Sample treatment liquid for nucleic acid release and detection kit
By optimizing the composition and concentration of the sample treatment solution, rapid and efficient nucleic acid cleavage is achieved, solving the problems of complex operation of traditional methods and long real-time PCR detection time, and achieving rapid on-site pathogen nucleic acid detection.
Patent Information
- Application Number
- CN202510588914.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, the traditional isolation and culture method is troublesome and inefficient. Real-time fluorescence quantitative PCR requires precise temperature control and is not suitable for rapid on-site detection. The sample nucleic acid needs to be purified before amplification, resulting in a long detection time.
A sample treatment solution for nucleic acid release is provided, including buffer, guanidine hydrochloride or guanidine isothiocyanate, imidazole and other components. By optimizing the concentration ratio, combining sodium dodecyl sulfate (SDS) and nucleic acid protective agent, rapid and efficient nucleic acid cleavage is achieved, eliminating purification steps.
Rapid detection of pathogen nucleic acids is achieved within 35 minutes, and is suitable for hospitals, clinics, customs, centers of disease control and other places, improving detection efficiency and accuracy.
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Figure CN120290547A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of detection reagents, and particularly to a sample treatment solution for nucleic acid release and a detection kit. Background Art
[0002] Nucleic acid testing is to search for the nucleic acids of foreign invading viruses in respiratory specimens, blood specimens or fecal specimens to determine whether a person is infected with a virus.
[0003] In vitro diagnostic methods for viruses are mainly divided into two categories: traditional isolation and culture methods and molecular biology methods. The traditional isolation and culture method requires the use of host cells to achieve virus reproduction, with cumbersome operations and low efficiency, and cannot complete the diagnosis quickly and accurately. Therefore, molecular biology methods are now mainly used for virus detection.
[0004] Among the detection methods of molecular biology, the real-time fluorescence quantitative PCR technology has the advantages of high sensitivity, strong specificity, and the ability to achieve multiplex target detection, and is the most commonly used. However, real-time fluorescence quantitative PCR requires precise temperature control, and the sample nucleic acid needs to be purified before amplification detection. Therefore, the detection process needs to be carried out in a PCR laboratory and takes at least 1-2 hours, which is not suitable for on-site rapid detection. In view of this limitation, isothermal amplification technology has gradually gained favor due to its advantages such as rapidity, high efficiency, no need for repeated temperature changes, and strong tolerance to amplification inhibitors.
[0005] For nucleic acid detection by isothermal amplification, it is necessary to go through sample lysis, amplification, and then detection. Among them, the lysis efficiency and quality of the sample are related to the accuracy of detection and the detection time-consuming, and are one of the research focuses in this field. Summary of the Invention
[0006] To solve the above technical problems, the first object of the present invention is to provide a sample treatment solution for nucleic acid release; the second object of the present invention is to provide a detection kit; the sample treatment solution for nucleic acid release and the detection kit provided by the present application can rapidly and efficiently lyse various pathogen samples such as respiratory pathogens, release nucleic acids, can eliminate the step of nucleic acid purification, and can achieve rapid detection of pathogen nucleic acids within 35 minutes.
[0007] The technical solution provided by the present invention is as follows: A sample treatment solution for nucleic acid release, comprising: a buffer solution, guanidine hydrochloride or guanidine isothiocyanate, and imidazole; Wherein, the concentration of imidazole is greater than 0 and less than or equal to 90 mmol / L.
[0008] Preferably, the concentration of imidazole is 50-70 mmol / L.
[0009] Preferably, the concentration of the buffer solution is 50 - 500 mmol / L; and / or, the concentration of guanidine hydrochloride or guanidine isothiocyanate is 50 - 200 mmol / L.
[0010] Preferably, the concentration of guanidine hydrochloride or guanidine isothiocyanate is 100 - 150 mmol / L.
[0011] Preferably, it further includes sodium dodecyl sulfate.
[0012] Preferably, the mass percentage concentration of sodium dodecyl sulfate is 0.001 - 0.2%.
[0013] Preferably, the buffer solution is any one of Tris-HCl buffer solution, boric acid buffer solution, and HEPES buffer solution; and / or, the pH of the buffer solution is 8.0 - 8.6.
[0014] Preferably, it further includes any one or more of polar organic solvents, mucolytic agents, and nucleic acid protectants; the mass percentage concentration of the polar organic solvent is 0.1 - 3%; the concentration of the mucolytic agent is 10 - 30 mmol / L; the concentration of the nucleic acid protectant is 30 - 200 mmol / L.
