An alcohol-free, proteinase K-free nucleic acid extraction reagent and a rapid nucleic acid extraction method

Abstract

The application provides an alcohol-free and proteinase K-free nucleic acid extraction reagent and a nucleic acid rapid extraction method, and relates to the technical field of molecules.The extraction reagent comprises a lysis solution, and the lysis solution comprises guanidine isothiocyanate with a molar concentration of 2-4 M, a thiol reducing agent with a volume fraction of 1%-5% v / v, sodium erythorbate with a mass fraction of 0.02%-0.5% w / v and a surfactant with a mass fraction of 1%-10% w / v; and the lysis solution has a pH of 7-8.5.The lysis solution can be stably stored at room temperature for a long time; in application, only one washing solution is matched, one-step washing is performed, the complexity of nucleic acid extraction can be reduced, and the extraction time is shortened; and the lysis solution does not contain alcohol reagents, and there is no need to dry before nucleic acid elution.The technical problems that, in the prior art, the lysis solution has complex components, proteinase K needs to be matched, many impurities are introduced, multiple washing is needed, the steps are complicated, and a long time is consumed are solved.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to an alcohol-free, proteinase K-free nucleic acid extraction reagent and a rapid nucleic acid extraction method. Background Technology

[0002] Nucleic acid extraction is the most fundamental step in molecular biology techniques and methods, and it also has a crucial impact on the sensitivity and accuracy of subsequent nucleic acid detection. The purpose of nucleic acid extraction is to efficiently isolate DNA or RNA from various types of biological samples. Currently, nucleic acid extraction reagents based on magnetic beads typically include a lysis buffer, at least two washing buffers, an elution buffer, and magnetic beads. Proteinase K is often added to remove interference from proteins in the sample. The addition of proteinase K introduces more impurities during nucleic acid extraction, so multiple washings are required. The washing buffer usually contains alcohols to enhance impurity removal. The entire process is cumbersome, complex, and time-consuming.

[0003] For example, Chinese patent application number 202410691882.4 discloses a rapid nucleic acid extraction reagent, a nucleic acid extraction method, and its application. The rapid nucleic acid extraction reagent includes a protein denaturant and a protein solubilizer. When preparing nucleic acids, centrifugation is sufficient, eliminating the need for washing and purification steps, making the extraction process simple and fast. However, it requires the use of proteinase K and cannot be stably stored at room temperature; it also requires a centrifuge, making automated nucleic acid extraction impossible. Chinese patent application number 202110657003.2 discloses a rapid viral genome nucleic acid extraction kit and its usage method. The kit consists of solution I, solution II, solution III, Eluent solution, and a preparation tube with a silica membrane column. It eliminates the need for phenol-chloroform or Trizol extraction; it can simultaneously extract trace amounts of DNA / RNA from any liquid; the kit has high nucleic acid extraction efficiency, superior to the Trizol method, with minimal nucleic acid loss during extraction; and the extracted DNA / RNA has high purity. However, its drawback is that the reagent contains isopropanol and ethanol, which can easily cause alcohol residues in the extraction product, inhibiting downstream applications. Extraction using silica membrane columns requires a centrifuge, which is not conducive to automated nucleic acid extraction. For example, Chinese patent application number 202410404302.9 discloses a rapid nucleic acid extraction method and kit for biological samples based on magnetic beads, solving the problem of difficult digestion of sputum samples. However, the reagent also contains alcohols, which can easily lead to alcohol residues in the extracted product, inhibiting downstream applications. The lysis time is long, washing needs to be repeated multiple times, and drying requires a long time due to the alcohol content, resulting in a multi-step and time-consuming extraction process. It is only suitable for sputum samples; for other types of samples, the extraction speed is slow.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide an alcohol-free, proteinase K-free nucleic acid extraction reagent to solve the technical problems in the prior art, which are that the lysis buffer components are relatively complex and require the addition of proteinase K, thus introducing more impurities, requiring multiple washings, and involving cumbersome and time-consuming steps.

[0006] The second objective of this invention is to provide a rapid nucleic acid extraction method.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides an alcohol-free proteinase K-free nucleic acid extraction reagent, the extraction reagent comprising a lysis buffer comprising guanidine isothiocyanate at a molar concentration of 2-4 M, a thiol reducing agent at 1%-5% v / v, sodium isoascorbate at 0.02%-0.5% w / v, and a surfactant at 1%-10% w / v. The pH of the lysis solution is 7-8.5.

