How to choose the right nucleic acid extraction system

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

HERO-96

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

HERO 32

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.

What are the misunderstandings of the magnetic bead method for nucleic acid extraction?

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

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.

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 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.

Carboxyl magnetic beads

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.

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 group, different coating density, different functional group arm length, 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 extraction system; some magnetic beads have slow settling speed but long magnetic response time, and are more suitable for pipette automatic extraction system.

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.

Introduction to the whole process of laboratory nucleic acid detection

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 .

Pre-packed nucleic acid extraction kit

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.

Third Party Inspection

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.

Forensic Laboratory

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:

What is the process of COVID-19 nucleic acid testing?

How to improve the accuracy of COVID-19 nucleic acid testing?

Summary of nucleic acid separation and purification methods

With the recent rebound of the global epidemic, nucleic acid testing has become the primary way to detect viruses, and it has also deepened everyone’s understanding of nucleic acids. In fact, the separation and purification of nucleic acid is the primary problem that needs to be solved in genetic engineering or protein engineering research. It is the basic step to initiate other downstream activities (such as sequencing, amplification, hybridization, ligation, cloning and biological detection), and is a modern biological detection experiment. The basic means of technology. There are many commonly used methods for DNA extraction. Here we mainly introduce the phenol-chloroform extraction method, the high salt precipitation method, and the silica medium adsorption method.

The phenol-chloroform extraction method uses phenolic reagents as protein denaturants. The sample is first lysed, then extracted (phenol/chloroform) and then precipitated (absolute ethanol). The role of chloroform is to remove excess phenol and promote the separation of the aqueous and organic phases. The extraction method requires multiple centrifugation, and the steps are complicated and easy to cause cross-contamination. In addition, due to the addition of organic solvents such as benzene/chloroform, there will be residues in the final product, which will affect the subsequent downstream applications of genomic DNA.

The high-salt precipitation method removes protein impurities and separates DNA by adding various proteases. This method effectively eliminates the contamination of reagents, and the extracted DNA has a larger yield and higher purity, but the disadvantage is that the process of digesting the protease takes more time.

Pre-packed nucleic acid extraction kit

Silicon media adsorption is the dehydration of the phosphodiester skeleton in the DNA molecule under the action of chaotropic salt, so that the phosphoric acid group is exposed and at the same time it is reversibly adsorbed to the silica gel. Electrostatic force and hydrogen bond play a key role in the adsorption of silica gel and nucleic acid. This method has certain limitations on the chain length of deoxyribonucleic acid, and smaller DNA fragments (<100 bp) are not easy to be effectively adsorbed on the medium. The silica medium adsorption purification method allows the lysate to pass through the filter membrane by centrifugation and vacuum pressure, which can effectively extract trace amounts of DNA.

The above three methods have relatively strict requirements on the personal operation ability of the experimenter due to the cumbersome process, high time cost, and the addition of harmful reagents. All these make it impossible to achieve high-purity, high-return, and automated extraction of DNA, especially for the purification process of large samples. Taking the establishment of a gene sample bank as an example, the workload is relatively large, the required time period is relatively long, and the project budget is relatively large.

Magnetic solid phase extraction (MSPE) has attracted much attention in the pretreatment of biological samples. The ability of magnetic materials to bind to the target is the key to sample pretreatment. As an alternative to traditional extraction methods, MSPE has been increasingly used to extract genomic DNA from bacteria or cell lysates due to its fast processing time, reduced organic solvent requirements and ease of implementation, and is suitable for various biological samples DNA extraction (such as bacteria, viruses, semen, saliva, urine, plants, etc.).

First, the magnetic material is combined with the target after a certain operation (adsorption); then, a certain magnetic field is applied to the magnetic material to effectively separate it from the sample solution and remove impurities; finally, select appropriate reagents The target is eluted on the surface of the material.

MSPE has many advantages in the separation and purification of biological samples: the method steps are relatively simple; it avoids the destruction of proteins, nucleic acids and other substances; it can directly operate on existing biological samples without adding other operations; it can be used repeatedly under certain circumstances .

