Air drying reagent for nucleic acid amplification reaction as well as preparation method and application of air drying reagent

Abstract

The invention provides an air drying reagent for nucleic acid amplification reaction as well as a preparation method and application thereof, and relates to the technical field of PCR (Polymerase Chain Reaction). The air drying reagent for the nucleic acid amplification reaction comprises an air drying protective agent, a buffer solution, a hot start Taq enzyme and a specific nucleic acid amplification reaction reagent, the air drying protective agent is prepared from the following components: trehalose, dextran-40, polysaccharide, amino acid, bovine serum albumin, a surfactant, single-chain binding protein and tetramethylammonium chloride or Ectoine. The reagent provided by the invention is simple and convenient in preparation process, does not need complex operation steps and expensive professional equipment, and is good in redissolvability after being air-dried. The micro-fluidic chip can be used in combination with a micro-fluidic chip, is applied to on-site rapid detection, and can be conveniently matched with a full-automatic nucleic acid detector for use.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of PCR technology, and particularly relates to a dried reagent for nucleic acid amplification reaction and a preparation method and application thereof. BACKGROUND

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technology for amplifying specific nucleic acid sequences in vitro. It includes conventional PCR, real-time fluorescent quantitative PCR, isothermal nucleic acid amplification technology, etc. Due to its high specificity and high sensitivity, it has been widely used in the fields of pathogen detection, plant and animal quarantine, disease detection and environmental detection. The reagents involved in the traditional PCR amplification reaction have high temperature requirements, and usually need to be stored and transported under low temperature conditions. Moreover, repeated freezing and thawing of the reagents will have a great impact on the detection results. Therefore, the reagents need to be dried for easy storage and transportation.

[0003] The commonly used drying method at present is freeze-drying, which can ensure the storage and transportation of the reaction reagents at room temperature while maintaining their amplification activity. However, the relatively complex operation process and professional freeze-drying equipment involved in the freeze-drying process result in high cost. Compared with freeze-drying, air-drying technology has the advantages of simple operation, low cost and time saving. Only a conventional drying oven or similar equipment is needed to complete the entire process in a short time, and the reaction reagents can be directly air-dried on a microfluidic chip, eliminating the complex dispensing and using process.

[0004] At present, freeze-drying technology has become mature in China, while air-drying technology has few reports. The present application provides a novel air-dried reagent for nucleic acid amplification reaction and its matching preparation method, which provides a new idea for nucleic acid detection, especially for on-site rapid detection.

[0005] Chinese patent CN116479100A provides a protective agent and its application in preparing reverse transcription PCR dried reagent. The composition includes sucrose, trehalose, formamide and bovine serum albumin. The stabilizer can maintain the high efficiency of the reverse transcription PCR system under high temperature air-drying treatment, but the air-drying time needs 16 hours, which is too long.

[0006] Chinese patent CN116200469A provides a kind of air-dried qPCR reagent and its preparation method, air-dried qPCR reagent includes air-dried protective agent, DNA polymerase, reverse transcriptase, RNA enzyme inhibitor, dNTPs, air-dried buffer, upstream and downstream primers and fluorescent probe.Among them, air-dried protective agent includes the following components: BSA, pullulan, trehalose, collagen and DTT.However, when using the protective agent, air-drying needs to use air-drying instrument, and the air-drying condition is 16 DEG C, 3h, the instrument, air-drying temperature has requirement, and air-drying time is also relatively long. SUMMARY

[0007] To solve the above problems, the present application provides an air-dried reagent for nucleic acid amplification reaction and its preparation method and application.

[0008] In one aspect, the present application provides an air-dried reagent for nucleic acid amplification reaction.

[0009] Specifically, the air-dried reagent includes air-dried protective agent, buffer, hot-start Taq enzyme and specific nucleic acid amplification reaction reagent.

[0010] Specifically, the air-dried protective agent includes the following components: trehalose, dextran-40, polysaccharide, amino acid, bovine serum albumin, surfactant, single-strand binding protein and tetramethylammonium chloride or icodextrin.

