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

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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?

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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

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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?

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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?

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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.

What are the nucleic acid purification technologies?

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At present, nucleic acid purification technologies widely used in scientific research can be divided into two categories: those that use media and those that do not use media. If media is used, nucleic acid is separated from all other impurities at one time; if media is not used, the first step is to separate the nucleic acid from all other impurities. Nucleic acid and salt are separated from macromolecular impurities, and nucleic acid is separated from salt by precipitation of nucleic acid.

1) Classical purification technology using phenol/chloroform extraction

After the cells are lysed, the aqueous phase containing nucleic acid is separated by centrifugation, and an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1 volume) mixture is added. According to the application purpose, the two phases are vortexed and mixed (suitable for the separation of small molecular weight nucleic acids) or simply inverted and mixed (suitable for the separation of high molecular weight nucleic acids) and then centrifuged. Hydrophobic proteins are partitioned into the organic phase, and nucleic acids are retained in the upper aqueous phase.

Phenol is an organic solvent. It should be saturated with STE buffer beforehand. Unsaturated phenol will absorb the water phase and take away a part of nucleic acid. Phenol is also easy to oxidize and turn yellow, and oxidized phenol can cause the phosphodiester bond in the nucleic acid chain to break or crosslink the nucleic acid chain; therefore, a special substance should be added when preparing the phenol saturated solution to prevent phenol oxidation. Chloroform can remove fat and denature more protein, thereby improving extraction efficiency. Isoamyl alcohol can reduce bubbles generated during operation.

Nucleic acid salt can be precipitated by some organic solvents, nucleic acid can be concentrated by precipitation, the type of nucleic acid dissolution buffer can be changed and some impurity molecules can be removed. A typical example is precipitation with ethanol after phenol and chloroform extraction. After adding pH 5.0 to 5.5 in the aqueous phase containing nucleic acid, the final concentration of NaAc or KAc at a final concentration of 0.3M will neutralize the sodium ions on the nucleic acid phosphate backbone. The negative charge promotes the hydrophobic renaturation of nucleic acids in an acidic environment. Then add 2 to 2.5 times the volume of ethanol, after a certain period of incubation, the nucleic acid can be effectively precipitated. Some other organic solvents (isopropanol, polyethylene glycol (PEG), etc.) and salts (10.0mol/L ammonium acetate, 8.0mol/L lithium chloride, magnesium chloride and low-concentration zinc chloride, etc.) ) Is also used for the precipitation of nucleic acids.

Nucleic acid purification magnetic beads NGS screening

2) Purification technology using ion exchange media

The lysate is passed through the column, and the nucleic acid is bound to the ion exchange medium; after washing to remove residual impurities, the nucleic acid is eluted from the medium with a high-salt buffer. After standard ethanol/isopropanol precipitation, ethanol washing, drying and other operations, the pure nucleic acid is obtained and dissolved in a suitable buffer.

3) Purification technology using adsorption media

The lysate is passed through the column, and the nucleic acid is selectively adsorbed by the adsorption medium; after washing to remove residual impurities, the nucleic acid is eluted from the medium with water or a suitable low-salt buffer, and it can be directly used in subsequent experiments.

4) Density gradient centrifugation

Density gradient centrifugation is also used for the separation and analysis of nucleic acids. Double-stranded DNA, single-stranded DNA, RNA and protein have different densities, so pure sample zones of different densities can be formed by density gradient centrifugation. This method is suitable for the preparation of a large number of nucleic acid samples, in which cesium chloride 2 ethyl bromide Spindle gradient equilibrium centrifugation is considered to be the preferred method for purification of large amounts of plasmid DNA. Cesium chloride is the standard medium for nucleic acid density gradient centrifugation. The ethidium bromide in the gradient solution combines with nucleic acid. The nucleic acid zone formed after centrifugation is irradiated by an ultraviolet lamp to produce fluorescence and be detected. Dialysis or ethanol precipitation removes cesium chloride to obtain purified nucleic acid.

