CN109486907B - Loop-mediated isothermal amplification reagent capable of being transported at normal temperature, preparation method and application - Google Patents
Loop-mediated isothermal amplification reagent capable of being transported at normal temperature, preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a loop-mediated isothermal amplification reagent capable of being transported at normal temperature, a preparation method and application thereof, wherein the reagent comprises Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent; the components are mixed, and are frozen and dried for 8-16 h at the temperature of-70 to-80 ℃ to obtain the loop-mediated isothermal amplification reagent. The loop-mediated isothermal amplification reagent for nucleic acid detection is prepared, can be transported and stored at normal temperature, is convenient to transport and has good stability; meanwhile, the problem that the promotion of the LAMP technology is limited by aerosol pollution is solved, and the application and promotion difficulty of the LAMP technology is greatly reduced. Bst DNA Polymerase activity in the reagent is 150%, the reagent can be stored for more than one year at normal temperature, only buffer solution needs to be added when the reagent is used, the operation is simple, the DNA Polymerase activity is high, the detection sensitivity is not lost, the interference of a protective agent is small, and the cost is low.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a loop-mediated isothermal amplification reagent capable of being transported at normal temperature, a preparation method and application thereof.
Background
Loop-mediated isothermal amplification (LAMP) is an in vitro amplification technique of nucleic acid invented by the Nippon Rongyan Kabushiki Kaisha, 4 specific primers are designed according to 6 regions of a target gene, and 10 specific primers can be completed within 1 hour at the temperature of 60-65 ℃ by using the strand displacement effect of Bst DNA Polymerase9～1010And (5) amplifying. The LAMP amplification process depends on 6 independent areas for identifying target sequences, so the reaction specificity is very strong, the nucleic acid amplification process is carried out under the constant temperature condition, and the reaction requirements can be met by a common water bath or equipment with a stable heat source. Compared with the common PCR technology, the LAMP technology has the advantages of simple operation, rapid detection, high specificity, low cost and the like.
At present, LAMP has been successfully applied to the detection of Mycobacterium tuberculosis, and is accepted by WHO and widely popularized. However, Bst DNA Polymerase, dNTP, primers and other components required for LAMP reaction need to be transported and stored under low temperature conditions, and the products need to be operated by professional technicians according to the instructions. Meanwhile, aerosol pollution of the LAMP technology easily causes false positive results, and application and popularization difficulty of the technology is further increased.
The freeze drying technology is also called sublimation drying, and is a drying method for directly removing water from a water-containing material under the conditions of below freezing point and higher vacuum. Can avoid the biological, chemical or physical change of the heat-sensitive substances and maintain the activity, physical and chemical properties of the raw materials to the maximum extent. The substance is then detected. At present, disaccharide such as trehalose, sucrose, lactose and the like and biomacromolecules are mostly combined for a detection reagent after freeze drying of bioactive raw materials. However, when the product is used in LAMP technology, the concentration is high, the non-reducing disaccharide needs to be more than 200mM to effectively play a role, and the subsequent reaction can be inhibited, so that the downstream detection of the non-reducing disaccharide is often influenced to a certain extent; and aerosol pollution can be caused, and the detection result is influenced.
Disclosure of Invention
Aiming at the limitations in the prior art, the invention aims to provide a loop-mediated isothermal amplification reagent capable of being transported at normal temperature, a preparation method and application thereof, and solves the problems that the existing reagent is high in use concentration, easily causes aerogel pollution and affects downstream detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a loop-mediated isothermal amplification reagent capable of being transported at normal temperature comprises Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventing agent;
the primer mixture comprises primers FIP, BIP, LF, LB, F3 and B3;
the indicator comprises HNB solution and Gelgreen solution;
the protective agent comprises bovine serum albumin, soybean lecithin, mannitol and hydroxyethyl starch;
the aerosol pollution preventing agent comprises glycyrrhizin, sodium alginate and tert-butyl alcohol.
Specifically, Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent are mixed according to the volume ratio of 1-2: 3-7: 1-2: 5-8: 3-7.
Specifically, the FIP, the LF and the F3 are mixed according to the mass concentration ratio of 4-8: 2-4: 1-2, the BIP concentration of the primer is the same as that of the FIP, the LB concentration of the primer is the same as that of the LF, and the F3 concentration of the primer is the same as that of the B3.
Specifically, the concentration of the HNB solution is 5-10 mM, and the HNB solution, the Gelgreen solution and deionized water are mixed according to the volume ratio of 5-10: 1-2.