[0008] Furthermore, the lysis buffer also includes a buffer solution; Preferably, the buffer solution is a 50-200 mM Tris-HCl buffer or a Tricine buffer.

[0009] Furthermore, the thiol reducing agent includes at least one of thioglycerol, hydroxylamine hydrochloride, DTE or DTT, preferably thioglycerol; Furthermore, the surfactant includes at least one of Brij35, Triton X-100, or NP40.

[0010] Furthermore, the extraction reagent also includes a washing solution, an elution solution, and magnetic beads; Preferably, the washing solution comprises 1%~5% w / v trisodium citrate dihydrate and 0.01%~0.05% w / v preservative; Preferably, the pH of the washing solution is 5-6; Preferably, the preservative is P-300.

[0011] Furthermore, the eluent comprises Tris-HCl with a molar concentration of 5-15 mM and sodium azide with a volume percentage of 0.01%-0.05% v / v; Preferably, the magnetic beads are silanol magnetic beads.

[0012] Thirdly, the present invention provides a rapid nucleic acid extraction method, the method using the aforementioned nucleic acid extraction reagent, comprising the following steps: A. Mix the sample with the lysis buffer and magnetic bead suspension for lysis, magnetically attract the sample, discard the liquid, and add washing solution to clean the magnetic beads; B. Elute the magnetic beads obtained in step A with elution buffer, and collect the liquid to obtain the nucleic acid extraction product.

[0013] Furthermore, the amount of the lysis buffer used is 1.5 to 2 times the sample volume; Preferably, the concentration of the magnetic bead suspension is 5~20 mg / ml; Preferably, the amount of the magnetic bead suspension is 25~150μl, more preferably 50μl; Preferably, the pyrolysis includes pyrolysis at 20~50℃ for 2~5 minutes, more preferably pyrolysis at 37℃ for 2 minutes; Preferably, the magnetic attraction time in step A is 20~60s, more preferably 30s; Preferably, the step of adding washing liquid to clean the magnetic beads includes adding washing liquid, mixing evenly, magnetically attracting the beads, and then discarding the liquid; Preferably, the amount of the washing solution used is 0.6~1.2ml, more preferably 1ml; Preferably, the time for magnetic attraction after uniform mixing is 20-60 seconds, and more preferably 30 seconds.

[0014] Furthermore, step B includes adding eluent, mixing thoroughly, incubating for elution, magnetically aspirating, and removing the liquid. Preferably, the amount of the eluent is 50~150 μl; Preferably, the temperature for the incubation and elution is 50~80℃; Preferably, the incubation and elution time is 1-5 minutes, more preferably 3 minutes; Preferably, the magnetic attraction time in step B is 20~60s, more preferably 30s.

[0015] Furthermore, the sample includes at least one of swab washing fluid, serum, plasma, or bronchoalveolar lavage fluid.

[0016] This invention provides an alcohol-free, proteinase K-free nucleic acid extraction reagent. The lysis buffer contains a thiol reducing agent and sodium isoascorbate as reducing agents, while maintaining a slightly alkaline pH. It achieves effective extraction of various sample types without the need for proteinase K or alcohols. It can be stably stored at room temperature for extended periods. In application, only one washing solution is used, requiring only one washing step, reducing the complexity of nucleic acid extraction and shortening the extraction time. Since neither the lysis buffer nor the washing solution contains alcohols, drying before nucleic acid elution is unnecessary, avoiding the risk of alcohol residue inhibiting subsequent nucleic acid amplification. This invention solves the technical problems of existing technologies where the lysis buffer composition is complex, requiring proteinase K, introducing numerous impurities, necessitating multiple washing steps, and resulting in cumbersome and time-consuming processes. Detailed Implementation Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0017] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The present invention provides an alcohol-free proteinase K-free nucleic acid extraction reagent, wherein the extraction reagent comprises a lysis buffer, the lysis buffer comprising guanidine isothiocyanate at a molar concentration of 2-4 M, a thiol reducing agent at 1%-5% v / v, sodium isoascorbate at 0.02%-0.5% w / v, and a surfactant at 1%-10% w / v. The pH of the lysis solution is 7-8.5.