What is the process of COVID-19 nucleic acid testing?

Recently, as the COVID-19 strain has mutated, its toxicity and infectivity have become stronger, and the global epidemic situation has become extremely serious. To prevent the spread of the virus, in addition to prevention, it is more important to control the source of infection. Nucleic acid testing can The virus lurking in the human body is detected at the first time. Nucleic acid testing is to find out whether there are nucleic acids of foreign viruses in the respiratory specimens, blood or feces of patients to determine whether they are infected by COVID-19. Therefore, once the test is “positive” for nucleic acid, it can prove that there is a virus in the patient’s body.

After COVID-19 infects the human body, it will first reproduce in the respiratory system. Therefore, the virus nucleic acid in sputum and nasopharyngeal swabs can be tested to determine whether the human body is infected with the virus. Therefore, a positive nucleic acid test can be used as a new standard for the diagnosis of COVID-19 infection.

Pre-packed nucleic acid extraction kit

Principles of COVID-19 nucleic acid detection

The most common method to detect the specific sequence of COVID-19 is fluorescence quantitative PCR (polymerase chain reaction). Since the PCR reaction template is only DNA, the COVID-19 nucleic acid (RNA) should be reverse transcribed into DNA before the PCR reaction. In the PCR reaction system, a pair of specific primers and a Taqman probe are included. The probe is a specific oligonucleotide sequence with a reporter fluorophore and a quencher fluorophore labeled at both ends. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the enzyme’s exonuclease activity to transfer the probe along the template. Enzyme digestion and degradation, the reporter group is separated from the quenching group and emits fluorescence. Every time a DNA strand is amplified, a fluorescent molecule is produced. The fluorescent quantitative PCR instrument can monitor that the number of cycles (Ct value) at which the fluorescence reaches the preset threshold is related to the concentration of viral nucleic acid. The higher the concentration of viral nucleic acid, the smaller the Ct value. The products of different manufacturers will determine the positive judgment value of this product based on the performance of their own products.

COVID-19 nucleic acid detection process

For the nucleic acid detection of COVID-19, first according to the sample requirements of the nucleic acid extraction kit instructions, some samples are collected. The conventional sample types include throat swabs, nasal swabs, sputum, bronchial lavage fluid, alveolar lavage fluid, etc.

After obtaining the patient sample, the test should be carried out as soon as possible. If the sample that needs to be transported cannot be tested immediately, it should be packaged at a low temperature according to the instructions and sent to a special testing agency for testing. After receiving the sample, the testing institution shall perform nucleic acid extraction on the sample, and the nucleic acid extraction reagent shall use the nucleic acid extraction kit specified in the approved product manual.

Viral RNA needs to be reverse transcribed into cDNA first, and then amplified and tested. PCR amplification and detection should use the fluorescent quantitative PCR instrument specified in the approved product manual. The Ct value of the sample obtained by fluorescent quantitative PCR can be used to determine whether the patient sample contains COVID-19.
In the above-mentioned process, the collection, storage, and transportation of samples, the extraction and detection of sample nucleic acids, and the interpretation of results must all be carried out in strict accordance with the requirements of the kit instructions.

Manual nucleic acid extraction kit

“False positive” and “false negative” in nucleic acid testing

The “false positive” of the nucleic acid test means that the patient has not been infected with the new coronavirus, but the nucleic acid test has a positive result. We have also introduced to you how to improve the accuracy of the COVID-19 nucleic acid test. The occurrence of “false positives” is usually caused by cross-contamination between specimens or laboratory nucleic acid contamination during laboratory testing. On the technical level, as long as the laboratory strictly implements the quality control work, the occurrence of “false positives” can be effectively avoided.

The “false negative” of the nucleic acid test means that the patient’s clinical symptoms, lung imaging results and even epidemiological history support the new coronary pneumonia, but the patient’s viral nucleic acid test result is “negative”, and the test result is inconsistent with the clinical.

