and the recovery rate of nucleic acid is low, and the loss is large. Due to the large operating system, the operation repeatability of different experimenters is poor. , It is not conducive to protecting RNA, and it is difficult to carry out miniaturization operations.

The advantage is that it uses common reagents and medicines in laboratories, and the cost is relatively low.

2. Spin column purification

This method utilizes the surface of nucleic acid to be covered with a thin film composed of water molecules. In a high-salt environment, this hydrophilic membrane will be destroyed so that nucleic acid can be adsorbed on the spin column, while other impurities such as protein and metabolism Products etc. will be separated from nucleic acid by centrifugal precipitation.

But even so, the spin column purification method still has shortcomings:

(1) Highly dependent on the binding capacity of the adsorption membrane;

(2) Incomplete elution leads to the loss of nucleic acid;

(3) Nucleic acid cannot be extracted in large quantities.

3. Magnetic bead method for nucleic acid separation technology

The positively charged magnetic beads are easy to adsorb negatively charged nucleic acids and are mainly used to extract DNA and RNA from human serum or plasma samples. Generally speaking, the sample is mixed with the binding buffer and magnetic beads. After the nucleic acid is combined with the magnetic beads, it is washed and captured by the magnetic field several times. The eluted nucleic acid can be detected by PCR or other specific methods to detect specific DNA or RNA. .

Magnetic separation is also widely used in the diagnostic industry, and can achieve high-throughput and automated nucleic acid extraction methods.

Different types of genetic testing will involve nucleic acid extraction and purification steps. Among the methods of nucleic acid extraction and purification, both the liquid method and the spin column method involve the use of toxic organic substances, which are harmful to the environment and experiment operators.

The magnetic bead method requires corresponding specially formulated magnetic bead buffer and specially modified magnetic beads. From the cost and operation aspects, both have high cost and cumbersome operation steps. At the same time, it also limits the automation process and the promotion of applications. . Therefore, the development of a rapid nucleic acid release method that can quickly release nucleic acid without affecting subsequent genetic testing experiments has become an urgent problem to be solved.

4. Gene nucleic acid rapid release technology

The DNA release kit is dedicated to quickly release constant temperature fluorescence amplified DNA from various common samples. It has the following characteristics:

1. The nucleic acid release agent can quickly lyse the sample and free the DNA of the sample in the solution. There is no need to prepare any reagents by yourself, and no toxic reagents such as phenol and chloroform are used.

2. The operation is simple, it only takes one step (about 3 minutes) to obtain the DNA template for constant temperature fluorescence amplification, without any operation steps such as centrifugation and extraction.

3. The whole process is only completed in one centrifuge tube, which avoids the loss of trace samples and is not easy to cross contamination. It is simpler and more convenient than commonly used extraction kits.

4. Wide compatibility, suitable for common molecular biology samples, including bacteria, insects, various animal tissues, oral cells, hair, bacteria and viruses in tissues, etc.

The nucleic acid extraction system is an instrument that automatically completes the nucleic acid extraction work of samples by applying matching nucleic acid extraction reagents. It is widely used in various fields such as the Center for Disease Control, clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbiological testing, food safety testing, animal husbandry and molecular biology research.

Nucleic acid extraction system classification

1. Divided according to the size of the instrument model

1) Automatic liquid workstation

The automatic liquid workstation is a very powerful device, which automatically completes liquid dispensing and aspiration, and can even realize full automation of specimen extraction, amplification, and detection by integrating functions such as amplification and detection. Nucleic acid extraction is only one application of its function, and it is not suitable for routine laboratory extraction of nucleic acid. It is generally applied to the experimental needs of a single type of specimen and a very large amount of specimens (at least 96, generally several hundred) at a time. The platform establishment and operation of automatic workstations require relatively large funds.

2) Small-scale automatic nucleic acid extraction system The small-scale automatic instrument achieves the purpose of automatic nucleic acid extraction through the particularity of the operating structure, and can be used in any laboratory.

2. Differ according to the extraction principle

1) Instruments using spin column method

The centrifugal column method nucleic acid extraction system mainly uses a combination of a centrifuge and an automatic pipetting device. The throughput is generally 1-12 samples. The operation time is about the same as manual extraction. It does not improve the actual work efficiency and is expensive. Different models The consumables of the instrument are not universal, and are only suitable for large-scale laboratories with sufficient funds.