Specifically, bovine serum albumin, soybean lecithin, mannitol and hydroxyethyl starch are mixed according to the mass ratio of 50-150: 1-40: 10-50: 5-25.
Specifically, glycyrrhizin, sodium alginate and tert-butyl alcohol are mixed according to the mass ratio of 1-10: 1-20: 1-50.
The invention also discloses a preparation method of the loop-mediated isothermal amplification reagent capable of being transported at normal temperature, which comprises the following steps: bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent are mixed according to the proportion, and the mixture is frozen and dried for 8 to 16 hours at the temperature of between 70 ℃ below zero and 80 ℃ below zero to obtain the loop-mediated isothermal amplification reagent.
The invention also discloses the application of the loop-mediated isothermal amplification reagent capable of being transported at normal temperature in nucleic acid detection.
Specifically, a loop-mediated isothermal amplification reagent and a buffer are mixed, a template to be detected is added, then the mixture is placed in a constant temperature condition of 63-67 ℃ for reaction for 30-60 min, and then the temperature is raised to 83-87 ℃ for reaction for 5 min;
wherein the buffer solution is selected from betaine, KCl, Tris-HCl, (NH)4)2SO4、MgSO4The Tris-HCl-based water-based oil-based water-based paint is prepared by mixing 50-100: 2-5: 1-2: 0.8-1 by mass, and the pH value of Tris-HCl is 6.8-8.8.
Compared with the prior art, the invention has the beneficial effects that:
(1) the Bst DNA Polymerase, dNTP, the primer, the indicator and the protective agent which need to be stored and transported at low temperature are mixed, and the loop-mediated isothermal amplification reagent for nucleic acid detection is prepared under the protection action of the protective agent, can be transported and stored at normal temperature, and is convenient to transport and good in stability; meanwhile, the problem of aerosol pollution which is the biggest trouble in the process of limiting the popularization of the LAMP technology is solved, and the application and popularization difficulty of the LAMP technology is greatly reduced.
(2) The Bst DNA Polymerase activity in the reagent is 150%, the reagent can be stored at normal temperature for more than one year, only buffer solution needs to be added when the reagent is used, the operation is simple, the DNA Polymerase activity is high, the detection sensitivity is not lost, the interference of a protective agent is small, and the cost is low.
(3) The protective agent of the invention takes BSA, soybean lecithin, mannitol and hydroxyethyl starch as main components, can effectively ensure the activity of Bst DNA Polymerase under the condition of lower concentration, and avoids the influence on the detection result caused by adopting polymer with higher concentration, non-reducing disaccharide and the like.
(4) The HNB and the GelGreen are mixed for use, so that the color change of the GelGreen is easier to distinguish by utilizing the color reaction of the HNB, the qualitative detection capability of the HNB is integrated with the quantitative detection system of the GelGreen, and the detection mode can be selected automatically according to different requirements.
(5) The invention uses glycyrrhizin as excipient and aerosol pollution preventer, and simultaneously, glycyrrhizin and hydroxyethyl starch play a role in shaping in the freeze drying process, thus avoiding the use of non-reducing disaccharide and polymer; meanwhile, the tert-butyl alcohol in the aerosol pollution preventing agent increases the solubility of glycyrrhizin and improves the freeze-drying efficiency.
Drawings
FIG. 1 is the effect of lyophilization of the reagent prepared in example 1.
Fig. 2 is a graph showing the effect of lyophilization of the reagent prepared in comparative example 1.
FIG. 3 is a graph of the cold drying effect of reagents prepared for protectant components from groups A through I, respectively, of Table 1.
FIG. 4 is a graph showing the effect of various lyoprotectants on Bst DNA Polymerase activity.
FIG. 5 is a graph showing the color development results of different indicators, wherein (A) is the color development result of the reagent of comparative example 2 under a blue light lamp, (B) is the color development result of the reagent of example 1 under a blue light lamp, and (C) is the color development result of the reagent of example 1 under a white light lamp.
FIG. 6 is a graph showing the effect of different concentrations of t-butanol on the freeze-drying time and the time of the amplification peak.
FIG. 7 is a graph of the effect of different concentrations of glycyrrhizin on the rate of reconstitution and the time to peak amplification.
The invention is described in detail below with reference to the drawings and the detailed description.