The usual causes of “false negatives” are:

(1) In the initial stage of virus invading the human body, the amount of virus in the human body has not yet reached a detectable level. In different periods of viral incubation, mild symptoms, and severe symptoms, the viral load of different parts of the human body (such as the nasopharynx, oropharynx, trachea, bronchi, and alveoli) will vary. Therefore, different sampling timing and sampling locations may result in insufficient virus in the collected specimens;

(2) Any test reagent has its lower limit of detection (ie sensitivity). If the virus in the patient’s specimen does not reach the lower limit of detection of the reagent used, a false negative will occur;

(3) Poor performance of laboratory equipment and personnel, poor quality management, etc. will also produce “false negatives”;

(4) Irregular sampling, improper collection location, and atypical collection of specimens have resulted in too few or no virus-infected cells in the specimens. That may cause “false negatives.”

The above is an introduction to the current principles and procedures of nucleic acid detection for COVID-19. It is hoped that the source of infection can be controlled to the greatest extent through nucleic acid detection and a method to suppress the virus can be found as soon as possible.

How to improve the accuracy of COVID-19 nucleic acid testing?

With the rebound of the COVID-19 epidemic, the demand for nucleic acid testing has increased sharply. However, the accuracy of nucleic acid testing results has attracted widespread attention. The “missing” situation has caused some clinicians and the public to raise questions about the quality of test kits. doubt. So, what are the factors that may affect the current accuracy of nucleic acid detection? As a professional nucleic acid extraction kit supplier, Ascent shared with us how to improve the accuracy of COVID-19 nucleic acid detection from specimen collection, specimen storage and transportation, nucleic acid extraction, and amplification testing.

1. Specimen collection

Correct sampling is the key to the success of the experiment. It is understood that due to the limitation of maneuverability, the most common sampling method is to use nasopharyngeal swabs, sputum or alveolar lavage fluid. The most common method is to collect nasopharyngeal swab specimens. However, the sampling technique of the nasopharyngeal swab will affect the accuracy of the test. It is best to have a trained nurse sample the patient. It is necessary to collect enough effective cells from the patient’s nasopharynx. Inexperienced nurses may not be able to obtain qualified specimens. .

In addition, the nasopharyngeal swabs and preservation solutions used for specimen collection will also affect the quality and preservation of the collected specimens. It is recommended to use sterile flocked swabs that can absorb and release more samples and free of free DNA. The cell preservation solution of enzyme and RNase can better preserve viral nucleic acid and avoid COVID-19 RNA degradation.

2. Specimen storage and transportation

COVID-19 is an RNA virus. Special attention should be paid to the storage temperature and the corresponding length of time when specimens are stored and transported to avoid nucleic acid degradation and false negatives. Specimens should be sent for inspection as soon as possible after collection, and sent to the laboratory within 2h to 4h after collection. Blood specimens should be transported at room temperature, and other specimens should be transported at 2°C to 8°C; if the transport time exceeds 24 hours, the specimens should be stored at -70°C or lower, and short-distance transport should be carried out at 2°C to 8°C. Dry ice transportation shall be used for distance transfer. Avoid repeated freezing and thawing during specimen transportation and storage.

3. Nucleic acid extraction

Nucleic acid testing has high requirements for laboratories, equipment, personnel and reagents. Special PCR laboratory partitions are needed to ensure the accuracy of the test results. Nucleic acid extraction is a crucial step in nucleic acid detection.

The currently approved kit only contains the amplification reagents required for COVID-19 nucleic acid detection, and does not contain nucleic acid extraction reagents. There may be differences in the extraction efficiency of different nucleic acid extraction reagents or nucleic acid extraction systems. It is recommended to use verified extraction methods and extraction reagents for nucleic acid extraction to ensure the accuracy of the test results.

In order to ensure the safety of inspectors and the purity and efficiency of nucleic acid extraction, an automated nucleic acid extraction method based on magnetic bead adsorption can be used. Many authoritative laboratories and related experts recommend using a small-sized semi-automatic nucleic acid extraction instrument, put it in a biological safety cabinet, and complete all the steps of sample loading and nucleic acid extraction in the safety cabinet, which is simple and efficient, and protects the safety of operators.