[0011] Further specifically, the content of each component of the air-dried protective agent is: trehalose mass concentration 4%-20%, dextran-40 concentration 0.1%-5%, polysaccharide mass concentration 0.1%-5%, amino acid 0.1-20mM, tetramethylammonium chloride concentration 1-60mM, bovine serum albumin 0.1-2mg / mL, surfactant mass concentration 0.01%-2%, and single-strand binding protein concentration 10-300ng / μL.

[0012] Further specifically, the content of each component of the air-dried protective agent is: trehalose mass concentration 4%-20%, dextran-40 concentration 0.1%-5%, polysaccharide mass concentration 0.1%-5%, amino acid 0.1-20mM, icodextrin concentration 1-60mM, bovine serum albumin 0.1-2mg / mL, surfactant mass concentration 0.01%-2%, and single-strand binding protein concentration 10-300ng / μL.

[0013] In some embodiments of the present application, the content of each component of the air-drying protective agent is as follows: trehalose mass concentration of 6%-12%, dextran-40 concentration of 1%-2%, polysaccharide mass concentration of 1%-4%, amino acid 10-20 mM, tetramethylammonium chloride concentration of 20-60 mM, bovine serum albumin 1-2 mg / mL, surfactant mass concentration of 0.01%-0.05%, and single-stranded binding protein concentration of 80-160 ng / μL.

[0014] In some embodiments of the present application, the content of each component of the air-drying protective agent is as follows: trehalose mass concentration of 6%-12%, dextran-40 concentration of 1%-2%, polysaccharide mass concentration of 1%-4%, amino acid 10-20 mM, tetramethylammonium chloride concentration of 20-60 mM, bovine serum albumin 1-2 mg / mL, surfactant mass concentration of 0.01%-0.05%, and single-stranded binding protein concentration of 80-160 ng / μL.

[0015] Further specifically, the polysaccharide includes at least one of maltose cyclodextrin, 2-hydroxypropan-β-cyclodextrin, and / or hydroxyethyl starch.

[0016] Further specifically, the amino acid includes at least one of L-histidine, L-lysine, L-proline, glycine, and / or arginine.

[0017] Preferably, the amino acid is L-histidine or L-lysine.

[0018] Further specifically, the surfactant includes at least one of Tween-20, Tween-80, Triton x-100, Brij-L23, PVP-40, and / or PEG 8000.

[0019] Preferably, the surfactant includes Tween-20, Tween-80, or Triton x-100.

[0020] Specifically, the buffer includes at least one of Tris-HCl buffer, HEPES buffer, potassium chloride, magnesium chloride, ammonium sulfate, or magnesium sulfate.

[0021] Further specifically, the buffer includes HEPES buffer, potassium chloride, and magnesium chloride.

[0022] In some embodiments of the present application, the buffer includes 600 mM HEPES buffer, 500 mM potassium chloride, and 35 mM magnesium chloride.

[0023] Specifically, the specific nucleic acid amplification reaction reagent includes at least one of a primer, a probe, a sample, a fluorescent dye, and dNTPs.

[0024] Further specifically, the specific nucleic acid amplification reaction reagent comprises dNTPs, primers and probes.

[0025] In some specific embodiments of the present application, the specific nucleic acid amplification reaction reagent comprises 0.2 mM dNTPs and 0.2 μM upstream and downstream primers and probes.

[0026] In another aspect, the present application provides a preparation method of the above-mentioned air-dried reagent.

[0027] Specifically, the preparation method comprises the following steps: (1) weighing all components according to the formula, mixing uniformly, preparing an air-dried protective agent; (2) weighing all components according to the formula, mixing uniformly, preparing a buffer and a specific nucleic acid amplification reaction reagent: (3) uniformly mixing the air-dried protective agent with the buffer, the specific nucleic acid amplification reaction reagent and DNA polymerase, and supplementing with nuclease-free water to 8-15 μL to obtain a mixed solution; (4) air-drying the mixed solution at 30-50 ℃ for 1-3 h.