Manual nucleic acid extraction kit

Evaluation of purification methods

PC (P is the abbreviation of English phenol, C is the abbreviation of English chloroform) extraction/alcohol precipitation method is a method that will never be outdated. Stable, reliable, economical and convenient. PC extraction can completely remove proteins, and alcohol precipitation can remove salts. For ordinary clean samples (impurities are proteins), this method can completely obtain high-quality nucleic acids. Although each PC extraction will lose a part of the nucleic acid (because it is impossible to remove all of the water phase), and the low concentration of nucleic acid alcohol precipitation efficiency is low, but these problems can be solved by operating adjustments or reduce the impact.

The biggest problem of this method is that it is not suitable for large-scale extraction. PC extraction is a very effective means to remove protein. Phenol can denature the protein, and the denatured protein is separated from the water phase, in the phenol or between the phenol/water phase. The key to PC extraction is to mix thoroughly and to use enough. Thorough mixing can ensure sufficient contact between phenol and protein and completely denature the protein. Many people always worry about whether the violent mixing will cause damage to nucleic acids, especially genomic DNA. In fact, there is no need to be so careful.

Vigorous mixing operation will partially disrupt the genomic DNA of large molecules, but the damage will not be so strong that the DNA becomes a small fragment within 10kb. After shaking and mixing vigorously, most of the genomic DNA fragments will be larger than 20kb. This size, except for some special requirements, is completely suitable for PCR and restriction digestion. If the required fragments are very large, such as to construct a library, you cannot use a violent mixing method, but only gently inverted and mixed back and forth.

The key at this time is: the ratio of the lysis solution should be large enough to make the system not too viscous. The amount should be sufficient because phenol has a certain degree of saturation to remove protein. If the saturation is exceeded, the protein in the lysis system will not be removed at one time, and it must be extracted multiple times before it can be completely removed. In addition, the disadvantage of the system being too viscous is that the protein is difficult to completely remove, and the genomic DNA will be broken more severely, so pay attention to the ratio of the lysate to the sample. The 4C centrifugal operation is conducive to more thorough removal of protein. Another use of PC extraction is to use acidic phenol to partially remove DNA, and to obtain RNA with minimal DNA residue during RNA extraction. However, one thing to remind is that some plant samples cannot be extracted with PC before some impurities are removed, otherwise the nucleic acid will be degraded.

The high-salt precipitation protein/alcohol precipitation method is also a very good method. Compared with the PC extraction method, this method almost overcomes all the shortcomings of PC extraction, except that the purity stability may be lower. The benefit of faster and easier removal of protein is that it can be used for large-scale extraction, but the disadvantage is that the purity (protein residue) is not stable enough. The precipitation efficiency of protein is better at 4C.

The media purification method is a method that has received more and more attention. Its biggest feature is that it is very suitable for large-scale nucleic acid extraction, and because it is not affected by human factors, the stability of the purity is high. Its Achilles’ heel is the excessive amount of sample. The medium can be divided into two categories, one is column type, that is, the medium is pre-filled in the column below it; the other type is granular. The purification operation of the granular medium is not much different from the classic alcohol precipitation. It is through the process of adding liquid and pouring liquid several times. After drying, the purified nucleic acid can be obtained by dissolving it.

Although the operation of column purification also has the process of adding liquid and pouring liquid, because the added liquid will enter another centrifuge tube after centrifugation, and it is completely separated from the column containing nucleic acid, so the washing is more thorough and the operation is more labor-saving. However, the cost of the media purification method is the highest.

Application of immunomagnetic beads in the field of biomedicine

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Immunomagnetic bead technology is a technical method that appeared in the 1980s. Based on immunology, it has penetrated into various fields such as pathology, physiology, pharmacology, microbiology, biochemistry, and molecular genetics. It has become more and more widely used in immunoassays, cell separation, purification of biological macromolecules, and molecular biology.

Structure of immunomagnetic beads

Magnetic beads are composed of core metal particles (Fe2O3, Fe3O4), a polymer material (such as polystyrene, polyvinyl chloride) wrapped in the core outer layer, and the outermost functional ligand (such as -NH2, -COOH, -OH, -CHO) composition.