2) Instruments using magnetic bead method

Using magnetic beads as a carrier, using the principle of magnetic beads adsorbing nucleic acids under high salt and low pH values, and separating them from nucleic acids under low salt and high pH values, the entire nucleic acid extraction and purification process is realized by moving the magnetic beads or transferring the liquid. Due to its unique principle, it can be designed into a variety of fluxes. It can be extracted from a single tube or 8-96 samples, and its operation is simple and fast. It only takes 30-45min to extract 96 samples, which greatly improves The efficiency of the experiment and the low cost allow it to be used in different laboratories. It is currently the mainstream instrument on the market.

The magnetic bead method automatic nucleic acid extraction system is divided into the magnetic rod method and the suction method.

Let me talk about the suction method first: as the name implies, the suction method is carried out by an automatic pipetting device. The lysis solution is added through the automatic pipetting device and the magnetic beads are adsorbed; the lysis solution is removed, the rinsing solution is added, the magnetic beads are adsorbed, and the rinsing is removed. Solution, add elution buffer. There seems to be no problem from the steps, but the biggest problem with the suction method is that in order not to remove the magnetic beads when removing the waste liquid, the pipette should not be too close to the magnetic beads to prevent the magnetic beads and the waste liquid from being sucked out. There is always a small part of the waste liquid that cannot be completely removed each time, especially the device with the magnet at the bottom. Because the magnetic beads are attracted to the bottom by the magnet, the pipette should not be too close to the bottom, so that the rinsing solution cannot be completely removed. Removal, the residual salt and ethanol in the rinsing solution will affect the subsequent elution efficiency and PCR success rate; the magnet on the side of the device will have less residual fluid, but the elution efficiency is not good, relying on the self-dissolving of the DNA to wash In the de-buffered solution, unless you wait for more than half an hour, some 96 automatic nucleic acid extraction workstations require 150 minutes for one round of extraction. The same time for the 32-magnet method can be used for 5 rounds.

Let’s talk about the magnetic rod method nucleic acid extraction system: flux usually has 8, 16, 32, 96 channels. Compared with the suction method, the advantage of the magnetic rod method is that the liquid in each step does not remain, because the magnetic rod only takes away the magnetic beads and transfers Go to the corresponding reaction wells in the next step. In addition, the extraction system of the magnetic rod method is better to have more complete functions, such as heating, and each heating tank should be independently temperature controlled, so that heating, pyrolysis, and elution can be set. Different temperatures, another very important point is that the motor that drives the magnetic rod must be able to drive the magnetic rod to quickly mix and stir the liquid, so as to facilitate automation and facilitate the lysis and thorough rinsing. The flux selection of the magnetic rod method is best to be 32 channels. The 96 channels seem to have higher flux, but there is a very practical problem. When 96 magnetic rods are transferred from one plate to another, the magnetic rods What to do if the liquid drops halfway, it will drip into other holes and cause pollution, and the 32-channel magnetic rod does not pass through the sky above other samples, so there will be no cross-contamination.

The basic principle of magnetic bead nucleic acid extraction system

1. The suction method

1. The suction method, also called the pipetting method, is to extract nucleic acid by fixing magnetic beads and transferring liquid. Generally, the operation system controls the mechanical arm to realize the transfer. The extraction process is as follows:

1) Lysis: Add the lysis solution to the sample, realize the mixing and full reaction of the reaction solution through mechanical movement and heating, cell lysis, and release of nucleic acid.

2) Adsorption: Add magnetic beads to the sample lysing solution and mix them thoroughly. The magnetic beads have a strong affinity for nucleic acids under high salt and low pH values ​​to adsorb nucleic acids. Under the action of an external magnetic field, the magnetic beads are separated from the solution. , Use the tip to remove the liquid and discard it to the waste tank, and discard the tip.

3) Washing: Remove the external magnetic field, replace with a new tip, add washing buffer solution, mix thoroughly to remove impurities, and remove the liquid under the action of an external magnetic field.

4) Elution: Remove the external magnetic field, replace with a new tip, add elution buffer, and mix thoroughly. The bound nucleic acid is separated from the magnetic beads to obtain purified nucleic acid.

2. Magnetic rod method

The magnetic rod method realizes the separation of nucleic acids by fixing the liquid and transferring the magnetic beads. The principle and process are the same as the suction method, but the difference is the method of separating the magnetic beads and the liquid. The magnetic rod method is to separate the magnetic beads from the waste liquid through the adsorption of the magnetic beads on the magnetic rod, and put them into the next liquid to realize the extraction of nucleic acid.