[0015] Preferably, the polar organic solvent is any one or more of dimethyl sulfoxide, hexamethylphosphoramide, and tetramethylethylenediamine; the mucolytic agent is any one or more of bromhexine hydrochloride, ambroxol, acetylcysteine, and erdosteine; the nucleic acid protectant is any one or more of trehalose, sucrose, RNase inhibitor, and aurintricarboxylic acid.
[0016] A detection kit includes the sample treatment solution for nucleic acid release described in any one of the above.
[0017] This application first provides a sample treatment solution for nucleic acid release, including: a buffer solution, guanidine hydrochloride or guanidine isothiocyanate, and imidazole; wherein, the concentration of imidazole is greater than 0 and less than or equal to 90 mmol / L. The sample treatment solution for nucleic acid release provided by this application is used to release nucleic acid in a sample. By adding imidazole, the lysis efficiency can be improved, facilitating subsequent amplification. The product treated with the sample treatment solution of this application can be used for PCR or CRISPR detection.
[0018] The sample treatment solution for nucleic acid release provided by this application can rapidly and efficiently lyse various pathogen samples such as respiratory pathogens to release nucleic acids, eliminating the need for nucleic acid purification steps. After passing through the conventional nucleic acid amplification and detection steps in this field, rapid detection of pathogen nucleic acids can be achieved within 35 minutes, and the applicable places can include hospitals, clinics, customs, disease control centers, etc.
[0019] The applicant also found that adding sodium dodecyl sulfate (SDS) to the sample treatment solution containing imidazole in this application has a better lysis effect. Preferably, the mass percentage concentration of sodium dodecyl sulfate is 0.001 - 0.2%, and more preferably the concentration is 0.08 - 0.12%.
[0020] This application also provides a detection kit, including the above-mentioned sample treatment solution for nucleic acid release. The detection kit may also include conventional nucleic acid amplification reagents in this field, as well as reagents for CRISPR reaction or PCR reaction.
[0021] Among them, the nucleic acid amplification reagents may include nucleic acid amplification Buffer reagents, amplification-related enzymes, isothermal amplification substrates, and isothermal amplification primers, etc. Among them, the nucleic acid amplification Buffer reagents may include buffer reagents (such as Tricine), salt ions (such as potassium salts), reducing agents (such as DTT), crowding reagents (such as PEG20000), amplification enhancers (such as BSA), etc.; amplification-related enzymes may include reverse transcriptase, polymerase, recombinase protein, single-stranded binding protein, recombinase protein auxiliary protein, helicase, creatine kinase, RNA enzyme inhibitor and other conventional enzymes in this field; the isothermal amplification substrates may include ATP, phosphocreatine, and dNTP, etc.; the isothermal amplification primers are selected according to the target pathogen to be detected. The CRISPR reaction reagents may include CRISPR reaction Buffer reagents, related enzymes, reaction substrates, fluorescence reporting systems, etc. The CRISPR reaction Buffer reagents may include buffer reagents (such as Tris-HCl), salt ions (such as magnesium salts), reducing agents (such as DTT), enhancers (such as BSA, betaine), etc. The related enzymes may include Cas13a protein, T7 RNA polymerase, RNA enzyme inhibitor and other conventional enzymes in this field; the reaction substrates may include NTP and crRNA; the fluorescence reporting system may be single-stranded RNA labeled with a fluorescent group and a quenching group, such as FAM-UUUUUU-BHQ1, etc. The PCR reaction reagents may be commercially available conventional detection kit products based on the RT-PCR method in this field.
[0022] In this application, the nucleic acid protector can use "RNA enzyme inhibitor", which is the commercial name of the reagent and is also called RNase inhibitor, and is a protein-based RNA protector. Description of the Drawings
[0023] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings described below are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0024] Figure 1 It is the graph of the constant-temperature amplification-CRISPR detection results at different imidazole concentrations in Example 1 of the present invention; Figure 2 It is the graph of the RT-PCR detection results at different imidazole concentrations in Example 1 of the present invention; Figure 3 It is the graph of the influence results of different concentrations of SDS in Example 2 of the present invention; Figure 4 It is the graph of the 45°C stability results in Example 3 of the present invention; Figure 5 It is the graph of the influenza virus nucleic acid detection results in Example 4 of the present invention. Detailed implementation manners
[0025] In order to enable those skilled in the art to better understand the technical solutions in the present application, the following will clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.