Detailed Description
Aiming at the problems existing in the LAMP technology at present, the loop-mediated isothermal amplification reagent capable of being transported at normal temperature disclosed by the invention comprises Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent. Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution prevention agent are mixed according to the volume ratio of 1-2: 3-7: 1-2: 5-8: 3-7.
Wherein the primer mixture comprises FIP, BIP, LF, LB, F3 and B3; FIP, LF and F3 are mixed according to the mass concentration ratio of 4-8: 2-4: 1-2, the concentration of BIP is the same as that of FIP, the concentration of LB is the same as that of LF, and the concentration of F3 is the same as that of B3. The concentration of the primers FIP, LF, F3, LB, F3 and B3 is 100-200 mu M, and six primers are prepared into mixed liquor according to different proportions when the primer is used. The primers of the present invention can be used to detect different bacteria and viruses by replacing different primer sequences as needed.
The indicator comprises HNB solution and GelGreen solution, wherein the concentration of the HNB solution is 5-10 mM (mmol/L), and 100 multiplied by GelGreen to 1000 multiplied by GelGreen can be sold in the market. The indicator is formed by mixing HNB solution, GelGreen solution and deionized water according to the volume ratio of 5-10: 1-2. The preparation process of the indicator comprises the following steps: respectively dissolving HNB and GelGreen in deionized water to obtain HNB solution and GelGreen solution, and then mixing the HNB solution and GelGreen solution. In the following embodiments of the present invention, the Gelgreen solution used is 10000 × Gelgreen solution commercially available, the specific concentration is adjusted according to actual needs, and the Gelgreen solution is diluted to 100-1000 × Gelgreen in the present invention.
The protective agent comprises bovine serum albumin, soybean lecithin, mannitol and hydroxyethyl starch; bovine serum albumin, soybean lecithin, mannitol and hydroxyethyl starch are mixed according to the mass ratio of 50-150: 1-40: 10-50: 5-25. The preparation process of the protective agent comprises the following steps: bovine serum albumin, soybean lecithin, mannitol and hydroxyethyl starch are mixed according to a required proportion and then added into deionized water to obtain the soybean milk. Bovine serum albumin is added into the protective agent to improve the protein concentration of a freeze-drying system, hydroxyethyl starch reduces surface tension, the possibility that Bst DNA Polymerase is inactivated due to low protein concentration and large surface tension is effectively reduced, and the Bst DNA Polymerase is well protected.
The aerosol pollution preventing agent comprises glycyrrhizin, sodium alginate and tert-butyl alcohol, wherein the glycyrrhizin, the sodium alginate and the tert-butyl alcohol are mixed according to the mass ratio of 1-10: 1-20: 1-50. The preparation process of the aerosol pollution preventing agent comprises the following steps: mixing glycyrrhizin, sodium alginate and tert-butyl alcohol according to a required proportion, adding deionized water, and storing in a water bath at 50-85 ℃. The glycyrrhizin is used as an excipient and an aerosol pollution preventer, and simultaneously, the glycyrrhizin and hydroxyethyl starch play an excipient role together in the freeze drying process; moreover, the tertiary butanol increases the solubility of glycyrrhizin and improves the efficiency of freeze-drying.
The preparation method of the reagent provided by the invention specifically comprises the following steps: bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent are mixed according to the proportion, and the mixture is frozen and dried for 8 to 16 hours at the temperature of between 70 ℃ below zero and 80 ℃ below zero to obtain the loop-mediated isothermal amplification reagent.
The reagent of the invention can be transported under normal temperature condition and can be used for detecting nucleic acid. The general specific detection process is as follows: mixing the loop-mediated isothermal amplification reagent and a buffer solution, adding 1-2 mul of a template to be detected (namely nucleic acid), then placing the template to be detected at the constant temperature of 63-67 ℃ for reaction for 30-60 min, and then heating to 83-87 ℃ for reaction for 5 min; and finally, placing the reaction tube under a blue light lamp for observation, wherein the bright green is positive, and the brick red is negative. Wherein the buffer solution is selected from betaine, KCl, Tris-HCl, (NH)4)2SO4、MgSO4According to the mass ratio of 50-100: 2-5: 1-2: 0.8-1, and the pH value of Tris-HCl is 6.8-8.8;
or placing the reaction system on a real-time fluorescent quantitative PCR instrument, and setting the amplification conditions as follows: amplification at 65 ℃ for 45 sec; fluorescence was collected at 65 ℃ for 15 sec; the reaction is stopped at 85 ℃ for 5min after 60 cycles, amplification curves are positive, amplification curves are negative, and the samples can be quantitatively analyzed through the amplification curves.