Application of immunomagnetic beads in the field of biomedicine

Nucleic acid purification magnetic beads NGS screening

1. Cell sorting

Immunomagnetic bead cell sorting can separate very high purity cells from complex cell mixtures within a few minutes. When using nano-scale magnetic beads for cell sorting, the size of the magnetic beads and its composition make it biodegradable without activating the cells or affecting the function and vitality of the cells, and the physiological functions of the cells are also unchanged. Magnetically labeled cells can be used immediately for analysis and subsequent experiments.

Cell sorting can be divided into

a) Positive sorting (positive sorting): The cells bound by magnetic beads are the cells to be separated, which is suitable for flow analysis and cell-based analysis.

b) Negative sorting (negative sorting): The magnetic beads bind to unwanted cells, and the cells free in the supernatant are the desired cells.

Positive selection (left) and negative selection (right) are as follows:

1. Important indicators for magnetic separation of cells

Purity and yield depend on the specificity of the monoclonal antibody connected to the magnetic beads and the size (magnetic) of the magnetic beads. However, the yield of magnetic beads that are too small is not high, and magnetic beads that are too large will affect cell viability and cannot directly Upstream.

2. Protein/antibody separation and purification

The application of antibody (protein)-coated immunomagnetic bead purification technology does not require complicated chromatography equipment, and has no limitation on the clarity of the sample. It only requires a simple magnetic adsorption step to easily separate the monoclonal antibody from the monoclonal antibody expression product. Effectively solve the shortcomings of traditional chromatography technology.

3. Nucleic acid separation and purification

The binding of nucleic acid to magnetic beads mainly relies on electrostatic, hydrophobic and hydrogen bonding. The DNA/RNA in the cell or tissue is released under the action of the lysis solution. At this time, the surface-modified superparamagnetic silica nanomagnetic beads “specifically bind” with nucleic acid to form a “nucleic acid-magnetic bead complex”. Then under the action of an external magnetic field, the complex is separated.

It can be widely used in genome research in molecular biology, molecular evolution research, genetic disease research in medicine, mutation gene detection, tumor screening, HPV detection, HLA typing, transplantation matching, etc., forensic biological samples The detection of blood spots, fine spots, hair, cigarette butts and other on-site evidence, and judicial paternity testing, blood relationship identification, etc. provide evidence, archeology, biological experiments in universities, middle schools, and many other fields.

Amino Magnetic Beads

Magnetic bead method to extract nucleic acid:

Traditional nucleic acid extraction method Magnetic Bead Extraction of Nucleic Acid
Not technically difficult High quality, high yield, high throughput
Manual extraction Realize process automation
The operation is cumbersome, time-consuming and laborious Eliminates complicated manual extraction procedures
Not suitable for nucleic acid extraction of a large number of samples Reduce the damage of phenols, chloroform and other organic reagents to operators
Not suitable for clinical molecular diagnosis Greatly meet today’s market requirements for nucleic acid extraction efficiency

4. Immunoassay

Because of its small particle size and large specific surface area, immunomagnetic beads can capture more analytes, and directly perform enzyme color, fluorescence or isotope display on their surface, thus establishing a series of fast detection speed, high specificity, and sensitivity. High and reproducible immunoassay method.

5. Other applications

Under a high gradient magnetic field, the immunomagnetic bead method is used to separate B and T lymphocytes in abdominal blood or veins. The separated lymphocytes are then widely used in the rapid selection of clinical organ transplantation donors and recipients. Perform HLA-I type II antigen typing.

Magnetic bead technology, as a product developed by the convergence of interdisciplinary subjects, plays a huge role in biological and medical laboratory testing. With the rapid development of second-generation sequencing, the consumables involved in second-generation sequencing have also ushered in its spring.

Whether it is magnetic beads used to extract nucleic acids or immunomagnetic beads used in cell sorting and immunoassays, they are still a subdivision of the industry in the field of molecular biology, and the technology is still in continuous progress and development. As more and more companies join, the prices of products developed by them will also become cheaper.

