RT-qPCR air drying reagent as well as preparation method and application thereof

Abstract

The invention provides an RT-qPCR air drying reagent as well as a preparation method and application thereof, and relates to the technical field of PCR. The air drying reagent for RT-qPCR amplification reaction comprises an air drying protective agent, a buffer solution, an enzyme reaction solution and a specific nucleic acid amplification reaction reagent. The air drying protective agent comprises trehalose, polysaccharide, amino acid, bovine serum albumin, a surfactant, water bear protein, ammonium sulfate and DTT. 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] This invention belongs to the field of PCR technology, specifically relating to an air-drying reagent for RT-qPCR, its preparation method, and its application. Background Technology

[0002] Quantitative reverse transcription PCR (RT-qPCR) is a commonly used molecular biology technique that is widely applied in fields such as pathogen detection, animal and plant quarantine, disease detection, and environmental monitoring. It has advantages such as high specificity and high sensitivity.

[0003] However, PCR amplification reactions involve active molecules such as enzymes, which are highly sensitive to temperature changes and typically require a low-temperature environment to maintain their activity. This leads to high costs for reagent storage and transportation. To reduce costs and increase reagent stability during storage and transportation, PCR amplification reagents are usually prepared into lyophilized beads or lyophilized sheets using freeze-drying. The resulting lyophilized reagents can maintain their activity for a longer period, facilitating storage and transportation. However, freeze-drying technology still has certain limitations. The resulting lyophilized beads are easily broken and susceptible to moisture, leading to reagent inactivation. Furthermore, the complex operational procedures and specialized freeze-drying equipment involved in freeze-drying contribute to the high cost of this technology.

[0004] Compared to freeze drying, air drying technology has advantages such as simple operation, low cost, and time saving. It only requires a conventional drying oven or similar equipment to complete the entire preparation process in a short time, and the reaction reagents can be air dried directly on the microfluidic chip, eliminating the need for complicated dispensing and use processes.

[0005] Chinese patent CN116411054A provides a composition for improving the stability of PCR air-drying reagents and its application. The composition includes sucrose, trehalose, dextran, polyvinylpyrrolidone, and raffinose. This stabilizer can maintain comparable detection performance of a fully premixed PCR system before and after air-drying, but the air-drying time is 16 hours, which is too long.

[0006] Chinese patent CN116200469A discloses an air-dried qPCR reagent and its preparation method. The air-dried qPCR reagent includes a drying protectant, DNA polymerase, reverse transcriptase, RNase inhibitor, dNTPs, drying buffer, upstream and downstream primers, and fluorescent probes. The drying protectant includes the following components: BSA, pullulan, trehalose, collagen, and DTT. However, using this protectant requires an air dryer at 16°C for 3 hours, placing specific requirements on the instrument and drying temperature, and the drying time is relatively long. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides an air-drying reagent for RT-qPCR, its preparation method, and its application.

[0008] On the one hand, the present invention provides an air-drying reagent for RT-qPCR.

[0009] Specifically, the air-drying reagent includes: air-drying protectant, buffer solution, enzyme reaction solution, and specific nucleic acid amplification reaction reagent.

[0010] Specifically, the air-drying protectant comprises the following components: trehalose (4%-20% by mass), polysaccharide (0.1%-5% by mass), amino acids (0.1-20mM), bovine serum albumin (0.1-4mg / mL), surfactant (0.01%-2% by mass), tardigrade protein (1-100ng / μL), ammonium sulfate (1-20mM), and DTT (1-20mM).

[0011] More specifically, the polysaccharide includes at least one of 2-hydroxypropyl-β-cyclodextrin and pullulan.

[0012] More specifically, the amino acid includes at least one selected from L-histidine, L-lysine, L-histidine, L-proline, glycine, or arginine.

[0013] More specifically, the surfactant includes at least one of Tween-20, Tween-80, PVP-40, or PEG 8000.