The following embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention are within the protection scope of the present invention.
Example 1
Firstly, preparing indicator mixed liquor, and dissolving 62mg of HNB in 10ml of deionized water to obtain 10mM HNB solution; 100. mu.l of 10000 XGelGreen was added to 900. mu.l of deionized water to obtain 1000 XGelGreen solution. Then, 375. mu.l of 10mM HNB, 35. mu.l of 1000 XGelgreen and 590. mu.l of water were taken to prepare a 25 Xindicator mixture.
Preparing a protectant mixture, mixing 75 μ l of 2% bovine serum albumin, 75 μ l of 0.05% soybean lecithin, 50 μ l of 100mM mannitol and 50 μ l of 0.25% hydroxyethyl starch, and adding deionized water to 500 μ l to obtain a 25 × protectant mixture.
Preparing aerosol pollution preventing agent, taking 100 μ l of 0.25% glycyrrhizin, 100 μ l of 0.5% sodium alginate and 100 μ l of 2.5% tert-butyl alcohol, then supplementing 1ml with 700 μ l of deionized water, and storing in 50-85 deg.C water bath.
Then, 0.6. mu.l of water, 1.4. mu.l of 10mM dNTP, 0.4. mu.l of a primer mixture, 0.4. mu.l of an indicator mixture, 1. mu.l of a protectant mixture, 1. mu.l of an aerosol contamination preventive, 0.4. mu.l of Bst DNA Polymerase and 2. mu.l of deionized water were sequentially added, mixed uniformly, and stored in an ice bath.
Wherein, the primer sequence used is as follows:
F3：TGGCATTCTGATCCACGATT B3：CGGGAACTCAAAGGAGACTG FIP：CGAGAGCAAGCGGACCTCATAACATGGAGTCGAGTTGCAGAC BIP：TATGCGCCATTGTAGCACGTGTAGGAAGGTGGGGATGACG LF：TGCGTCGTAGTCCGGATTGGA LB：CTGGTCGTAAGGGCCATGATGAC
and (3) subpackaging the prefabricated freeze-drying system into eight tubes, each tube is 5 mu l, quickly placing the tubes into a PCR plate, pre-cooling the tubes for 2h in a refrigerator at the temperature of-80 ℃, and then carrying out freeze-drying for 8h to obtain the loop-mediated isothermal amplification reagent.
FIG. 1 shows that the loop-mediated isothermal amplification reagent obtained in this example shows that the materials are completely distributed at the bottom of the tube, the product is clean and tidy, the consistency is good, and water molecules are thoroughly removed.
The application comprises the following steps: the loop-mediated isothermal amplification reagent prepared in this example was mixed with a buffer containing 0.8M betaine, 0.05M KCl, 20mM Tris-HCl, 10mM (NH)4)2SO4、8mM MgSO4. Adding 8 mul of the buffer solution and 2 mul of the Escherichia coli DNA template into a freeze-drying tube, fully mixing uniformly, placing in a real-time fluorescence quantitative PCR instrument, amplifying for 60min at 65 ℃, simultaneously collecting fluorescence signals, and stopping reaction at 85 ℃ for 5 min. Or reacting the reaction system at the constant temperature of 65 ℃ for 60min, terminating the reaction at the temperature of 85 ℃ for 5min, and performing qualitative analysis under a blue light lamp.
The invention was also tested for the performance of the different components of the protective agent according to example 1, as shown in table 1 below,
TABLE 1 protectant composition of the different components
| Grouping | Bovine serum albumin | Soybean lecithin | Mannitol | Hydroxyethyl starch | Deionized |
| A | 50μl | ||||
| 2％ | 50μl 0.05％ | 10μl 100mM | 50μl 0.25％ | 340μl | |
| B | 50μl 2％ | 75μl 0.05％ | 25μl 100mM | 100μl 0.25％ | 250μl |
| C | 50μl 2％ | 150μl 0.05％ | 50μl 100mM | 200μl 0.25％ | 50μl |
| D | 75μl 2％ | 50μl 0.05％ | 25μl 100mM | 200μl 0.25％ | 150μl |
| E | 75μl 2％ | 75μl 0.05％ | 50μl 100mM | 50μl 0.25％ | 250μl |
| F | 75μl 2％ | 150μl 0.05％ | 10μl 100mM | 100μl 0.25％ | 165μl |
| G | 150μl 2％ | 50μl 0.05％ | 50μl 100mM | 100μl 0.25％ | 150μl |
| H | 150μl 2％ | 75μl 0.05％ | 10μl 100mM | 200μl 0.25％ | 65μl |
| I | 150μl 2％ | 150μl 0.05％ | 25μl 100mM | 50μl 0.25％ | 125μl |
Group E in table 1 is the composition of the protectant in example 1. Fig. 3 shows the reagents prepared in groups a-I, with the reagents of group A, C, E, G, I remaining substantially at the bottom of the tube, and the remaining lyophilized groups all "splashed" to a slight degree, but with more complete water removal. The remaining lyophilised groups all "splashed" to a slight extent, but the water was removed more thoroughly than in comparative example 1.