How to perform nucleic acid extraction

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The common difficulty in nucleic acid extraction lies in the difficulty of extracting high-quality RNA from some plant tissues, which is related to the rich ingredients in these plant tissues, such as phenolic compounds, polysaccharides, and some unidentified secondary metabolites. In addition, there are tissues that are rich in higher activity. In an intact cell, these substances are separated from nucleic acids. Once the cell is broken, these substances will interact with RNA.

Nucleic acid extraction method:

1. Quickly inactivate endogenous RNase to prevent RNA degradation. The following 3 methods can effectively inactivate endogenous RNase:

1) Harvest the sample with a cell lysate containing chaotropic (such as guanidine salt) and homogenize immediately.

2) Instantly freeze the sample with liquid nitrogen. It is worth noting that: the tissue pieces must be small enough to be frozen the moment they are immersed in liquid nitrogen to ensure instant inactivation of RNase.

3) Immediately place the sample in the liquid nitrogen-free RNA sample storage solution. It is an aqueous, non-toxic collection reagent that can immediately stabilize and protect RNA in intact, unfrozen tissue and cell samples. The key point is that the tissue sample slice must be thin enough (<0.5 cm) so that it can quickly penetrate into the tissue block before the RNase destroys the RNA.

2. Use the correct cell or tissue storage conditions

After the sample is frozen instantaneously with liquid nitrogen, it should be stored at -80°C and must not be thawed. Even a short thawing before homogenization in a lysate containing guanidine salt will result in degradation and loss of RNA. Instantly frozen tissues should be ground into powder under ultra-low temperature, and then placed in the lysis solution for homogenization.

Manual nucleic acid extraction kit

3. Choose a good RNA isolation method

The numerous RNA isolation methods available may be difficult to choose. The current simple and safe method is column separation. For example, it is loved by everyone because of its simple operation, short time and high purity. It does not require DNase to digest DNA, which saves time and avoids RNA degradation, thereby increasing the yield of nucleic acid extraction; Relative to non-column separation, it removes proteins and other impurities cleanly, improves purity, and is very suitable for cells, tissues, and general plants.

Plant nucleic acid extraction is more difficult to choose. Plant RNA nucleic acid extraction kits is affected by the content of phenols, polysaccharides, protein impurities, and secondary metabolites. You can choose the polysaccharide and polyphenol plant total RNA nucleic acid extraction kits (narcissus, pepper, carrot, corn, lily, wheat, Tomatoes, cauliflower, rapeseed, etc.) and general plant RNA nucleic acid extraction kits (suitable for the extraction of most plants, including: apple, grape, strawberry, banana, longan, lychee, lawn plant, pine, cedar, white birch, purple pine cone, Coleus, poinsettia, oleander, Ficus benjamina, violet, rose, geranium, morning glory, etc.); it is worth mentioning that blood (including serum, plasma, cerebrospinal fluid, other liquids) nucleic acid extraction TRIZOL and red blood cells The lysis method is not effective. The red blood cell lysate does not contain RNase inhibitors. In this process, RNA is easily degraded. It is recommended to use a blood total RNA nucleic acid extraction kits.

What are the precautions for the nucleic acid extraction system when doing separation experiments?

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The nucleic acid extraction system is a high-tech product that uses the universal magnetic bead method to extract nucleic acids. It has the advantages of high automation, fast extraction speed, stable results, and easy operation. In almost every laboratory, the separation and purification work related to biomolecules is very important.

However, it is quite difficult to purify multiple samples. It is not only necessary to select a suitable purification technology, but also the workload is particularly large, which is difficult to meet the current rapid development of high-throughput sample extraction and purification needs.

The magnetic bead method of nucleic acid extraction system is very suitable for genomics research. Regardless of whether the source of the extracted sample is microorganisms, animals, plants or viruses, combined with a whole blood genomic DNA extraction kit specially optimized for magnetic bead extraction and purification systems, a white blood cell layer whole blood genome extraction kit, and animal tissue/cell genomic DNA extraction reagents The box can quickly purify DNA or RNA in sufficient quantity and purity.