[0014] In certain specific embodiments of the present invention, the air-drying protectant comprises the following components: trehalose (12% by mass), 2-hydroxypropyl-β-cyclodextrin (2% by mass), pullulan (0.1% by mass), L-histidine (10mM), bovine serum albumin (1mg / mL), Tween-80 (0.05% by mass), tardigrade protein (40ng / μL), ammonium sulfate (1mM), and DTT (1mM).

[0015] Specifically, the buffer solution includes at least one of Tris-HCl buffer, HEPES buffer, potassium chloride, magnesium chloride, ammonium sulfate, or magnesium sulfate.

[0016] More specifically, the buffer solution includes HEPES buffer, potassium chloride, and magnesium chloride.

[0017] In some specific embodiments of the present invention, the buffer solution comprises 300 mM HEPSE buffer, 500 mM potassium chloride and 35 mM magnesium chloride.

[0018] Specifically, the enzyme reaction solution includes at least one of hot-start Taq enzyme, reverse transcriptase, RNA polymerase, helicase, RNase inhibitor and / or single-stranded binding protein.

[0019] More specifically, the enzyme reaction solution includes a hot-start Taq enzyme, a reverse transcriptase, and an RNase inhibitor.

[0020] In some specific embodiments of the present invention, the enzyme reaction solution comprises 1 U / μL of hot-start Taq enzyme, 0.2 U / μL of reverse transcriptase and 0.5 U / μL of RNase inhibitor. Specifically, the specific nucleic acid amplification reaction reagent includes at least one of primers, probes, samples, fluorescent dyes, and dNTPs.

[0021] More specifically, the specific nucleic acid amplification reaction reagents include primers, probes, and dNTPs.

[0022] In some specific embodiments of the present invention, the specific nucleic acid amplification reaction reagent includes 0.2 mM dNTPs and 0.2 μM upstream and downstream primers and probes.

[0023] On the other hand, the present invention provides a method for preparing the above-mentioned air-drying reagent.

[0024] Specifically, the preparation method includes the following steps: (1) Weigh all components according to the formula, mix them evenly, and prepare the air-drying protectant; (2) Weigh all components according to the formula, mix them evenly, and prepare buffer solution and specific nucleic acid amplification reaction reagents: (3) Weigh all components according to the formula, mix them evenly, and prepare the enzyme reaction solution: (4) Mix the air-drying protectant with the buffer, specific nucleic acid amplification reaction reagent and enzyme reaction solution evenly, and add nuclease-free water to make up to 8-15 μL to obtain the mixture; (5) Air dry the mixture at 30-50℃ for 1-3 hours.

[0025] On another front, the present invention provides the application of the above-mentioned air-dried reagent in rapid on-site testing.

[0026] Specifically, the air-drying reagent can be used in conjunction with a microfluidic chip and / or a fully automated nucleic acid testing instrument.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention does not require complicated operating steps or expensive professional equipment. The preparation of air-dried reagents can be completed in a short time through simple operation.

[0028] 2. The operation process of this invention is simple, the reagents have good reconstitution after air drying, it can be used in conjunction with microfluidic chips, applied to rapid on-site detection, and is convenient to use with fully automated nucleic acid detection instruments. Attached Figure Description

[0029] Figure 1 The amplification results are for groups 1-7 in Example 1.

[0030] Figure 2 The results show the sensitivity of the air-dried reagent for RT-qPCR.

[0031] Figure 3 The results show the comparison between the air-dried RT-qPCR reagent and the conventional RT-qPCR amplification system. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0033] The following sources of raw materials are provided as examples: Table 1

[0034] Tardigrade protein: prepared in the laboratory using the same method as Yamaguchi A, Tanaka S, Yamaguchi S, et al. Two novel heat-soluble protein families abundantly expressed in ananhydrobiotic tardigrade[J]. 2012. Hot start Taq enzyme: Self-made in the laboratory, the amino acid sequence is as shown in SEQ ID NO:9: MEEMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDSVIVVFDAKAPSFRHEFYEGYKARRAPTPEDFPRQLALIKELVDLLGLVRLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKHGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLERLLKNLDRLRPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAAREGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWERLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTNLKNTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFESFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE。

[0035] Reverse transcriptase: prepared in the laboratory, the amino acid sequence is shown in SEQ ID NO:10.