[0028] In yet another aspect, the present application provides an application of the above-mentioned air-dried reagent in on-site rapid detection.

[0029] Specifically, the air-dried reagent can be used in combination with a microfluidic chip and / or a full-automatic nucleic acid detector.

[0030] Compared with the prior art, the present application has the following beneficial effects: 1. The present application does not require complex operation steps and expensive professional equipment, and the air-dried reagent can be transported at room temperature, thereby reducing the cost.

[0031] 2. The air-dried reagent prepared by the present application has good stability and high sensitivity, and still has good amplification effect after being stored at room temperature for a long time.

[0032] 3. The operation process of the present application is simple, the air-dried reagent has good reconstitution property, can be used in combination with a microfluidic chip, applied to on-site rapid detection, and conveniently used in combination with a full-automatic nucleic acid detector. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is the sensitivity experimental result of the air-dried reagent.

[0034] Figure 2 is the comparison of amplification of the air-dried reagent and the conventional reagent. DETAILED DESCRIPTION

[0035] The application will be further described in conjunction with specific examples. The following examples are not intended to limit the application, but merely to illustrate the application. Unless otherwise specified, the experimental procedures used in the following examples were carried out according to conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources and used without further purification.

[0036] The following sources of raw materials are exemplary: Table 1

[0037] Thermostable Taq enzyme: laboratory-made, amino acid sequence as shown in SEQ ID NO: 8: MEEMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDSVIVVFDAKAPSFRHEFYEGYKARRAPTPEDFPRQLALIKELVDLLGLVRLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKHGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLERLLKNLDRLRPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAAREGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWERLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTNLKNTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFESFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE.

[0038] Example 1 (1) Prepare the air-drying protectant, buffer, and specific nucleic acid amplification reaction reagent according to the formulations in Tables 2-5, wherein the solvent of the air-drying protectant is nuclease-free water.

[0039] Table 2

[0040] Table 3

[0041] Table 4

[0042] Table 5

[0043] (2) Prepare the PCR reaction solution in the reactor, including: 4 μL of air-drying protectant, 2 μL of buffer, 0.2 μL of hot-start Taq enzyme and 1 μL of specific nucleic acid amplification reaction reagent, and make up to 8 μL with nuclease-free water.

[0044] (3) Place the reactor in a forced-air drying oven and set the drying conditions to 50°C for 1.5 hours.

[0045] (4) After air drying, tighten the reactor lid and seal it in an aluminum foil bag containing desiccant.

[0046] Example 2 (1) Prepare the air-drying protectant, buffer solution and specific nucleic acid amplification reaction reagent according to the formulas in Tables 6-9. The solvent for the air-drying protectant is nuclease-free water.

[0047] Table 6

[0048] Table 7

[0049] Table 8

[0050] Table 9

[0051] (2) Prepare the PCR reaction solution in the reactor, including: 4 μL of air-drying protectant, 2 μL of buffer, 0.2 μL of hot-start Taq enzyme and 1 μL of specific nucleic acid amplification reaction reagent, and make up to 8 μL with nuclease-free water; (3) Air drying: Place the reactor in a forced-air drying oven, set the air drying conditions to 50℃, and set the air drying time to 1.5h; (4) After air drying, tighten the reactor lid and seal it in an aluminum foil bag containing desiccant.

[0052] Example 3 The difference from Example 1 is that the concentration of trehalose in the air-drying protectant is 6%, the concentration of dextran-40 is 2%, and the concentration of 2-hydroxypropyl-β-cyclodextrin is 4%. Other operations and steps are the same as in Example 1.

[0053] Example 4 The difference from Example 2 is that the concentration of trehalose in the air-drying protectant is 6%, the concentration of dextran-40 is 2%, and the concentration of maltodextrin is 2%. Other operations and steps are the same as in Example 2.

