Hot-start visual loop-mediated isothermal amplification reaction kit and preparation method thereof

Abstract

The invention relates to the field, in particular to a preparation method of a visual loop-mediated isothermal amplification reaction kit capable of being hot-started, which comprises the following steps: preparing primer freeze-dried microspheres; treating the reaction tube: coating the inner wall of the reaction tube with an ethanol solution of 2% gamma-glycidyl ether oxypropyl trimethoxy silane, and curing at 90 DEG C for 30 minutes; respectively sealing the base solution, the color developing agent and the DNA large fragment polymerase by using a sealing solution, adding the primer freeze-dried microspheres, and closing a reaction tube cover; compared with the prior art, the inner wall of the reaction tube is treated to improve the interface bonding force between the sealing layer and the reaction wall, so that the failure of the kit caused by the displacement of the paraffin layer in the transportation and storage process of the kit is reduced; the primer is subjected to freeze-drying treatment, and the color developing agent, the DNA large fragment polymerase and the basic reaction liquid are sealed by the sealing liquid, so that the work of operators is simplified, and the reaction environment is prevented from being polluted by aerosol generated by reaction in the detection process.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a hot-start visual loop-mediated isothermal amplification reaction kit and its preparation method. Background Technology

[0002] Isothermal amplification is a type of in vitro nucleic acid amplification technique. Its advantage lies in eliminating the need for temperature variations required for denaturation, annealing, and extension steps, requiring only a constant heat source to initiate the reaction. However, traditional isothermal amplification methods still heavily rely on manual operation by specialized technicians. These technicians must add basic reactants, chromogenic agents, and key enzyme reagents during the detection process. This results in a heavy workload for the technicians, and the amplification product aerosols released during the detection process can contaminate the testing environment, leading to false positives in subsequent tests.

[0003] Patent No. ZL202111084753.1 discloses a method for preserving RNA-based nucleic acid detection reagents, a kit, and a method for using them. Specifically, it discloses the use of paraffin sheets with different melting points to isolate a first mixture, a second mixture, and a third mixture. The first mixture consists of reverse transcriptase, 50% glycerol, and an RNA inhibitor; the second mixture consists of downstream primers, a detection probe, and an amplification reaction solution; and the third mixture consists of Taq DNA polymerase, an upstream primer, and a reaction solution. Separating the different components of the reagent with paraffin allows for stable long-term preservation at room temperature and ensures that the detection effect does not decrease. However, this method is a PCR reaction, and the reverse transcription, pre-denaturation, and PCR reaction conditions are relatively harsh. Furthermore, after the first and second mixtures react, the mixture needs to be heated to fuse the products of the first and second mixtures with the third mixture for a second round of reaction.

[0004] To solve the above-mentioned technical problems, the present invention provides a hot-start visible ring-mediated isothermal amplification reaction tube that requires only one reaction and produces good results. Summary of the Invention

[0005] To achieve the above technical solution, the present invention provides a hot-start, visually-guided loop-mediated isothermal amplification reaction kit and its preparation method, the preparation method comprising: S1, Preparation of primer lyophilized microspheres; S2, Reaction tube treatment: Coat the inner wall of the reaction tube with an ethanol solution of 2-5% γ-glycidyl etheroxypropyltrimethoxysilane and cure at 90-100℃ for 30 minutes. S3, add the base solution to the reaction tube of step S2, add blocking solution to form the first blocking layer, add colorimetric reagent and blocking solution to the first blocking layer, solidify the blocking solution to form the second blocking layer, add DNA large fragment polymerase and blocking solution to the second blocking layer to form the third blocking layer, add primer lyophilized microspheres to the third blocking layer, and close the reaction tube cap.