[0036] Example 1: Preparation of air-dried RT-qPCR reagent (1) Prepare the air-drying protectant, buffer solution, enzyme reaction solution and specific nucleic acid amplification reaction reagent according to Tables 2-5 respectively. The solvent of the air-drying protectant is nuclease-free water.

[0037] Table 2

[0038] Table 3

[0039] Table 4

[0040] Table 5

[0041] (2) Prepare the RT-qPCR reaction solution in the reactor, including: 5 μL of air-drying protectant, 2 μL of buffer, 1 μL of enzyme reaction solution and 1 μL of specific nucleic acid amplification reaction reagent; (3) Place the reactor in a forced-air drying oven, set the drying conditions to 40℃, and the drying time to 2h; (4) After air drying, tighten the reactor lid and seal it in an aluminum foil bag containing desiccant.

[0042] Example 2 The difference from Example 1 is that the concentration of trehalose in the air-drying protectant is 8%, the concentration of 2-hydroxypropyl-β-cyclodextrin is 1%, and L-histidine is replaced with L-lysine. Other components remain unchanged, and the operation and steps are the same as in Example 1.

[0043] Example 3 The difference from Example 1 is that the concentration of trehalose in the air-drying protectant is 10%, the concentration of 2-hydroxypropyl-β-cyclodextrin is 1%, and Tween-80 is replaced with Tween-20. Other components remain unchanged, and the operation and steps are the same as in Example 1.

[0044] Example 4 The difference from Example 1 is that the concentration of bovine serum albumin in the air-drying protectant is 2 mg / mL, while other components remain unchanged, and the operation and steps are the same as in Example 1.

[0045] Example 5 The difference from Example 1 is that the concentration of ammonium sulfate in the air-drying protectant is 10 mM, while the other components remain unchanged, and the operation and steps are the same as in Example 1.

[0046] Example 6 The difference from Example 1 is that the air drying conditions are set to 30°C and the air drying time is set to 3 hours, while the other operations and steps are the same as in Example 1.

[0047] Example 7 The difference from Example 1 is that the air drying conditions are set to 50°C and the air drying time is set to 1 hour, while other operations and steps are the same as in Example 1.

[0048] Experiment Example 1: Air-dried reagents used for amplification (1) Prepare air-dried RT-qPCR reaction solutions according to Examples 1-7. The primer and probe sequences in the above-mentioned specific nucleic acid amplification reaction reagents are shown in Table 6: Table 6

[0049] (2) Air drying: Air dry according to the air drying conditions in Examples 1-7; (3) Add 20 μL of each containing 5 × 10 3 The novel coronavirus and internal quality control samples (sequences shown in SEQ ID NO:7) were added to PCR reaction tubes, 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: 55℃ for 5 min; 95℃ for 1 min; 40 cycles (95℃ for 10 s, 60℃ for 30 s). The Ct values ​​of the RT-qPCR results are shown in Table 7, and the amplification curves are shown in Table 8. Figure 1 As shown: Table 7

[0050] As can be seen from Table 7, the air-dried RT-qPCR reagent has good amplification effect.

[0051] Experiment Example 2 Sensitivity Detection (1) The air-dried RT-qPCR reaction solution was prepared in the same way as in Example 1; the primers and probes in the above-mentioned specific nucleic acid amplification reaction reagents were 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 5 5×10 4 5×10 3 5×10 2 5×10 1 Novel coronavirus samples were added to PCR reaction tubes, 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: 55℃ for 5 min; 95℃ for 1 min; 40 cycles (95℃ for 10 s, 60℃ for 30 s). The Ct values ​​of the RT-qPCR results are shown in Table 8, and the amplification curves are shown in Table 8. Figure 2 As shown: Table 8

[0052] From Table 8 and Figure 2 The results show that the RT-qPCR air-dried reagent has good sensitivity.