[0020] The lysis buffer contains thiol reducing agents and sodium isoascorbate as reducing agents, while maintaining a slightly alkaline pH. This allows for effective extraction of various sample types without the need for proteinase K or alcohols. It can be stably stored at room temperature for extended periods. Only one washing buffer is used, requiring only one washing step, reducing the complexity and time of nucleic acid extraction. The alcohol-free lysis buffer eliminates the need for drying before nucleic acid elution, avoiding the risk of alcohol residue inhibiting subsequent nucleic acid amplification. This solution overcomes the technical problems of existing technologies where the lysis buffer composition is complex, requiring proteinase K and thus introducing numerous impurities, necessitating multiple washing steps, and resulting in cumbersome and time-consuming processes.

[0021] Because the lysis buffer has a strong ability to lyse and remove impurities, lysis can be performed at room temperature without heating, shortening the lysis time. Furthermore, only one washing is required, significantly reducing the total time of the nucleic acid extraction process. This allows for rapid nucleic acid extraction with high yield and purity. When used with magnetic beads, centrifugation is unnecessary, enabling large-scale rapid nucleic acid extraction and detection on fully automated instruments.

[0022] The molar concentration of guanidine isothiocyanate in the lysis solution can be, but is not limited to, 2M, 2.2M, 2.4M, 2.6M, 2.8M, 3M, 3.2M, 3.4M, 3.6M, 3.8M or 4M, or any value between 2 and 4M.

[0023] The volume percentage of the thiol reducing agent in the lysis solution can be, but is not limited to, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, or any value between 1% and 5%.

[0024] The weight-to-volume ratio of sodium isoascorbate in the lysis solution can be, but is not limited to, 0.02%, 0.05%, 0.1%, 0.12%, 0.15%, 0.2%, 0.22%, 0.25%, 0.3%, 0.32%, 0.35%, 0.4%, 0.42%, 0.45%, or 0.5%, or any value between 0.02% and 0.5%.

[0025] The surfactant in the lysis solution can be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or any value between 1% and 10%.

[0026] The pH of the lysis solution can be, but is not limited to, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4 or 8.5, or any value between 7 and 8.5.

[0027] In some specific embodiments, the lysis buffer further includes a buffer solution; in some specific embodiments, the buffer solution is a 50-200 mM Tris-HCl buffer or a Tricine buffer.

[0028] The molar concentration of the Tris-HCl buffer solution can be, but is not limited to, 50mM, 70mM, 90mM, 100mM, 120mM, 140mM, 160mM, 180mM or 200mM, or any value between 50 and 200mM.

[0029] In some specific embodiments, the thioglycerol reducing agent includes at least one of thioglycerol, hydroxylamine hydrochloride, DTE or DTT, preferably thioglycerol.

[0030] Among them, thiol reducing agents refer to a class of compounds containing thiol (-SH, also known as thiohydrogen group) functional groups and having strong reducing properties. They exert their reducing effect through the chemical activity of the thiol group.

[0031] In some specific embodiments, the surfactant includes at least one of Brij35, Triton X-100, or NP40.

[0032] In some specific implementations, it also includes a washing solution, an eluent, and magnetic beads.

[0033] In some specific embodiments, the washing solution includes 1%~5% w / v trisodium citrate dihydrate and 0.01%~0.05% w / v preservative; in some specific embodiments, the pH of the washing solution is 5~6; in some specific embodiments, the preservative is P-300.

[0034] In some specific embodiments, the eluent comprises Tris-HCl at a molar concentration of 5-15 mM and sodium azide at 0.01%-0.05% v / v.

[0035] In some specific embodiments, the magnetic beads are siloxane magnetic beads.

[0036] According to another aspect of the present invention, a rapid nucleic acid extraction method is also provided, the method using the above-described nucleic acid extraction reagent, comprising the following steps: A. Mix the sample with the lysis buffer and magnetic bead suspension for lysis, magnetically attract the sample, discard the liquid, and add washing solution to clean the magnetic beads; B. Elute the magnetic beads obtained in step A with elution buffer, and collect the liquid to obtain the nucleic acid extraction product.