HERO96 magnetic bead method nucleic acid extraction system

 

What should we pay attention to when using a nucleic acid purifier for separation experiments?

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

2. Remove the pollution of other biological macromolecules (protein, carbohydrate, lipid molecules).

3. Remove the contamination of other nucleic acid molecules (extract DNA and remove RNA contamination).

4. Remove organic solvents and high-concentration metal ions that inhibit enzymes.

5. Simplify the operation steps and shorten the extraction process.

HERO 32 Magnetic Bead Method Nucleic Acid Extraction System

6. Prevent the degradation of nucleic acid by physical and chemical factors (shear force, high temperature, strong acid and strong base).

7. Prevent the degradation of nucleic acid by biological factors (nuclease).

The nucleic acid purification instrument has stable measurement results, avoids differences and errors caused by manual operation, and has good temperature and repeatability. The purifier can optimize the purification scheme according to the reagents and cooperate with the precise incubation time to achieve higher extraction efficiency.The extracted DNA/RNA has high purity and can be directly used for PCR and RT-PCR.

This product has a powerful program editing function; it can define your application flexibly and efficiently to meet the requirements of different reagents. The lysis and elution temperature can be customized according to the needs. Built-in engineering computer, no need to connect to a personal computer; stand-alone operation saves more space and energy, and provides a highly stable automatic control system.

Basic magnetic beads common questions and answers

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Magnetic beads is a newly developed immunological technology in recent years. It combines the unique advantages of curing reagents with the high specificity of immunological reaction. Based on immunology, it penetrates into pathology, physiology, pharmacology, microorganisms, biochemistry and molecules. Genetics and other fields, and have been more and more widely used in immunological detection, cell separation, biological macromolecule purification and molecular biology. Common types of magnetic beads include hydroxyl magnetic beads, amino magnetic beads and carboxyl magnetic beads. Different types of magnetic beads have different application problems. Next, the editor of Aisen will share with you some common questions about basic magnetic beads and the answers to these questions.

Frequently Asked Questions about Hydroxyl Magnetic Beads

1. What is the dispersion of each type of magnetic beads?

70301 Mag OH-500 has uniform particle size and monodisperse;

70302 Mag OH-1000 amorphous, non-uniform particle size, average particle size 1 micron, polydisperse;

70802 Magrose OH spherical, particle size 30-150 microns, polydisperse.

2. How much is the nucleic acid binding capacity?

The binding force is greater than 20μg DNA/mg magnetic beads.

3. Application areas of Magrose OH

The surface modification of Magrose OH magnetic beads is used in the field of protein purification.

4. Is there any instructions for using hydroxy magnetic beads for nucleic acid extraction?

Hydroxyl magnetic beads need different buffers for nucleic acid extraction according to different samples. Therefore, there is no relevant instructions, and customers need to choose the buffer by themselves. If you need a matching kit, you can choose the relevant nucleic acid extraction kit.

Frequently Asked Questions about Amino Magnetic Beads

1. 15% glutaraldehyde solution preparation method and matters needing attention

Use PBS buffer for glutaraldehyde, pay attention to the current use.

2. How to deal with biological ligands such as IgG

IgG is prepared with PBS buffer. If there are other small molecules containing carboxyl or amino groups, remove them as much as possible to avoid competing reactions with magnetic beads.

Common problems of carboxyl magnetic beads

1. What is the amount of antibody coupling?

About 2.5-3μg/mg magnetic beads.

2. After carboxyl magnetic beads are coupled to IgG, which magnetic bead storage solution is more suitable?

The preservation solution can be PBST or tris-HCl.

The above is the introduction to the common questions and answers of basic magnetic beads. Different magnetic beads will always encounter various problems in the application process. It is necessary to understand the simple application methods and techniques of various magnetic beads to ensure that the magnetic beads are normal. use. The magnetic beads produced by Aisen Biotechnology are diverse in variety and excellent in performance. Customers in need are welcome to call for consultation.