[0053] Experimental Example 3: Comparison of Air-Dried RT-qPCR and Conventional RT-qPCR Methods (1) The air-dried RT-qPCR reaction solution was prepared in the same manner as in Example 1; (2) The primers and probes in the above-mentioned specific nucleic acid amplification reaction reagents are the novel coronavirus N gene, and the sequence is the same as that in Experiment 1; (3) The air-drying conditions were the same as in Example 1; (4) The conventional RT-qPCR reaction solution was prepared using the conventional commercial RT-qPCR mix method; (5) Add 20 μL of each containing 5 × 10 3 The novel coronavirus sample was placed into a PCR reaction tube, and two replicates were set up; (6) After mixing thoroughly, centrifuge and perform real-time quantitative PCR using an ABI 7500. The reaction conditions are set as follows: 55℃ for 5 min; 95℃ for 1 min; 40 cycles (95℃ for 10 s, 60℃ for 30 s). (7) The Ct values ​​of the real-time quantitative PCR results are shown in Table 9, and the amplification curves are shown in Table 9. Figure 3 As shown: Table 9

[0054] From Table 9 Figure 3 It can be seen that when using the reagents of this invention, air drying has no effect on the Ct value of RT-qPCR.

[0055] Experiment Example 4: Accelerated Experiment The air-dried RT-qPCR reagent was placed in a 37°C incubator for 30 days, and samples were taken every 7 days to verify the stability of the air-dried reagent. The results of the accelerated experiment are shown in Table 10. The results show that there is no significant difference in the Ct value after being placed at 37℃ for 30 days, indicating that the air-dried reagent has good stability.

[0056] Table 10

[0057] 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, an enzyme reaction solution, and a specific nucleic acid amplification reaction reagent. The air-drying protectant includes the following components: trehalose, polysaccharides, amino acids, bovine serum albumin, surfactants, tardigrade protein, ammonium sulfate, and DTT.

2. The air-drying reagent according to claim 1, characterized in that, The air-drying protectant comprises the following components: trehalose (4%-20% by mass), polysaccharide (0.1%-5% by mass), amino acids (0.1-20mM), bovine serum albumin (0.1-4mg / mL), surfactant (0.01%-2% by mass), tardigrade protein (1-100ng / μL), ammonium sulfate (1-20mM), and DTT (1-20mM).

3. The air-drying reagent according to claim 2, characterized in that, The polysaccharide includes at least one of 2-hydroxypropyl-β-cyclodextrin and pullulan; the amino acid includes at least one of L-histidine, L-lysine, L-proline, glycine, or arginine; and the surfactant includes at least one of Tween-20, Tween-80, PVP-40, or PEG 8000.

4. The air-drying reagent according to claim 3, characterized in that, The air-drying protectant comprises the following components: trehalose (4%-20% by mass), 2-hydroxypropyl-β-cyclodextrin (0.1%-5% by mass), pullulan (0.1%-5% by mass), L-histidine (0.1-20mM), bovine serum albumin (0.1-4mg / mL), Tween-80 (0.01%-2% by mass), tardigrade protein (1-100ng / μL), ammonium sulfate (1-20mM), and DTT (1-20mM).

5. 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, or magnesium sulfate.

6. The air-drying reagent according to claim 1, characterized in that, The enzyme reaction solution also includes at least one of the following: hot-start Taq enzyme, reverse transcriptase, RNA polymerase, helicase, RNase inhibitor, or single-stranded binding protein.

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, 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, 2-hydroxypropyl-β-cyclodextrin, L-histidine, bovine serum albumin, Tween-80, tardigrade protein, ammonium sulfate and DTT and mix them evenly; (2) Preparation of buffer solution and specific nucleic acid amplification reaction reagents; (3) Prepare the enzyme reaction solution; (4) Mix the air-drying protectant with the buffer, specific nucleic acid amplification reaction reagent and enzyme reaction solution evenly, and add nuclease-free water to make up to 8-15 μL to obtain the mixture; (5) Air dry the mixture at 30-50℃ for 1-3 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 is used in conjunction with a microfluidic chip and / or a fully automated nucleic acid detection instrument.

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