[0006] According to the above technical solution, the hot-start visible loop-mediated isothermal amplification reaction kit prepared by this invention uses lyophilized microspheres containing a 55℃ solid paraffin blocking solution, chromogenic agent, DNA fragment polymerase, and primers. The effects of this hot-start reaction are as follows: First, during sample addition, the pipette tip does not contact the reaction solution. Residual liquid on the pipette tip undergoes amplification at room temperature, forming aerosols that contaminate the reaction environment, leading to false positives in subsequent tests. Second, after adding the sample, heating to 65℃ causes the lyophilized microspheres to release primers, dissolving the blocking layers, allowing the sample, primers, reaction solution, and DNA fragment polymerase to react. Third, throughout and after the reaction, the paraffin layer and tube cap completely seal the reaction solution inside the tube, preventing aerosol contamination of the reaction environment. Fourth, it avoids using high primer concentrations and large numbers of primers. If the solution is prepared and the enzyme is added at room temperature, the primers themselves are prone to forming dimers and hairpin structures, causing non-specific amplification under the action of the enzyme.

[0007] Preferably, in the above technical solution, the step of preparing primer lyophilized microspheres in step S1 is as follows: S11, synthesis, purification, and obtaining specific primers; S12, melt tert-butanol and acetonitrile, add the specific primer and primer protection solution to the melted tert-butanol and acetonitrile to prepare a dispersed phase, slowly drop the dispersed phase into the continuous phase, homogenize at 8000-8500 rpm for 2-5 minutes, and obtain a uniform droplet by high pressure homogenizer; S13, the droplet is pre-frozen at -80~-75℃ for 2-3 hours, then freeze-dried at -40~-35℃ for 5-6 hours, and then dried again at 20-25℃ to form solid microspheres.

[0008] Preferably, in the above technical solution, the continuous phase comprises 80-85% palm oil, 10-15% sorbitan monooleate and 5-10% cholesterol.

[0009] Preferably, in the above technical solution, in step S12, the volume ratio of the dispersed phase to the continuous phase is 1:7-9.

[0010] Preferably, in the above technical solution, in step S12, the primer protection solution includes trehalose, mannitol, polyethylene glycol and L-histidine.

[0011] Preferably, in the above technical solution, the primer protection solution comprises, by mass percentage, 65-78% trehalose, 15-25% mannitol, 3-8% polyethylene glycol and 0.5-2% L-histidine.

[0012] Preferably, in the above technical solution, the volume ratio of tert-butanol to acetonitrile is 5-7:3.

[0013] Preferably, in the above technical solution, the sealing liquid is solid paraffin with a melting point of 55°C.

[0014] Preferably, in the above technical solution, the DNA large fragment polymerase is Bst DNAase.

[0015] A visible loop-mediated isothermal amplification reaction kit prepared by the method described above, the kit being used to detect Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) It is a hot-start reaction, meaning that the reaction can only occur after the temperature is reached and the paraffin melts and the reagents are mixed. Secondly, the sample tip does not come into contact with the reaction solution, thus avoiding environmental pollution caused by residual reaction solution on the tip.

[0017] (2) After adding the sample, heat to 65°C to release the primers from the freeze-dried microspheres and dissolve each sealing layer. The sample, primers, reaction solution and DNA fragment polymerase react. Third, throughout the reaction and after the reaction, the paraffin layer and the tube cap are combined to completely seal the reaction solution in the tube to avoid aerosol contamination of the reaction environment.

[0018] (3) Improve the interfacial bonding force between the solid paraffin layer and the reaction tube, and reduce the failure of the kit due to the displacement of the paraffin layer during transportation and storage. Attached Figure Description

[0019] Figure 1 This is a diagram of one embodiment of the present invention; Figure 2 This is a diagram of the gel electrophoresis of the PCR products of this invention; Figure 3 This is a graph of the fluorescent PCR amplification curve of the present invention; Figure 4 This is a diagram of the LAMP product gel electrophoresis of the present invention; Figure 5 This is a diagram of the hot-start visual LAMP of the paraffin segmented blocking reagent of the present invention; Figure 6 This is a diagram illustrating the detection of common intestinal pathogens using paraffin-based segmented blocking reagents in a hot-start visual LAMP assay, as described in this invention. Detailed Implementation