[0037] In some specific embodiments, the amount of the lysis buffer is 1.5 to 2 times the sample volume; in some specific embodiments, the concentration of the magnetic bead suspension is 5 to 20 mg / ml; in some specific embodiments, the amount of the magnetic bead suspension is 25 to 100 μl, preferably 50 μl.

[0038] In some specific embodiments, the pyrolysis includes pyrolysis at 20~50℃ for 2~5 minutes, preferably pyrolysis at 37℃ for 2 minutes; in some specific embodiments, the magnetic attraction time in step A is 20~60 seconds, preferably 30 seconds.

[0039] In some specific embodiments, the pyrolysis process lasts for 2.5 minutes. In some specific embodiments, the step of adding washing solution to clean the magnetic beads includes adding washing solution, mixing evenly, magnetically attracting the beads, and then discarding the liquid.

[0040] In some specific embodiments, the amount of the washing liquid is 0.6~1.2ml, preferably 1ml; in some specific embodiments, the magnetic adsorption time after mixing is 20~60s, preferably 30s.

[0041] In some specific embodiments, step B includes adding eluent, mixing evenly, incubating for elution, magnetically aspirating, and collecting the liquid.

[0042] In some specific embodiments, the amount of the eluent used is 50~150 μl.

[0043] In some specific embodiments, the temperature for incubation and elution is 50~80℃; in some specific embodiments, the time for incubation and elution is 1~5min, preferably 3min.

[0044] In some specific implementations, the magnetic attraction time in step B is 20~60s, preferably 30s.

[0045] In some specific implementations, the total time for the nucleic acid extraction process is 5 minutes.

[0046] In some specific embodiments, the sample includes at least one of swab washing fluid, serum, plasma, or bronchoalveolar lavage fluid.

[0047] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0048] I. A lysis buffer for nucleic acid extraction The components and amounts of the lysis buffers in each embodiment and comparative example are shown in Tables 1 to 3.

[0049] Table 1

[0050] Table 2

[0051] Table 3

[0052] II. Nucleic Acid Extraction Reagents Examples 6-10 A nucleic acid extraction reagent includes a lysis buffer, a washing buffer, an elution buffer, and magnetic beads, wherein the lysis buffer is selected from the lysis buffers described in Examples 1 to 5; The washing solution consists of 1% w / v trisodium citrate dihydrate and 0.01% v / v P-300, with the pH adjusted to 5.0.

[0053] The eluent consists of 5 mM Tris-HCl (pH 8.0) and 0.01% v / v sodium azide.

[0054] The magnetic beads used are silanol magnetic beads, and the concentration of the magnetic bead suspension is 5 mg / ml.

[0055] Example 11 Unlike Example 7, the washing solution included 5% w / v trisodium citrate dihydrate and 0.05% v / v P-300, with the pH adjusted to 6.0.

[0056] The eluent consists of 15 mM Tris-HCl (pH 8.8) and 0.05% v / v sodium azide.

[0057] The magnetic beads used are silanol magnetic beads, and the concentration of the magnetic bead suspension is 20 mg / ml.

[0058] Example 12 Unlike Example 7, the washing solution included 3% w / v trisodium citrate dihydrate and 0.03% v / v P-300, with the pH adjusted to 5.5.

[0059] The eluent consists of 10 mM Tris-HCl (pH 8.5) and 0.03% v / v sodium azide.

[0060] The magnetic beads used are silanol magnetic beads, and the concentration of the magnetic bead suspension is 10 mg / ml.

[0061] Comparative Examples 11-20 Unlike Example 7, the lysis buffer used was selected from Comparative Examples 1 to 10.

[0062] Comparative Example 21 Unlike Example 7, the lysis buffer used is the lysis buffer from Example 1 of the rapid extraction kit for viral genome nucleic acid and its usage method disclosed in patent application number 202110657003.2.

[0063] Comparative Example 22 Unlike Example 7, the washing solution is a 70% ethanol solution.