The invention also carries out real-time fluorescence quantitative amplification experiments on the reagents of the groups A to I, and Bst DNA Polymerase stored at the temperature of minus 20 ℃ is used as a control group (CK group) to compare the activity of the Bst DNA Polymerase in the reagents, and the results are shown in figure 4, wherein the Bst DNA Polymerase activity of the groups E and A is obviously higher than 100 percent (J group), and the activity of the group E is higher than that of the group A; the effect of the rest of the freeze-drying protective agents was less than 100%, and no amplification occurred in group H.
Example 2
This example differs from example 1 in that: the concentration of tert-butanol was 0.1% and the amount added was 100. mu.l.
The loop-mediated isothermal amplification reagent prepared in this example was prepared as a buffer, and then mixed with an E.coli DNA template, and its lyophilization efficiency was measured, as shown in FIG. 6.
Example 3
This example differs from example 1 in that: the concentration of tert-butanol was 5% and the amount added was 100. mu.l.
The loop-mediated isothermal amplification reagent prepared in this example was prepared as a buffer, and then mixed with an E.coli DNA template, and its lyophilization efficiency was measured, as shown in FIG. 6.
According to example 1, the present invention also compares the acceleration effect of tert-butanol of different concentrations during the freeze-drying process, and verifies the freeze-drying efficiency at different concentrations by adding 100. mu.l of tert-butanol of 0.5%, 1%, 1.5%, 2%, 3%, 5% respectively. FIG. 6 shows the freeze-drying efficiency of t-butanol in example 1 and the concentrations described above.
It can be seen from the above that when the concentration of the tert-butyl alcohol reaches 2.5% (the concentration of example 1), the freeze-drying efficiency is significantly improved, and the freeze-drying efficiency is basically stable and close to the limit value by continuously increasing the concentration of the tert-butyl alcohol to 5%. Meanwhile, whether the LAMP amplification reaction is inhibited or not by different concentrations of the tert-butanol is verified, and the result is shown in FIG. 6, the Bst DNA Polymerase activity in the reagent with the tert-butanol content of less than 3% after freeze-drying is greater than that stored at-20 ℃, which may be that the structure of the Bst DNA Polymerase is changed to a certain extent during the freeze-drying process. However, when the concentration of t-butanol was higher than 4%, although the freeze-drying rate was increased, the activity of Bst DNA Polymerase was significantly inhibited.
Example 4
This example differs from example 1 in that: the glycyrrhizin concentration was 0.1%.
The loop-mediated isothermal amplification reagent prepared in this example was prepared as a buffer, and then mixed with an E.coli DNA template, and its lyophilization efficiency was measured, as shown in FIG. 7.
Example 5
This example differs from example 1 in that: in a 50-microliter reaction system, the concentration of glycyrrhizin is 1%; the reaction system is the whole reaction system after the reagent is mixed with the buffer solution and the Escherichia coli DNA template.
The loop-mediated isothermal amplification reagent prepared in this example was prepared as a buffer, and then mixed with an E.coli DNA template, and its lyophilization efficiency was measured, as shown in FIG. 7.
In example 1, the concentration of glycyrrhizin in 50. mu.l of the reaction system was 2.5%. According to example 1, the present inventors also compared the dissolution rates of glycyrrhizin at concentrations of 0.5% and 0.75% at 65 ℃ in a 50. mu.l reaction system, respectively, and as a result, as shown in FIG. 7, it can be seen that the dissolution time of glycyrrhizin gel is significantly prolonged when the concentration of glycyrrhizin is more than 0.25%. Meanwhile, when the glycyrrhizin concentration is detected to be higher than 0.25%, the amplification peak time is obviously delayed, and the repeatability is poor, because a reaction system is not rapidly homogenized in 3-4 min before reaction, the reaction degree is not uniform, and the detection repeatability is further influenced.