[0020] The technical solutions described in the embodiments of this invention are clear and complete. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Example 1

[0021] A hot-start, visually-guided loop-mediated isothermal amplification reaction method, the reaction method comprising: S1, Preparation of primer lyophilized microspheres: Synthesize and purify specific primers, melt tert-butanol and acetonitrile at 50°C, add the specific primers, trehalose, mannitol, polyethylene glycol and L-histidine to the melted tert-butanol and acetonitrile to prepare a dispersed phase, slowly drop the dispersed phase into the continuous phase (volume ratio 1:9), homogenize at 8000 rpm for 2 minutes, obtain a uniform droplet by high pressure homogenizer, pre-freeze the droplet at -78°C for 2 hours, freeze-dry at -38°C for 6 hours, and then freeze-dry at 22°C for a second time to form solid microspheres, wherein the continuous phase is made of 85% palm oil, 10% sorbitan monooleate and 5% cholesterol; S2, Reaction tube treatment: Coat the inner wall of the reaction tube with an ethanol solution of 2% γ-glycidyl etheroxypropyltrimethoxysilane and cure at 90°C for 30 minutes. S3, add the basic reaction solution to the reaction tube of step S2, add heated and liquefied paraffin to the reaction tube, and the paraffin solidifies to form a first sealing layer; add the color developer and liquefied paraffin to the reaction tube, and the paraffin layer solidifies to form a second sealing layer; add the DNA large fragment polymerase and the paraffin layer to the reaction tube, and the paraffin layer solidifies to form a third sealing layer; place the solid microspheres on the upper layer of the reaction tube; wherein, the melting point of the solid paraffin is 55°C.

[0022] The above-mentioned paraffin-segmented blocking reagent hot-start visual loop-mediated isothermal amplification (LAMP) reaction tube consists of 4 layers: the bottom layer is the basic reaction solution layer, the middle layer is the colorimetric reagent layer blocked by solid paraffin, the top layer is the DNA large fragment polymerase layer blocked by solid paraffin, and the primer lyophilized microspheres are placed on the top layer. The melting point of solid paraffin is 55°C. Example 2

[0023] A hot-start, visually-guided loop-mediated isothermal amplification reaction method, the reaction method comprising: S1, Preparation of primer lyophilized microspheres: Synthesize and purify specific primers, melt tert-butanol and acetonitrile at 50°C, add the specific primers, trehalose, mannitol, polyethylene glycol and L-histidine to the melted tert-butanol and acetonitrile to prepare a dispersed phase, slowly drop the dispersed phase into the continuous phase (volume ratio 1:9), homogenize at 8000 rpm for 2 minutes, obtain a uniform droplet by high pressure homogenization, pre-freeze the droplet at -77°C for 2 hours, freeze-dry at -37°C for 6 hours, and then freeze-dry at 23°C for a second time to form solid microspheres, wherein the continuous phase is made of 85% palm oil, 10% sorbitan monooleate and 5% cholesterol.

[0024] S2, Reaction tube treatment: Coat the inner wall of the reaction tube with an ethanol solution of 5% γ-glycidyl etheroxypropyltrimethoxysilane and cure at 100°C for 30 minutes. S3, add the basic reaction solution to the reaction tube of step S2, add heated and liquefied paraffin to the reaction tube, and the paraffin solidifies to form a first sealing layer; add the color developer and liquefied paraffin to the reaction tube, and the paraffin layer solidifies to form a second sealing layer; add the DNA large fragment polymerase and the paraffin layer to the reaction tube, and the paraffin layer solidifies to form a third sealing layer; place the solid microspheres on the upper layer of the reaction tube; wherein, the melting point of the solid paraffin is 55°C.

[0025] The above-mentioned paraffin-segmented blocking reagent hot-start visual loop-mediated isothermal amplification (LAMP) reaction tube consists of 4 layers: the bottom layer is the basic reaction solution layer, the middle layer is the colorimetric reagent layer blocked by solid paraffin, the top layer is the DNA large fragment polymerase layer blocked by solid paraffin, and the primer lyophilized microspheres are placed on the top layer. The melting point of solid paraffin is 55°C.