[0054] Example 5 The difference from Example 1 is that the concentration of bovine serum albumin in the air-drying protectant is 2 mg / mL, while the other operations and steps are the same as in Example 1.

[0055] Example 6 The difference from Example 1 is that tetramethylammonium chloride in the air-drying protectant is replaced with ectoine; the other operations and steps are the same as in Example 1.

[0056] Example 7 The difference from Example 1 is that Tween-20 in the air-drying protectant is replaced with Triton X-100, while the other operations and steps are the same as in Example 1.

[0057] Example 8 The difference from Example 1 is that the air drying conditions are set to 50°C and the air drying time is set to 3 hours, while the other operations and steps are the same as in Example 1.

[0058] Example 9 The difference from Example 1 is that the air drying conditions are set to 80°C and the air drying time is set to 0.5h, while the other operations and steps are the same as in Example 1.

[0059] Comparative Example 1 The difference from Example 1 is that the air-drying protectant contains only trehalose, while the other operations and steps are the same as in Example 1.

[0060] Comparative Example 2 The difference from Example 1 is that the air-drying protectant contains only trehalose and bovine serum albumin, while the other operations and steps are the same as in Example 1.

[0061] Comparative Example 3 The difference from Example 1 is that the air-drying protectant does not contain dextran-40 and 2-hydroxypropyl-β-cyclodextrin; the other operations and steps are the same as in Example 1.

[0062] Comparative Example 4 The difference from Example 1 is that the air-drying protectant does not contain L-histidine; the other operations and steps are the same as in Example 1.

[0063] Experiment 1: Detection of amplification results from Examples 1-9 and Comparative Examples 1-4 (1) PCR reaction reagents were prepared according to Examples 1-9 and Comparative Examples 1-4 respectively. The undried components of Example 1 were used as the undried control group. The primer and probe sequences used in the specific nucleic acid amplification reaction reagents are shown in Table 10. The sequence of the internal quality control is shown in SEQ ID NO:7.

[0064] Table 10

[0065] (2) Real-time quantitative PCR was performed using an ABI 7500. The reaction conditions were set as follows: 95℃ for 1 min; 40 cycles (95℃ for 10 s, 60℃ for 30 s). The Ct values ​​of real-time quantitative PCR are shown in Table 11: Table 11

[0066] As shown in Table 5, the Ct values ​​of the air-drying reagents prepared in Examples 1-9 of this invention are basically consistent with those before and after air-drying, indicating that the air-drying protectant prepared in this invention can effectively protect the enzyme activity during the air-drying process. In Comparative Examples 1-4, some components were reduced, and the Ct values ​​after air-drying were significantly delayed, indicating that reducing the necessary components could not effectively protect the enzyme activity during the air-drying process.

[0067] Experimental Example 2 Sensitivity (1) The preparation of the air-dried reagent reaction solution is the same as in Example 1; the primers and probes in the above-mentioned specific nucleic acid amplification reaction reagents are the same as in Example 1; (2) The air-drying conditions were the same as in Example 1; (3) Add 20 μL of each containing 5 × 10 4 5×10 3 5×10 2 5×10 1 Copy the Chlamydia trachomatis sample into a PCR reaction tube, with two replicates for each gradient; (4) After mixing thoroughly, centrifuge and perform real-time quantitative PCR using an ABI 7500. The reaction conditions are set as follows: 95℃ for 1 min; 45 cycles (95℃ for 10 s, 60℃ for 30 s). (5) The PCR results Ct values ​​are shown in Table 12, and the amplification curves are shown in Table 12. Figure 1 As shown: Table 12

[0068] The results show that the RT-qPCR air-dried reagent has good sensitivity.