Pre-packed nucleic acid extraction kit

4. Amplification detection

The nucleic acid detection of COVID-19 mainly targets the open reading frame 1ab (ORF1ab), nucleocapsid protein (N), and envelope protein (E) of the COVID-19 genome. To confirm that a case is positive in the laboratory, the following conditions are met: three targets (ORF1ab, N, E) specific real-time fluorescent RT-PCR test results in the same specimen are more than two (including two) positive. If there is a positive test result for a single target, re-sampling and re-testing are required.

Negative results cannot rule out new COVID-19 infections. Factors that may produce false negatives need to be ruled out, including: poor quality of specimens, such as respiratory samples from oropharynx and other parts; specimens collected too early or too late; improper storage and transportation And processing samples; the reasons for the technology itself, such as PCR inhibition, etc.

Due to the serious spread of the epidemic, the development of COVID-19 nucleic acid detection kits and emergency approval are urgent. There may be insufficient clinical sample verification data in the early stage, and there is a risk of insufficient sensitivity and specificity optimization. The reagent performance and positive detection rate of different manufacturers are different. Most kits only detect the two targets of ORF1ab and N gene, and the single target positive results of some reagents account for more than 20% of the positive results. For single target positive results and suspicious results, it is recommended to use different kits for review, or send CDC for confirmation, but it will increase the workload and delay the diagnosis time. From the public information, the currently approved reagents do not contain UNG enzyme and dUTP anti-pollution system, and there are risks of contamination and false positives.

What are the functions of immunomagnetic beads?

In recent years, immunomagnetic beads have attracted more and more attention from scientists, and their applications in the field of biomedicine have become more and more extensive. But in fact, immunomagnetic beads are not new products that have only recently developed. As early as the late 1980s, it has begun to play a huge role in the field of cell separation and enrichment.

Immunomagnetic beads generally have super strong paramagnetism. In the presence of an external electric field, the immunomagnetic beads will exhibit magnetism and be aggregated. After leaving the magnetic field, they can be uniformly dispersed like ordinary particles. The surface of immunomagnetic beads generally has abundant surface active groups, and biologically active molecules can be adsorbed or coupled to its surface, so as to realize its use in cell sorting, nucleic acid separation and extraction, immunoassay, biological macromolecule purification and enzyme immobilization, etc. Applications in multiple fields.

Nucleic acid extraction is one of the earliest applications of magnetic beads, and the method has not changed much in decades. By adjusting the pH value of the buffer, the nucleic acid binds to the magnetic beads through electrostatic adsorption. Then, under the condition of an external electric field, the waste liquid is removed and washed, and finally a pure nucleic acid sample can be obtained.

The magnetic bead method avoids steps such as vacuuming and high-speed centrifugation, and can reduce damage to the sample caused by external forces. The magnetic bead extraction method is easy to operate, simple reagents, and suitable for high-throughput automated operations in 96-well plates or 384-well plates.

With the advancement of technology, more and more manufacturers have developed different nucleic acid extraction kits based on immunomagnetic beads. A variety of chemical modifications on the surface of magnetic beads bring the possibility to realize complex experiments.

Immunomagnetic Beads Chemiluminescent Magnetic Beads

Cell sorting

Cell sorting is another popular application of immunomagnetic beads. Using immunomagnetic beads combined with markers on the surface of target cells, high-purity target cells can be separated from complex cell mixtures within a few minutes. The immunomagnetic beads will not activate the cells or affect the function and viability of the cells, and the physiological functions of the cells will not change, so the magnetically labeled cells can be used for analysis and subsequent experiments immediately.

Another common method for cell sorting is FACS. In the case of single cell sorting, multiple cell sorting at the same time, or sorting based on intracellular cell markers (such as GFP), FACS method is more advantageous. Compared with FACS, the operation of magnetic bead sorting is simpler and faster. For general cell sorting, the method of immunomagnetic bead can be preferred.