[0064] III. Nucleic Acid Extraction Examples 13-19 A rapid nucleic acid extraction method is provided, comprising plasma sample 1 from a clinically confirmed HBV patient with a concentration of 1000 IU / mL and plasma sample 2 from a clinically confirmed HBV patient with a concentration of 100 IU / mL, using the nucleic acid extraction reagents provided in Examples 6-12 respectively, and specifically following the steps below: (1) Take 400 μL of sample, add 800 μL of lysis buffer and 100 μL of silanol magnetic bead suspension with a particle size of 100~300 nm, and heat at 37°C for 2 minutes. (2) Place the centrifuge tube on the magnetic rack, let it adhere to the magnet for 30 seconds, and then discard the liquid; (3) Add 1 mL of cleaning solution to the centrifuge tube, mix by blowing or shaking, magnetically remove for 30 s, and discard the liquid; (4) Add 100 μL of elution buffer, mix by blowing or shaking, incubate at 50°C for 3 min, magnetically aspirate for 30 s, and aspirate the liquid to obtain the nucleic acid extraction product.

[0065] Example 20 Unlike Example 13, the samples were plasma samples from clinically confirmed HBV patients at two different concentrations (1000 IU / mL, 100 IU / mL), and the procedures were performed as follows: (1) Take 200 μL of sample, add 400 μL of lysis buffer and 25 μL of silanol magnetic bead suspension, and lyse at 20°C for 2 minutes; (2) Place the centrifuge tube on the magnetic rack, let it adhere to the magnet for 20 seconds, and then discard the liquid; (3) Add 0.6 mL of washing solution to the centrifuge tube, mix by blowing or shaking, magnetically remove for 20 s, and discard the liquid; (4) Add 50 μL of elution buffer, mix by blowing or shaking, heat at 80°C for 1 min, magnetically aspirate for 20 s, and aspirate the liquid to obtain the nucleic acid extraction product.

[0066] Example 21 Unlike Example 13, (1) 600 μL of sample was taken, 900 μL of lysis buffer and 100 μL of silanol magnetic bead suspension were added, and the mixture was heated at 37°C for 2 minutes. (2) Place the centrifuge tube on the magnetic rack, hold it magnetically for 60 seconds, and then discard the liquid; (3) Add 1.2 mL of washing solution to the centrifuge tube, mix by blowing or shaking, magnetically remove for 60 s, and discard the liquid; (4) Add 150 μL of elution buffer, mix by blowing or shaking, incubate at 65°C for 3 min, magnetically aspirate for 60 s, and aspirate the liquid to obtain the nucleic acid extraction product.

[0067] Comparative Examples 23-33 Unlike Example 13, the nucleic acid extraction reagents provided in Comparative Examples 11-21 were used.

[0068] Comparative Example 34 Unlike Example 20, in step (1), 200 μL of sample was taken, 400 μL of lysis buffer and 100 μL of silanol magnetic bead suspension were added, and then 30 μL of proteinase K was added. The mixture was heated at 50°C for 5 minutes.

[0069] Comparative Example 35 Unlike Example 13, in step (3), the washing solution is used to repeat the cleaning process twice.

[0070] Comparative Example 36 Unlike Example 13, the nucleic acid extraction reagent of Comparative Example 22 was used. In step (3), a washing solution containing 70% ethanol was used. After washing twice, the lid was opened and the container was air-dried for 5 minutes (to allow the residual ethanol to fully evaporate).

[0071] Experiment 1: Detection of Nucleic Acid Extraction Products Detection method: Take 50 μL of the extracted nucleic acid as a template and perform qPCR amplification according to the instructions of the Hepatitis B Virus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method) (National Medical Device Registration Certificate No. 20253400064). The reaction program is 50℃, 2 min; 95℃, 2 min; 95℃, 10 s; 60℃, 22 s (45 cycles).

[0072] 1. The effects of different lysis buffer formulations were compared, and the results of PCR amplification detection are shown in Table 4.

[0073] Table 4

[0074] Table 4 shows that the Ct values ​​of each embodiment in both samples were lower than those in the comparative examples, indicating that the HBV virus structure was sufficiently destroyed and the nucleic acid was completely released. Compared with Example 14, Comparative Example 23 removed the thiol reducing agent, and Comparative Example 25 removed sodium isoascorbate. The target Ct values ​​of the two lysis buffer formulations were significantly delayed (>1 Ct value), indicating that the thiol reducing agent and sodium isoascorbate had a synergistic effect on nucleic acid release in this scheme. The Ct value of Comparative Example 24 increased (>1 Ct value), indicating that when the pH of the lysis buffer was acidic, the HBV virus structure was not sufficiently destroyed and the nucleic acid was not completely released, resulting in a delayed Ct value during amplification. Although the Ct values ​​of Comparative Examples 26-29 were lower than those of Comparative Examples 23-25, they were still higher than those of Example 14, indicating that the 1%~5% v / v thiol reducing agent and 0.02%~0.5% w / v sodium isoascorbate had a synergistic effect in this scheme. Different reducing agents and guanidine salts were used in Comparative Examples 30-32, but their Ct values ​​were still higher than those in Example 14. This shows that not all reducing agents and guanidine salts have the same effect on nucleic acid release. Comparative Example 33 is an existing lysis buffer containing alcohols, salt ions, metal chelating agents, and other reagents. Compared with Comparative Example 33, this scheme has a simpler composition and a lower Ct value.