[0026] In the embodiments of the present application, the national reference product of the second-generation influenza A / B virus nucleic acid detection reagent is taken as an example for experimental research. The information of the national reference product of the second-generation influenza A / B virus nucleic acid detection reagent is shown in Table 1: Table 1
[0027] Note: The equal ratio (1:1) conversion of U / mL to copies / mL.
[0028] The components of the nucleic acid amplification reaction solution used include 100 mM Tricine (pH 8.6), 110 mM potassium acetate, 5 mM DTT, 5 mM ATP, 130 mM creatine phosphate, 1 mM dNTP, 50 ng / μL BSA, 8.3 (w / v)% PEG20000, 0.5 μM isothermal amplification primer, 15 mM magnesium acetate, 40 U reverse transcriptase, 500 ng / μL polymerase, 700 ng / μL recombinase protein, 885 ng / μL single-stranded binding protein, 80 ng / μL recombinase protein auxiliary protein, 500 ng / μL creatine kinase, 12 ng / μL RNase inhibitor, and the specification is 40 μL / T.
[0029] The CRISPR reaction solution used contains 72 mM Tris-HCl (pH 7.4), 7.2 mM magnesium chloride, 12 μg / mL BSA, 240 mM betaine, 18 mM DTT, 1 mM NTP, 50 U T7 RNA polymerase, 4 ng / μL RNase inhibitor, 24 nM Cas13a protein, 36 nM guide RNA, 15 nM reporter RNA, and the specification is 50 μL / T.
[0030] The specification refers to the reagent specification for one detection reaction, that is, the reagent volume in one reaction tube is 40 or 50 μL / Test.
[0031] The primer sequences for influenza A / B virus used are shown in Table 2: Table 2 Primer Sequences
[0032] The detection process is as follows: Add the sample to be tested into the sample treatment solution for nucleic acid release, and place it at room temperature (24 - 30 °C) for 3 - 5 minutes to release nucleic acid, obtaining a lysate. Add 10 μL of the lysate to the nucleic acid amplification reaction solution that has been pre-aliquoted at 40 μL in an 8-well tube, with a final volume of 50 μL; after mixing, incubate at a constant temperature of 37 °C using a metal bath or water bath for 20 min to obtain an amplification product. Take 10 μL of the amplification product and add it to the CRISPR reaction solution that has been pre-aliquoted at 50 μL in an 8-well tube, with a final volume of 60 μL. After incubating at 37 °C for 12 min, use an enzyme-labeled instrument to collect the endpoint fluorescence value. If the RFU value of the sample to be tested is higher than 3000, or use other detection instruments, and the fluorescence signal value of the sample to be tested is greater than or equal to 2 × the fluorescence value of the NTC (negative control), it is judged as positive, otherwise negative.
[0033] The function of the sample treatment solution is to lyse DNA or RNA. Example 1 was designed and implemented to verify whether imidazole can enhance the lysis efficiency of pathogens in the sample treatment solution. Example 1 set up two verification tests. One was to use the sample after treatment for detecting the final fluorescence value in the CRISPR kit of the present invention, and the other was to use a commercially available RT-PCR kit to detect the Ct value for verification.
[0034] Example 1 S1. Prepare sample treatment solutions with different concentrations of imidazole, including 300 mM Tris-HCl (pH 8.6), 120 mM guanidine hydrochloride, 1.3% DMSO, 0 - 90 mM imidazole, 18 ng / μL aurintricarboxylic acid (ATA), 20 mM bromhexine hydrochloride, 80 mM trehalose, with a specification of 400 μL / T.
[0035] S2. Use the sample treatment solution in step S1 to dilute the national reference product L3 (influenza A virus H1N1) to 5×10^4 Copies / mL, use the diluted enzyme-free water as a negative control, and place it at room temperature (24 - 30 °C) for 3 - 5 minutes to release nucleic acids, obtaining a lysis product.
[0036] S3. Add 10 μL of the lysis product in step S2 to the nucleic acid amplification reaction solution that has been pre-aliquoted 40 μL in an 8-well tube, with a final volume of 50 μL. After mixing, use a metal bath or water bath to incubate at a constant temperature of 37 °C for 20 min to obtain an amplification product.
[0037] S4. Take 10 μL of the amplification product in step S3 to the CRISPR reaction solution that has been pre-aliquoted 50 μL in an 8-well tube, with a final volume of 60 μL. After incubating in a metal bath at 37 °C for 12 min, use an enzyme-labeled instrument to collect the fluorescence value.