Antibody separation and purification

Traditional antibody separation and purification generally use chromatography, which is cumbersome, time-consuming, high in equipment requirements and high investment costs, while antibody magnetic purification based on protein-coated immunomagnetic beads can achieve expression of products from monoclonal antibodies through simple magnetic adsorption. The purpose of separating monoclonal antibodies. Compared with traditional separation methods, immunomagnetic beads can perform separation and enrichment at the same time, which effectively improves the separation speed and enrichment efficiency. The immunomagnetic beads can also realize automation and mass operation, meet the high-throughput operation requirements of biology, and have the characteristics of convenient use, simple operation, short time and low cost.

Cell stimulation

Immunomagnetic beads also play an important role in cell therapy. In the activation and expansion of T cells and NK cells, antibody or protein-coupled immunomagnetic beads can replace APC, which avoids the tedious operation of cell processing to a certain extent. After completing cell stimulation and activation, the immune magnetic beads can be completely removed by applying a magnetic field. Compared with directly adding antibodies or proteins for stimulation, the residual pollution of soluble antibodies or mitogens is avoided. The same immunomagnetic bead can be coupled to a variety of required antibodies and proteins, and the proximity effect can greatly optimize the effect of cell stimulation and improve the efficiency of cell expansion.

In Vitro Diagnosis

The use of immunomagnetic beads can combine the characteristics of target molecules, and its application methods in in vitro diagnosis are also increasing. First, immunomagnetic beads are used as substrates, which can be used to capture samples. Because the magnetic beads have good fluidity and large surface area, they can fully expose the ligand protein and greatly increase the capture efficiency. At the same time, the use of various detection methods such as fluorescence, electrochemistry, or chemiluminescence also brings a lot of freedom to the development of methodology. In addition, immunomagnetic beads can also be used as markers, and the magnetic properties of magnetic beads can be used to obtain signals. This method generally has extremely high sensitivity, which can reach fg/ml.

Introduction of Magnetic Bead Method of Nucleic Acid Extraction Technology

With the rising popularity of nucleic acid testing for the new coronavirus, nucleic acid testing has become deeply rooted in the hearts of the people and has become the “gold standard” for pathogen detection. Since the new crown epidemic, a large number of sample test results have been required in a short period of time, and the need for rapid and efficient nucleic acid testing has become more and more urgent. Nucleic acid extraction, as an important step in nucleic acid detection, also faces high-throughput and high-efficiency requirements.

Nucleic acid detection depends on the development of nucleic acid extraction technology. At present, the main clinical applications of nucleic acid extraction methods include magnetic bead method, spin column method, boiling method, and rapid nucleic acid release method. Magnetic bead method nucleic acid extraction method has been widely used. Realize the automatic nucleic acid extraction method, and the automatic magnetic bead extraction method mainly includes magnetic rod extraction and pipetting extraction.

1. The principle of magnetic bead method for nucleic acid extraction

The magnetic bead method of nucleic acid extraction technology is a new type of nucleic acid extraction technology that uses nano-biological magnetic beads as a carrier. Nucleic acid molecules can specifically recognize and bind to the silicon hydroxyl groups on the surface of the magnetic beads, and aggregate or disperse under the action of an external magnetic field. Completely get rid of manual operations such as centrifugation and supernatant extraction in the traditional nucleic acid extraction process, so as to realize the method of nucleic acid extraction and purification.

Most of the magnetic beads used at present have a “core-shell” structure, which uses superparamagnetic nanoparticles as the “core”, and then a layer of polymer organic compound and a functional base layer are wrapped on the surface of the core. As a “shell”, it has better suspension in the solution. It is magnetized in a magnetic field and gathered under the action of magnetic force. It is very easy to separate from non-magnetic materials. There is no need for centrifugal operations during the operation.