Features of magnetic bead method nucleic acid extractor

1. Able to realize automatic and high-throughput operation.

2. The operation is simple and fast.

3. Safety and environmental protection.

4. High purity and high yield.

5. No pollution and stable results.

6. Low cost, convenient for wide application.

7. Can process different types of samples at the same time.

Precautions

1. The installation environment of the instrument: normal atmospheric pressure (altitude should be lower than 3000m), temperature 20-35℃, typical use temperature 25℃, relative humidity 10%-80%, unobstructed air flow is 35℃ or below.

2. Avoid placing the instrument near a heat source, such as an electric heater; at the same time, to prevent short circuits of electronic components, avoid splashing water or other liquids into it.

3. The air inlet and exhaust vents are located on the back of the instrument, and at the same time, avoid dust or fibers from gathering at the air inlet to keep the air duct unobstructed.

4. The nucleic acid extractor is at least 10cm away from other vertical surfaces.

5. Instrument grounding: In order to avoid electric shock accidents, the input power cord of the instrument must be grounded.

6. Keep away from live circuits: Operators are not allowed to disassemble the instrument without authorization. Replacement of components or internal adjustments must be completed by qualified professional maintenance personnel. Do not replace components when the power is turned on.

Since August, the new crown pneumonia epidemic has once again become the “normal”. Epidemics of varying degrees and scales have appeared around the world, and there are thousands of new crown pneumonia nucleic acid samples waiting for testing every day. How are these samples detected in the laboratory? What does the P3 laboratory look like as the “first battlefield” for humans to fight the new crown virus, and what is the whole process of nucleic acid testing?

According to the level of risk, including the infectivity and harmfulness of infectious pathogens, biological laboratories are divided into four levels: P1, P2, P3, and P4 in the world. The work that can be undertaken by the P1-4 laboratory is also divided according to this safety level, with the strict level from low to high. P3 laboratory, also known as a protection laboratory, is suitable for processing highly hazardous to humans, animals, plants or the environment, through direct contact or aerosols that can infect people with serious or even fatal diseases, or highly harmful to animals, plants and the environment There are usually preventive and therapeutic measures for the pathogenic factors.

How is nucleic acid testing performed in the laboratory?

It can be roughly divided into: pre-test preparations-check sample information-sample inactivation-open the lid and add samples and nucleic acid extraction-PCR reaction system preparation-nucleic acid amplification testing-test completion-autoclave .

Before testing

Before opening the sample, the inspector needs to wear personal protective equipment, such as protective clothing, masks, goggles, face screens, double-layer medical latex gloves, and waterproof boots. Every step must not go wrong.

Checking

1. Use 75% alcohol to disinfect the outer surface of the sample delivery box in the core area of ​​the laboratory. After disinfection, check the name, age, gender and other information of the tested sample.

2. Put the sample in a water bath for half an hour to inactivate the virus protein at a high temperature of 56℃, so that the virus protein loses its physiological activity, and the virus loses its ability to infect, cause disease and reproduce without affecting the gene sequence of the virus protein. , To ensure the safety of detection.

3. When the experimenter gets the inactivated sample, the first step is to shake, try to let the virus on the swab elute in the medium solution, and the second step is to perform 5 minutes of precipitation. The third part is to open the cover and add samples. This step must be performed manually and requires a high degree of cooperation between two people. One person unscrews the lid of the sample tank, and the other uses a micropipette to suck a small amount of solution in the sample tank, put it in another extraction tube, and then screw on the tube cover.

Nucleic acid extraction can be assisted by instruments, but due to the need for comparison, manual operations are often required, and more than 10 steps such as centrifugation, addition of reagents, and washing are required to be repeated. Among them, there are more than 7 operations that need to open the lid of the tube, and it takes about 50 minutes to complete an artificial nucleic acid extraction.

4. Go out of the core test area, start the PCR reaction system preparation, and add the extracted viral nucleic acid to the nucleic acid amplification detection reagent.

5. Place the prepared PCR reaction on the fluorescent quantitative PCR machine. Set the PCR reaction conditions on the computer, run the instrument, and start nucleic acid amplification detection.

After testing

The tester needs to pay attention to the test situation in real time. After about 1.5 hours, the nucleic acid amplification is completed and the test result is interpreted. At this point, the entire process is completed, about 4 hours.

The last and very important step is to autoclave the contaminants. After the contaminants produced during the experiment are autoclaved, they are treated as ordinary medical waste.

It can be said that the process of nucleic acid detection is also a process of racing against time. Especially in scenarios where large-scale nucleic acid testing is required, transportation to a designated laboratory for testing obviously requires a high time cost. The use of mobile laboratories provides an effective method for improving the efficiency of nucleic acid detection.

For more information about COVID-19 virus nucleic acid testing, please click here:

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