[0026] Pre-set experiment 1: Sensitivity test of PCR, quantitative real-time PCR, conventional LAMP, and hot-start visual LAMP with paraffin segmented blocking reagent. Salmonella was used as a sample, and PCR, quantitative real-time PCR, conventional LAMP, and paraffin segmental blocking experiments were performed respectively. The PCR product gel electrophoresis results are as follows: Figure 2 As shown, the fluorescence PCR amplification curve is as follows: Figure 3 As shown, the LAMP product gel electrophoresis results are as follows: Figure 4 As shown, the paraffin-fragmented blocking reagent hot-start visual LAMP results are as follows: Figure 5 As shown.

[0027] Figure 2 The electrophoresis results of the PCR amplification product of Salmonella invA gene are shown. The product length is consistent with the target gene of 331 bp. The amount of PCR amplification product decreases as the amount of template decreases. The limit of detection is 60 cfu / reaction. Figure 3The amplification curve of Salmonella Ttr gene by real-time quantitative PCR is shown, with a detection limit of 6 CFU / reaction. Figure 4 and Figure 5 The results show that both traditional LAMP and visual LAMP can effectively amplify the Salmonella invA gene. Figure 4 The results showed that the agarose gel electrophoresis bands of the LAMP amplification products were arranged in a typical ladder pattern, with a detection limit of 6 CFU / reaction. Figure 5 The results showed that in the paraffin-blocked segmented blocking reagent hot-start visual LAMP, the negative tube remained blue-purple without change, while the positive tube turned cyan-blue. The limit of detection was 6 CFU / reaction, proving that the indicator hydroxynaphthol blue had no adverse effect on the LAMP reaction. In summary, these results indicate that the paraffin-blocked segmented blocking reagent hot-start visual LAMP method has comparable sensitivity to quantitative real-time PCR and traditional LAMP methods, all at 6 CFU / reaction, which is higher than the sensitivity of PCR (60 CFU / reaction). The results of the paraffin-blocked segmented blocking reagent hot-start visual LAMP method are easily and clearly determined visually, demonstrating significant technical advantages.

[0028] Pre-set experiment 2: Experiment on the preparation method of solid powder Preparation Method 1: Aqueous phase is prepared by mixing primers, trehalose, mannitol, polyethylene glycol, L-histidine, and purified water. Oil phase is prepared by mixing palm oil and 5% sodium lauryl sulfate. The aqueous phase is slowly added to the oil phase, and emulsified in an ice bath using a high-speed homogenizer (10,000 rpm) for 2 minutes. The emulsion is then dropped into liquid nitrogen (-196℃) and flash-frozen (30 seconds). Preparation Method 2: Triglycerides are melted at 50°C. The specific primer, trehalose, mannitol, polyethylene glycol, and L-histidine are added to the melted triglycerides to prepare a dispersed phase. The dispersed phase is slowly added dropwise to the continuous phase (volume ratio 1:9), homogenized at 8000 rpm for 2 minutes, and a uniform droplet is obtained by high-pressure homogenization. The droplet is pre-frozen at -80°C for 2 hours, freeze-dried at -40°C for 6 hours, and then dried again at 25°C. The continuous phase is composed of 85% palm oil, 10% sorbitan monooleate, and 5% cholesterol.

[0029] Preparation method 3: tert-butanol and acetonitrile are melted at 50°C. The specific primer, trehalose, mannitol, polyethylene glycol, and L-histidine are added to the melted tert-butanol and acetonitrile to prepare a dispersed phase. The dispersed phase is slowly added dropwise to the continuous phase (volume ratio 1:9), homogenized at 8000 rpm for 2 minutes, and a uniform droplet is obtained by high-pressure homogenization. The droplet is pre-frozen at -80°C for 2 hours, freeze-dried at -40°C for 6 hours, and then dried again at 25°C. The continuous phase is composed of 85% palm oil, 10% sorbitan monooleate, and 5% cholesterol.