Comparative example 1
This comparative example differs from example 1 in that: no protective agent is added.
As shown in FIG. 2, the reagent without protective agent has severe "splash" phenomenon on the tube wall, and the freeze-drying effect is poor, because disintegration occurs during the freeze-drying process.
Comparative example 2
This comparative example differs from example 1 in that: the indicator mixture of this comparative example used a Gelgreen solution alone.
As a result, as shown in fig. 5(a), Gelgreen alone can be used for quantitative analysis, but the degree of visual result discrimination under a blue light lamp is not high. While the HNB alone cannot realize quantitative detection.
Fig. 5(B) and 5(C) show color development results of reagents prepared by combining HNB and Gelgreen under blue light and white light, respectively, and it can be seen that combining HNB and Gelgreen increases the color difference of Gelgreen under blue light on the one hand without affecting the detection result, and on the other hand effectively combines the quantitative detection of HNB with the qualitative detection capability of Gelgreen, which is convenient for the detection personnel to select according to the experimental conditions.
Claims (4)
1. A loop-mediated isothermal amplification reagent capable of being transported at normal temperature is characterized by comprising Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent;
mixing Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent according to the volume ratio of 1-2: 3-7: 1-2: 5-8: 3-7;
the primer mixture comprises primers FIP, BIP, LF, LB, F3 and B3;
the indicator comprises HNB solution and Gelgreen solution; the concentration of the HNB solution is 5-10 mM, and the HNB solution, the Gelgreen solution and deionized water are mixed according to the volume ratio of 5-10: 1-2;
the protective agent consists of 50 mul 2% bovine serum albumin, 50 mul 0.05% soybean lecithin, 10 mul 100mM mannitol, 50 mul 0.25% hydroxyethyl starch and 340 mul deionized water or consists of 75 mul 2% bovine serum albumin, 75 mul 0.05% soybean lecithin, 50 mul 100mM mannitol, 50 mul 0.25% hydroxyethyl starch and 250 mul deionized water;
the aerosol pollution preventing agent consists of glycyrrhizin, sodium alginate and tert-butyl alcohol; the glycyrrhizin, the sodium alginate and the tert-butyl alcohol are mixed according to the mass ratio of 1-10: 1-20: 1-50.
2. The loop-mediated isothermal amplification reagent capable of being transported at normal temperature according to claim 1, wherein the primers FIP, LF and F3 are mixed according to the mass concentration ratio of 4-8: 2-4: 1-2, the concentration of the primer BIP is the same as FIP, the concentration of the primer LB is the same as LF, and the concentration of the primer F3 is the same as B3.
3. The normal-temperature transportable loop-mediated isothermal amplification reagent according to claim 1, wherein the loop-mediated isothermal amplification reagent is used by a method comprising: mixing a loop-mediated isothermal amplification reagent and a buffer solution, adding a template to be detected, then placing the template in a constant temperature condition of 63-67 ℃ for reaction for 30-60 min, and then heating to 83-87 ℃ for reaction for 5 min;
wherein the buffer solution is selected from betaine, KCl, Tris-HCl, (NH)4)2SO4、MgSO4The Tris-HCl-based water-based oil-based water-based paint is prepared by mixing 50-100: 2-5: 1-2: 0.8-1 by mass, and the pH value of Tris-HCl is 6.8-8.8.
4. The method for preparing a normal-temperature transportable loop-mediated isothermal amplification reagent according to any one of claims 1 to 3, wherein the Bst DNA Polymerase, dNTP, a primer mixture, an indicator, a protective agent and an aerosol pollution preventive agent are mixed, and freeze-dried at-70 to-80 ℃ for 8 to 16 hours to obtain the loop-mediated isothermal amplification reagent.
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| CN112481359B (en) * | 2020-11-30 | 2021-09-17 | 河南智泰生物科技有限公司 | Freeze-dried microsphere of LAMP isothermal amplification reagent and preparation method and application thereof |
| CN112442555B (en) * | 2020-12-09 | 2023-05-26 | 陕西师范大学 | Visual LAMP detection system for preventing aerosol pollution and preparation method, using method and application thereof |
| CN112342318B (en) * | 2020-12-09 | 2023-05-30 | 陕西师范大学 | Primer pair, reaction freeze-drying tube and kit for detecting novel coronavirus SARS-CoV2 |
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