Carboxyl magnetic beads

2. The difference of nucleic acid extraction by magnetic bead method

Magnetic bead nucleic acid extraction consists of lysis, binding, washing, elution and other steps. The magnetic bead nucleic acid extraction reagents produced by different manufacturers are different, mainly reflected in: (1) different sizes of magnetic beads; (2) magnetic bead modification Different methods; (3) Different washing methods; (4) Different sample sizes.

The magnetic beads have small particle size, large specific surface area, and strong nucleic acid capture ability. They can be tested on the machine with magnetic beads to improve detection sensitivity. The particle size is different, and there are different preferences for nucleic acid fragments of different sizes. Generally speaking, magnetic beads with a larger particle size have greater magnetic responsiveness and a greater ability to capture small nucleic acid fragments. Magnetic beads with a smaller particle size have lower magnetic responsiveness and a greater ability to capture large nucleic acids. Magnetic beads with different particle sizes have different specific surface areas and different nucleic acid capture capabilities. At the same time, magnetic beads with a larger particle size have a smaller specific surface area and have a slightly poorer ability to capture nucleic acids; magnetic beads with a smaller particle size have a larger specific surface area and have a slightly stronger ability to capture nucleic acids. The elution step directly affects the yield of nucleic acid. Of course, the magnetic beads have small particle size and strong light transmittance, and can also use magnetic beads on the machine to detect when they do not affect the detection signal collection of the fluorescent quantitative PCR instrument (add PCR reaction during elution) Nucleic acid detection is carried out in the manner of mixing the magnetic beads and detecting the nucleic acid. There is almost no loss of nucleic acid. All template nucleic acids are detected on the machine, which can improve the sensitivity of nucleic acid detection.

Functional group-targeting nucleic acid probe modification further improves the extraction efficiency of targeted nucleic acid and improves the sensitivity of targeted nucleic acid detection. The functional groups contained in the functional base layer of the magnetic bead shell structure are different, and the specific binding ability of the magnetic bead and nucleic acid is different. The commonly used modified functional groups are amino (-NH2), carboxyl (-COOH), and hydroxyl (-OH).

Because the functional groups are different, the binding buffers used are also different. The hydroxyl group is negative and alkaline, and can exist stably in alkaline solutions, while the carboxyl group is acidic and can exist stably in acidic solutions. The functional group-targeting nucleic acid probe modification method can improve the specific binding ability of the targeted nucleic acid sequence and further improve the efficiency of nucleic acid extraction.

The fewer washing times, the less nucleic acid loss and the higher the nucleic acid yield. Generally speaking, the more washing times, the greater the possibility of nucleic acid loss, and the lower the nucleic acid yield may be. The more steps will affect the stability of automated nucleic acid extraction, the more difficult it is to achieve automation.

Increase the sample size and improve the sensitivity of nucleic acid detection. Increasing the sample size is also the usual practice of some manufacturers. For example, Roche Diagnostics and Abbott Diagnostics’ hepatitis B virus nucleic acid quantitative detection kits can achieve the purpose of high-sensitivity detection by increasing the sample size and increasing the nucleic acid concentration, and its sensitivity can reach 10 IU/mL.

In short, the magnetic bead method of nucleic acid extraction technology is currently the most widely used clinically automated nucleic acid extraction technology on the market, and it plays a main role in the prevention and control of the new coronavirus epidemic. The magnetic bead method nucleic acid extraction reagents produced by different manufacturers have different nucleic acid lysis methods, magnetic bead binding capacity, washing methods, and elution methods. Only the nucleic acid lysis efficiency is high enough, the magnetic bead specific binding capacity is strong enough, and the number of washings is sufficient. Only a small amount and high enough elution efficiency (or the elution of the original sample nucleic acid) can guarantee a higher nucleic acid extraction efficiency, and a high-sensitivity or hyper-sensitivity nucleic acid detection can be guaranteed to be better. The stability of automated nucleic acid extraction.

How to do nucleic acid testing?

Now, due to the spread of the COVID-19 epidemic, we are required to do nucleic acid testing when we travel or travel to other countries or provinces. We need to have a negative nucleic acid test result. Then, what should we do?