[0030] The solid microspheres were refused and the control group (freshly prepared specific primers) were amplified under the same template concentration, Bst DNA enzyme and basic reaction conditions. The Ct values ​​of each group and the control group were compared as shown in Table 1.

[0031] Table 1: control group Preparation Method 1 Preparation Method 2 Preparation method three Ct value (Cycle) 29.65 36.49 33.51 33.84 Table 2: Solid powder formation Preparation Method 1 After freeze-drying, a solid is formed. Preparation Method 2 Unable to freeze dry to form a solid Preparation method three After freeze-drying, a solid is formed. Preparation method one uses purified water, trehalose, mannitol, polyethylene glycol, L-histidine, and primers to prepare an aqueous phase. Water molecules attack the phosphodiester bonds in the primers, causing partial inactivation of the primers and resulting in a higher Ct value, which affects the accuracy of the detection. Preparation method two obtains a Ct value that is closer to that of the control group, but it uses triglycerides as a polar oil phase. Triglycerides have a high boiling point and cannot form crystals through freeze-drying, resulting in the inability to obtain a solid powder of primers.

[0032] Pre-set experiment 3: Further optimization of the preparation of primer lyophilized microspheres using method three Using Ct value as an indicator, the effect of freezing temperature changes on primer lyophilized microspheres was investigated. The freezing temperature changes are as follows: Pre-freezing temperatures are: -85℃, -80℃, -75℃ and -70℃; The freeze-drying temperatures are: -50℃, -45℃, -40℃, and -35℃; The secondary drying temperatures were 10℃, 15℃, 20℃ and 25℃, and the specific experimental design is shown in Table 3.

[0033] Table 3. Effect of freezing temperature on Ct value of primer lyophilized microspheres Pre-freezing temperature Freeze-drying temperature Secondary drying temperature Ct value 1 -85℃ -50℃ 10℃ 35.87 2 -85℃ -45℃ 15℃ 34.72 3 -85℃ -40℃ 20℃ 34.41 4 -85℃ -35℃ 25℃ 33.7 5 -80℃ -50℃ 15℃ 33.94 6 -80℃ -45℃ 10℃ 34.89 7 -80℃ -40℃ 25℃ 32.82 8 -80℃ -35℃ 20℃ 33.25 9 -75℃ -50℃ 20℃ 32.73 10 -75℃ -45℃ 25℃ 33.21 11 -75℃ -40℃ 10℃ 33.67 12 -75℃ -35℃ 15℃ 34.18 13 -70℃ -50℃ 25℃ 35.91 14 -70℃ -45℃ 20℃ 34.73 15 -70℃ -40℃ 15℃ 33.48 16 -70℃ -35℃ 10℃ 34.26 ∆Ct value at -85℃ 34.675 ∆Ct value at -50℃ 34.6125 ∆Ct value at 10℃ 34.6725 ∆Ct value at -80℃ 33.725 ∆Ct value at -45℃ 34.6375 ∆Ct value at 15℃ 34.08 ∆Ct value at -75℃ 33.6975 ∆Ct value at -40℃ 33.595 ∆Ct value at 20℃ 33.78 ∆Ct value at -70℃ 34.595 ∆Ct value at -35℃ 33.8475 ∆Ct value at 25℃ 33.91 As can be seen from Table 3, the ∆Ct values ​​are lower when the pre-freezing temperature is -80℃ to -75℃, lower when the freeze-drying temperature is -40℃ to -35℃, and lower when the secondary drying temperature is 20℃ to 25℃. Based on this, a method for preparing specific primers with Ct values ​​closer to freshly prepared primers was obtained.

[0034] Pre-freezing temperatures are -79℃, -78℃, -77℃ and -76℃; The freeze-drying temperatures are -39℃, -38℃, -37℃ and -36℃; The secondary drying temperatures were 21℃, 22℃, 23℃ and 24℃, and the specific experimental design method is shown in Table 4.