[0069] Experiment Example 3: Comparison with Conventional PCR (1) The preparation of the air-dried PCR reaction solution is the same as in Example 1; (2) The air-drying conditions were the same as in Example 1; (3) The conventional PCR reaction solution was prepared using the conventional commercial PCR mix method; (4) Add 20 μL of solution containing 5 × 10⁻⁶ ppm. 3 5×10 2 Copy the Chlamydia trachomatis sample into a PCR reaction tube, with two replicates for each gradient; (5) After mixing evenly, centrifuge and perform real-time quantitative PCR using ABI 7500. The reaction conditions are set as follows: 95℃ for 1 min; 95℃ for 10 s, 60℃ for 30 s, for 40 cycles.

[0070] The Ct values ​​for real-time quantitative PCR are shown in Table 13, and the amplification curves are shown in Table 14. Figure 2 As shown: Table 13

[0071] Experimental results show that air drying has no effect on the Ct value of PCR results and can improve the fluorescence value of PCR results, thus achieving better results.

[0072] Experiment Example 4: Accelerated Experiment The air-dried PCR reagents were placed in a constant temperature incubator at 37°C for 60 days, and samples were taken every 7 days to verify the stability of the air-dried reagents. The experimental results are shown in Table 14.

[0073] Table 14

[0074] The experimental results show that there was no significant difference in Ct values ​​after 56 days of storage at 37℃, indicating that the air-dried reagent has good stability.

[0075] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A drying reagent for RT-qPCR, characterized in that, The air-drying reagent includes an air-drying protectant, a buffer solution, DNA polymerase, and specific nucleic acid amplification reaction reagents; the air-drying protectant includes: trehalose, dextran-40, polysaccharides, amino acids, bovine serum albumin, surfactants, single-stranded binding proteins, and tetramethylammonium chloride or ectoine.

2. The air-drying reagent according to claim 1, characterized in that, The components of the air-drying protectant have the following contents: trehalose mass concentration 4%-20%, dextran-40 mass concentration 0.1%-5%, polysaccharide mass concentration 0.1%-5%, amino acids 0.1-20mM, bovine serum albumin 0.1-2mg / mL, surfactant mass concentration 0.01%-2%, single-chain binding protein concentration 10-300ng / μL, and tetramethylammonium chloride or ectoine concentration 1-60mM.

3. The air-drying reagent according to claim 2, characterized in that, The polysaccharide includes at least one of maltodextrin, 2-hydroxypropyl-β-cyclodextrin, and / or hydroxyethyl starch.

4. The air-drying reagent according to claim 2, characterized in that, The amino acid includes at least one of L-histidine, L-lysine, L-proline, glycine, and / or arginine.

5. The air-drying reagent according to claim 2, characterized in that, The surfactant includes at least one of Tween-20, Tween-80, Triton X-100, Brij-L23, PVP-40 and / or PEG 8000.

6. The air-drying reagent according to claim 1, characterized in that, The buffer solution includes at least one of Tris-HCl buffer, HEPES buffer, potassium chloride, magnesium chloride, ammonium sulfate and / or magnesium sulfate.

7. The air-drying reagent according to claim 1, characterized in that, The specific nucleic acid amplification reaction reagents include at least one of primers, probes, and / or dNTPs.

8. The method for preparing the air-drying reagent according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Preparation of air-drying protectant: Take the formula amount of trehalose, dextran-40, polysaccharide, amino acid, bovine serum albumin, surfactant, single-chain binding protein and tetramethylammonium chloride or ectoine and mix them evenly; (2) Preparation of buffer solution and specific nucleic acid amplification reaction reagents; (3) Mix the air-drying protectant with the buffer, specific nucleic acid amplification reaction reagent and DNA polymerase evenly, and add nuclease-free water to make up to 8-15 μL to obtain the mixture; (4) Dry the mixture at 30-80℃ for 0.5-3 hours.

9. The application of the air-dried reagent according to any one of claims 1-7 in on-site rapid testing.

10. The application according to claim 9, characterized in that, The air-drying reagent can be used in conjunction with microfluidic chips and / or fully automated nucleic acid testing instruments.

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