1. Before testing:

(1) Try to avoid eating 2 hours before the nucleic acid test to avoid vomiting;

(2) Do not drink beverages (including water), smoke, drink, or chew gum 30 minutes before sampling;

(3) During the clinical examination, reduce swallowing movements and avoid clearing the throat (such as expectoration and spitting);

(4) Before collecting nasopharyngeal swabs, the person being tested should inform the collecting staff whether they have relevant past medical history or related matters. For example, history of nasal surgery, curvature of the nasal septum, blood disease, throat disease, or taking anticoagulants and other related risk factors;

(5) The tester needs to wear a mask correctly, remove the mask before the test, and wear it immediately after the test. A spare mask can be prepared, which can be easily replaced at any time after contamination.

Manual nucleic acid extraction kit

2. Testing:

(1) When taking an oropharyngeal swab, the subject tilts his head back and opens his mouth to make an “ah” sound, which helps to expose the throat, but symptoms such as dry cough, nausea, and vomiting may occur during the process. The subject may cooperate Collectors try to relax and breathe deeply;

(2) During the collection process, there may be a soreness of the nose and irritation to sneezing, which can be covered immediately with a paper towel (prepared in advance) or elbow;

3. After testing:

(1) Leave the collection site immediately after collection, and avoid spitting and vomiting around the collection site;
(2) Pay attention to hand hygiene before and after the nucleic acid test. You can use hand sanitizer or alcohol to wipe your hands.

(3) In addition, in order to improve efficiency, you can prepare in advance: due to the tight protection of medical staff, you may not be able to hear the speech. At the same time, it is also to improve efficiency. Please be sure to bring your ID card or household registration book and wear a mask (preferably two, of which One spare), with a package of tissues (collection may cause discomfort and tears), you can write a note in advance with your name, ID number, current address, and contact number (please keep the font neat when writing to prevent the staff from being unrecognizable) ) To the relevant personnel!

Pre-packed nucleic acid extraction kit (inner packaging)

Nucleic acid is actually genetic material, or genes in layman’s terms. Take viruses as an example. Their life forms are very simple, just composed of outer capsids and genetic material wrapped in them. The genetic material of a virus can be DNA or RNA, and it is divided into one strand and two strands, namely, single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. The genetic material of the new coronavirus is single-stranded RNA, which determines the outer capsid synthesis, as well as the pathogenicity and transmission of the virus.

Nucleic acid testing is an important means of epidemic prevention and control. Different viruses have different forms, all because they carry different nucleic acids. The nucleic acid of all viruses has its characteristic part. The nucleic acid test is to amplify the characteristic part of the virus gene in a large amount to reach the detection amount that can be identified. Therefore, a positive nucleic acid test of a certain virus can be used as the diagnosis of this virus infection. Basis. The COVID-19 virus nucleic acid test is to detect at least two characteristic fragments in its RNA, and if it is detected, it will be judged as positive.

How to choose a suitable nucleic acid extraction system, and what are the misunderstandings in the selection?

With the rapid development of biotechnology, with the application of PCR technology in various fields, including medical disease detection, agricultural genetic modification detection, and many other applications, high-throughput sample nucleic acid extraction has become extremely urgent, and many companies have launched With the automatic nucleic acid extraction instrument, there are various principles. How to choose a suitable nucleic acid automatic extraction instrument requires detailed analysis.

From the perspective of extraction principle, there are spin column method and magnetic bead method. The centrifugal column method automatic nucleic acid extraction system uses a centrifuge and an automatic pipetting device to combine the method. The throughput is generally 1-12 samples. The time is similar to manual extraction. It does not improve efficiency, and the price is not cheap. Not universal, suitable for labs with sufficient funds.

The magnetic bead method automatic nucleic acid extraction system is divided into the magnetic rod method and the suction method. First, the suction method. As the name suggests, the suction method is carried out by an automatic pipetting device, and the lysis solution and magnetic beads are added through the automatic pipetting device. Adsorption, aspirate the lysate, add the rinsing solution, magnetic bead adsorption, aspirate the rinsing solution, and add the elution buffer.