[0035] Table 4. Effect of freezing temperature on Ct value of primer lyophilized microspheres Pre-freezing temperature Freeze-drying temperature Secondary drying temperature Ct value 1 -79℃ -39℃ 21℃ 33.27 2 -79℃ -38℃ 22℃ 32.11 3 -79℃ -37℃ 23℃ 32.75 4 -79℃ -36℃ 24℃ 33.34 5 -78℃ -39℃ 22℃ 32.57 6 -78℃ -38℃ 21℃ 31.38 7 -78℃ -37℃ 24℃ 31.61 8 -78℃ -36℃ 23℃ 32.15 9 -77℃ -39℃ 23℃ 31.26 10 -77℃ -38℃ 24℃ 32.47 11 -77℃ -37℃ 21℃ 31.46 12 -77℃ -36℃ 22℃ 32.58 13 -76℃ -39℃ 24℃ 33.24 14 -76℃ -38℃ 23℃ 31.45 15 -76℃ -37℃ 22℃ 32.39 16 -76℃ -36℃ 21℃ 33.61 ∆Ct value at -80℃ 33.725 ∆Ct value at -40℃ 33.595 ∆Ct value at 20℃ 33.78 ∆Ct value at -79℃ 32.8675 ∆Ct value at -39℃ 32.585 ∆Ct value at 21℃ 32.43 ∆Ct value at -78℃ 31.9275 ∆Ct value at -38℃ 31.8525 ∆Ct value at 22℃ 32.4125 ∆Ct value at -77℃ 31.9425 ∆Ct value at -37℃ 32.0525 ∆Ct value at 23℃ 31.9025 ∆Ct value at -76℃ 32.6725 ∆Ct value at -36℃ 32.92 ∆Ct value at 24℃ 32.665 ∆Ct value at -75℃ 33.6975 ∆Ct value at -35℃ 33.8475 ∆Ct value at 25℃ 33.91 Table 4 shows that the freezing temperatures for obtaining primer freeze-dried microspheres are: pre-freezing at -78 to -77°C, freeze-drying at -38°C to -37°C, and secondary drying at 22°C to 23°C.

[0036] Pre-set experiment 4: Investigating the effect of different treatments on the inner wall of the reaction tube on the segmented sealing effect of paraffin. Blank treatment: The inner wall of the reaction tube was not treated in any way; Treatment 1: Polish the inner wall of the reaction tube with 200-grit sandpaper; Treatment 2: Coat the inner wall of the reaction tube with an ethanol solution of 2% γ-glycidyl etheroxypropyltrimethoxysilane and cure at 90°C for 30 minutes. After preparing 20 reaction tubes for each treatment, red water was placed into the reaction tubes, and heated and liquefied paraffin was added to the reaction tubes. After the reaction tubes were completely solidified, they were placed in a container and placed in a shaker and shaken vigorously for 20 minutes. The integrity of the solid paraffin layer in each treatment was then observed.

[0037] Table 5. Effects of different treatments on the inner wall of the reaction tube on the segmented sealing effect of paraffin. The cured paraffin layer did not shift. Blank processing 11 Process 1 17 Process 2 17 As can be seen from the table above, the number of reaction tubes treated with 200-grit sandpaper and γ-glycidoxypropyltrimethoxysilane that did not have the paraffin layer shifted was greater than that of the untreated reaction tubes. This prevents the paraffin layer from shifting during transportation and allows each layer of reaction liquid to be better sealed.

[0038] Pre-set experiment 5: Paraffin-coated segmented blocking reagent hot-start visual LAMP detection of common enteric pathogens The following bacteria were detected using a paraffin-embedded segmented blocking reagent hot-start visual LAMP assay: Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Salmonella typhi, Salmonella enteritidis, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, hemolytic streptococci, Enterococcus faecalis, Listeria monocytogenes, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Clostridium perfringens, Cronobacter sakazakii, Proteus mirabilis, Bacillus cereus, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Klebsiella pneumoniae, and a negative control. Results are as follows: Figure 6 As shown.