[0038] S5. Use the detection kit of Da'an Gene (Influenza A virus, Influenza B virus and novel coronavirus nucleic acid detection kit - PCR fluorescence probe method, product number DA1042) to detect the lysis product in step S2, compare the influence of the sample treatment solution in different detection methods, and perform the sample addition process and the upper machine program according to the instructions.
[0039] The influence results of imidazole concentration on the fluorescence value are shown in Table 3 and Figure 1 as follows: Table 3
[0040] The influence of imidazole concentration on the RT-PCR detection results is shown in Table 4 and Figure 2 as follows: Table 4
[0041] The single-factor optimization of the optimal concentration range of imidazole was carried out, and the detection kit of Da'an Gene and the isothermal amplification-CRISPR reagent product in the present invention were used for testing. The results all showed that compared with the condition without adding imidazole, the sample treatment solution added with an appropriate amount of imidazole could accelerate the deformation of the sample protein shell, promote the rapid and sufficient release of nucleic acid, and thus have a higher lysis efficiency. The optimal concentration could be 10 - 90 mM, and 50 mM was the best.
[0042] Example 2 S1. Prepare sample treatment solutions with different concentrations of sodium dodecyl sulfate (SDS), including 300 mM Tris-HCl (pH 8.6), 120 mM guanidine hydrochloride, 1.3% DMSO, 50 mM imidazole, 0 - 0.3 (w / v)% SDS, 18 ng / μL aurintricarboxylic acid (ATA), 20 mM bromhexine hydrochloride, 80 mM trehalose, and the specification is 400 μL / T.
[0043] S2. Dilute the national reference product L3 (influenza A virus H1N1) to 2×10^4 Copies / mL with the sample treatment solution in step S1, use the diluted enzyme-free water as a negative control, and place it at room temperature (24 - 30 °C) for 3 - 5 minutes to release nucleic acid to obtain a lysis product.
[0044] S3. Add 10 μL of the lysis product in step S2 to the nucleic acid amplification reaction solution that has been pre-aliquoted 40 μL in an 8-well tube, with a final volume of 50 μL. After mixing, incubate at a constant temperature of 37 °C for 20 min using a metal bath or a water bath to obtain an amplification product.
[0045] S4. Take 10 μL of the amplification product in step S3 to the CRISPR reaction solution that has been pre-aliquoted 50 μL in an 8-well tube, with a final volume of 60 μL. After incubating at 37 °C for 12 min in a metal bath, use an enzyme-labeled instrument to collect the fluorescence value.
[0046] The influence results of SDS concentration on the fluorescence value are shown in Table 5 and Figure 3 as follows: Table 5
[0047] As a protein denaturant, SDS can play a promoting role in the lysis and release of nucleic acid from viruses. The single-factor optimization of SDS concentration was carried out using isothermal amplification-CRISPR. The results showed that within the range of 0 < SDS ≤ 0.2%, the lysis efficiency of the lysis solution could be improved, and the optimal was 0.1%. Too high a concentration would inhibit isothermal amplification and instead affect the detection performance.
[0048] Example 3 Stability test of the sample treatment solution S1. Prepare three sample treatment solutions. Sample treatment solution A contains 300 mM Tris-HCl (pH 8.6), 120 mM guanidine hydrochloride, 1.3% DMSO, 50 mM imidazole, 18 ng / μL aurintricarboxylic acid (ATA), 20 mM bromhexine hydrochloride, and 80 mM trehalose, with a specification of 400 μL / T. Compared with sample treatment solution A, sample treatment solution B does not include imidazole and has an increased 0.1 (w / v)% SDS. Compared with sample treatment solution A, sample treatment solution C has an increased 0.1 (w / v)% SDS. Place sample treatment solutions A, B, and C at 45 °C for several days respectively.
[0049] S2. After restoring the sample treatment solutions in step S1 to room temperature, dilute the national reference product L3 (influenza A virus H1N1) to 2×10^4 Copies / mL, use diluted enzyme-free water as a negative control, and place it at room temperature (24 - 30 °C) for 3 - 5 minutes to release nucleic acids.
[0050] S3. Add 10 μL of the lysis product in step S2 to the nucleic acid amplification reaction solution that has been pre-aliquoted at 40 μL in an 8-well strip tube, with a final volume of 50 μL. After mixing, incubate at a constant temperature of 37 °C for 20 min using a metal bath or a water bath.