HERO 96

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 adsorbed to the bottom by the magnet, the pipette cannot 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 liquid, but the elution efficiency is not good, relying on the DNA to dissolve to the wash. In the de-buffered solution, unless you wait for more than half an hour, so in some 96 automatic nucleic acid extraction workstations, the extraction-round time takes 150 minutes, and the 32-magnet method can take 5 rounds in the same time.

Let’s talk about the magnetic rod method nucleic acid extraction system. The 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 well in the next step. In addition, the extraction instrument of the magnetic rod method should be more complete, such as heating, and each heating tank should be independently temperature controlled, so that heating, lysis, 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 help lysis and rinsing thoroughly.

The flux selection of the magnetic bar method is best to choose 32 channels. The 96 channels seem to have higher flux, but there is a very practical problem. When 96 magnetic bars are transferred from one plate to another, the magnetic bars are on the top. 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 above is the relevant introduction about the selection of nucleic acid extraction system, but many people are still easy to fall into the misunderstanding of selection. The following examples illustrate the two most common misunderstandings:

HERO 32

Misunderstanding 1: The difference between nucleic acid extraction system and workstation and the misunderstanding of selection

Many workstation manufacturers often tell their customers when they promote their workstations that the nucleic acid extraction system is fully automatic, while the nucleic acid extraction system is semi-automatic. Customers also subjectively think that the workstation can pipette, extract, and do anything, as if just put the sample in. Go in, you can get the nucleic acid you want without worrying about anything. When the workstation is in place, it turns out that the actual situation is not that way. Not only is it not as good as expected, but even the basic nucleic acid extraction is difficult. So what is the reason? ?

The first question is the problem of opening the lid. The lid structures of blood collection tubes, saliva storage tubes, and two-dimensional code cryopreservation tubes are different, the sizes are different, and the manufacturers are different. How to open the lid automatically at the workstation? This problem is solved No more.

The second problem is the entry of barcodes, barcodes and QR codes, as well as some manually affixed labels and handwritten labels, so the scanning of codes is still manual, which is difficult to automate.

The third problem is that the liquid addition is automated. The workstation can automatically add reagents, which seems to be an advantage, but it takes about 15-30 minutes to add a 96 deep-well plate, and extraction generally requires six reagents, that is, six plates. It takes about 1.5 hours. Many extraction reagents contain high concentration of ethanol. For such a long time, the concentration of ethanol will change, which will affect the extraction efficiency and sensitivity. Besides, many extraction instrument manufacturers have pre-installed kits that do not need to be added. liquid.

Misunderstanding 2: The difference between imported and domestically produced, this is the biggest misunderstanding of choice

Many people choose a nucleic acid extraction system based on habitual thinking and think that imported ones are better than domestic ones; however, nucleic acid extractors are application-oriented equipment, not basic experimental equipment, not the kind of equipment that can be used when plugged in. Nucleic acid extraction requires the overall cooperation of instruments, supporting kits and application programs. Manufacturers need to select the most suitable extraction kits and procedures according to the sample type. Reagents and procedures often need to be optimized. Without the manufacturer’s technical support, you cannot use it. . This explains well why many imported workstations and extraction instruments are collectively used by no one; the technical support of imported instruments cannot keep up, and the technical visits of imported instruments require expensive door-to-door fees, and there is no guarantee that they can be used. Help customers optimize the experimental plan (of course, the door-to-door fee must be charged).

On the whole, most nucleic acid extraction systems cannot keep up with domestic manufacturers in terms of instrument operating system convenience, instrument extraction sensitivity and yield, instrument kits, instrument upgrade and customization capabilities, etc., and the after-sales service charges are even more frustrating. There is no need to demonstrate the superiority of imports. Through the above discussion, we should be clear that when choosing a nucleic acid extraction system, it is best to conduct full research or trial, so as not to fall into the passive situation of buying a nucleic acid extraction system.