[0039] The results of the hot-start visual LAMP specificity test of the paraffin segmented blocking reagent show that Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei, all belonging to the same genus, showed positive reactions, while 19 other common enteropathogenic bacteria, such as Escherichia coli and Salmonella, showed negative reactions. This indicates that the hot-start visual LAMP of the paraffin segmented blocking reagent has strong specificity for Shigella.

[0040] Figure 6The labels in the table represent 1. Shigella dysenteriae, 2. Shigella flexneri, 3. Shigella boydii, 4. Shigella sonnei, 5. Salmonella typhi, 6. Salmonella enteritidis, 7. Escherichia coli, 8. Yersinia enterocolitica, 9. Campylobacter jejuni, 10. Hemolytic streptococci, 11. Enterococcus faecalis, 12. Listeria, 13. Enterococcus faecalis, 14. Staphylococcus aureus, 15. Staphylococcus epidermidis, 16. Pseudomonas aeruginosa, 17. Clostridium perfringens, 18. Cronobacter sakazakii, 19. Proteus mirabilis, 20. Bacillus cereus, 21. Pseudomonas aeruginosa, 22. Vibrio parahaemolyticus, 23. Klebsiella pneumoniae, and N. negative control.

[0041] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for preparing a hot-start, visually-guided loop-mediated isothermal amplification reaction kit, characterized in that, include: S1, Preparation of primer lyophilized microspheres; S2, Reaction tube treatment: Coat the inner wall of the reaction tube with an ethanol solution of 2-5% γ-glycidyl etheroxypropyltrimethoxysilane and cure at 90-100℃ for 30 minutes. S3, add the base solution to the reaction tube of step S2, add blocking solution to form the first blocking layer, add colorimetric reagent and blocking solution to the first blocking layer, solidify the blocking solution to form the second blocking layer, add DNA large fragment polymerase and blocking solution to the second blocking layer to form the third blocking layer, add primer lyophilized microspheres to the third blocking layer, and close the reaction tube cap.

2. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 1, characterized in that, In step S1, the specific steps for preparing primer lyophilized microspheres are as follows: S11, synthesis, purification, and obtaining specific primers; S12, melt tert-butanol and acetonitrile, add the specific primer and primer protection solution to the melted tert-butanol and acetonitrile to prepare a dispersed phase, slowly drop the dispersed phase into the continuous phase, homogenize at 8000-8500 rpm for 2-5 minutes, and obtain a uniform droplet by high pressure homogenizer; S13, the droplet is pre-frozen at -80~-75℃ for 2-3 hours, then freeze-dried at -40~-35℃ for 5-6 hours, and then dried again at 20-25℃ to form solid microspheres.

3. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 2, characterized in that, The continuous phase comprises 80-85% palm oil, 10-15% sorbitan monooleate, and 5-10% cholesterol.

4. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 2, characterized in that, In step S12, the dispersed phase is slowly dripped into the continuous phase, wherein the volume ratio of the dispersed phase to the continuous phase is 1:7-9.

5. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 2, characterized in that, In step S12, the primer protection solution includes trehalose, mannitol, polyethylene glycol, and L-histidine.

6. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 5, characterized in that, The primer protection solution comprises, by weight percentage, 65-78% trehalose, 15-25% mannitol, 3-8% polyethylene glycol, and 0.5-2% L-histidine.

7. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 1, characterized in that, The volume ratio of tert-butanol to acetonitrile is 5-7:

3.

8. The method for preparing the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 1, characterized in that, The sealing liquid is solid paraffin with a melting point of 55°C.

9. The preparation method of the hot-startable, visible ring-mediated isothermal amplification reaction kit as described in claim 1, characterized in that, The DNA fragment polymerase is Bst DNAase.

10. The visible ring-mediated isothermal amplification reaction kit prepared by any one of the preparation methods described in claims 1-9, characterized in that, The kit can be used to detect Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei.

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