[0051] S4. Take 10 μL of the amplification product in step S3 to the CRISPR reaction solution that has been pre-aliquoted at 50 μL in an 8-well strip tube, with a final volume of 60 μL. Incubate at 37 °C in a metal bath for 12 min, and then use an enzyme-linked immunosorbent assay (ELISA) reader to collect the fluorescence value.
[0052] The results are shown in Table 6 and Figure 4 as follows: Table 6
[0053] The results of the high-temperature accelerated stability test show that the combination of 50 mM imidazole and 0.1 (w / v)% SDS in the sample treatment solution can be stored at 45 °C for at least 8 days, and the reagent stability is better.
[0054] Example 4 Application test of the sample treatment solution of this application in the CRISPR detection of respiratory pathogen nucleic acids.
[0055] S1. Use sample treatment solution C in Example 3 to dilute the mixture of influenza A virus H3N2 and H1N1 and influenza B virus (Yamagata) to 2×10^4 Copies / mL. Both samples are replicated twice, use diluted enzyme-free water as a negative control, and place it at room temperature (24 - 30 °C) for 3 - 5 minutes to release nucleic acids to obtain the lysis product.
[0056] S2. Add 10 μL of the lysate from step S1 to the nucleic acid amplification reaction solution that has been pre-aliquoted at 40 μL in an 8-strip tube, with a final volume of 50 μL. After mixing, incubate at a constant temperature of 37 °C for 20 min using a metal bath or a water bath to obtain the amplification product.
[0057] S3. Take 10 μL of the amplification product from step S2 and add it to the CRISPR reaction solution that has been pre-aliquoted at 50 μL in an 8-strip tube, with a final volume of 60 μL. Incubate at 37 °C in a metal bath for 12 min, and then use a microplate reader to collect the fluorescence values.
[0058] The results are shown in Table 7 and Figure 5 as follows: Table 7
[0059] The results show that after the virus sample is lysed and directly amplified, it is judged to be positive by the highly specific CRISPR cleavage system. The overall detection time is 35 min. The virus sample does not require nucleic acid purification. The advantages of lysis and direct amplification and short detection time enable it to have a wider range of application scenarios.
[0060] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A sample treatment solution for nucleic acid release, characterized in that, Comprising: A buffer solution, guanidine hydrochloride or guanidine isothiocyanate, and imidazole; Wherein, the concentration of imidazole is greater than 0 and less than or equal to 90 mmol / L.
2. The sample treatment solution for nucleic acid release according to claim 1, wherein The concentration of imidazole is 50 - 70 mmol / L.
3. The sample processing solution for nucleic acid release according to claim 1, wherein The concentration of the buffer solution is 50 - 500 mmol / L; and / or, The concentration of guanidine hydrochloride or guanidine isothiocyanate is 50 - 200 mmol / L.
4. The sample treatment solution for nucleic acid release according to claim 3, characterized in that, The concentration of guanidine hydrochloride or guanidine isothiocyanate is 100 - 150 mmol / L.
5. The sample treatment solution for nucleic acid release according to any one of claims 1-4, characterized in that, Also includes sodium dodecyl sulfate.
6. The sample processing solution for nucleic acid release according to claim 5, wherein The mass percentage concentration of sodium dodecyl sulfate is 0.001 - 0.2%.
7. The sample treatment solution for nucleic acid release according to claim 1, wherein, The buffer solution is any one of Tris-HCl buffer solution, boric acid buffer solution, HEPES buffer solution; and / or, The pH of the buffer solution is 8.0 - 8.
6.
8. The sample processing solution for nucleic acid release according to any one of claims 1-4, 6-7, characterized in that Also includes any one or more of a polar organic solvent, a mucolytic agent, and a nucleic acid protecting agent; The mass percentage concentration of the polar organic solvent is 0.1 - 3%; The concentration of the mucolytic agent is 10 - 30 mmol / L; The concentration of the nucleic acid protecting agent is 30 - 200 mmol / L.
9. The sample treatment solution for nucleic acid release according to claim 8, wherein, The polar organic solvent is any one or more of dimethyl sulfoxide, hexamethylphosphoramide, and tetramethylethylenediamine; The mucolytic agent is any one or more of bromhexine hydrochloride, ambroxol, acetylcysteine, and erdosteine; The nucleic acid protecting agent is any one or more of trehalose, sucrose, RNase inhibitor, and aurintricarboxylic acid.
10. A detection kit, characterized in that, Comprising the sample treatment solution for nucleic acid release according to any one of claims 1 - 9.