[0075] 2. The effect of proteinase K on nucleic acid extraction efficiency and the results of PCR amplification detection are shown in Table 5.

[0076] Table 5

[0077] No significant difference was found; P-value > 0.05.

[0078] As shown in Table 5, the Ct value of the lysis buffer in the examples was not significantly different when it was not combined with proteinase K compared with that when it was combined with proteinase K. This indicates that the lysis buffer in the examples helps to lyse the sample and release nucleic acids, improves the efficiency of nucleic acid extraction, and can replace the auxiliary role of proteinase K in nucleic acid extraction.

[0079] 3. The results of PCR amplification detection are shown in Table 6, comparing the effects of one-step washing and two-step washing with extraction reagents, as well as washing with alcohol-containing solutions.

[0080] Table 6

[0081] Where ** indicates P < 0.01.

[0082] Compared with Example 11, Comparative Example 35 was washed twice and its Ct value did not change significantly, indicating that the reagent washing method can achieve the best effect with one wash. Comparative Example 36 used an alcohol-containing washing solution, but its Ct value increased, indicating that the alcohol-containing washing solution had a reverse effect on nucleic acid extraction.

[0083] Experiment 2: Stability of nucleic acid extraction reagents stored at room temperature Pharyngeal swab washing fluid from 5 patients clinically diagnosed with influenza A (numbered 1-5) was collected and used with nucleic acid extraction reagents stored at room temperature for 0 days, 1 month, 3 months, 6 months, 9 months, and 12 months, respectively, for nucleic acid extraction. The nucleic acid extraction reagents used in this experiment were those from Examples 6-12 and Comparative Examples 11-22, respectively. The specific procedures were performed as follows: (1) Take 200 μL of sample, add 400 μL of lysis buffer and 100 μL of silanol magnetic bead suspension, and heat at 37°C for 2 minutes; (2) Place the centrifuge tube on the magnetic rack, let it adhere to the magnet for 30 seconds, and then discard the liquid; (3) Add 1 mL of cleaning solution to the centrifuge tube, mix well, magnetically aspirate for 30 s, and discard the liquid; (4) Add 100 μL of elution buffer, mix by pipetting or shaking, incubate at 80℃ for 3 min, magnetically aspirate for 30 s, and aspirate the liquid to obtain the nucleic acid extraction product. Use 50 μL of the extracted nucleic acid as a template for qPCR amplification according to the instructions of the Influenza A Virus / Influenza B Virus / Respiratory Syncytial Virus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method) (National Medical Device Registration Certificate 20223401421). The reaction program is 50℃, 2 min; 60℃, 30 min; 95℃, 2 min; 95℃, 10 s; 60℃, 22 s (45 cycles). The PCR amplification results are shown in Table 7.

[0084] Table 7

[0085] Experimental results show that the performance of the nucleic acid extraction reagent in this invention did not change significantly after storage at room temperature for 0–12 months. This reagent exhibits good room temperature storage stability, making it more convenient for long-term storage and transportation. However, the reduced content of thiol reducing agent in Comparative Example 27 and the reduced content of sodium isoascorbate in Comparative Example 28 negatively impacted the stability of the nucleic acid extraction reagent, with Comparative Example 28 showing a significant change in stability (Ct value decreased by >1). This indicates that the synergistic effect of the thiol reducing agent and sodium isoascorbate not only promotes complete nucleic acid release but also positively contributes to the stability of the nucleic acid extraction reagent.

[0086] Example 22 Nucleic acid extraction from serum and bronchoalveolar lavage fluid samples This embodiment evaluates the nucleic acid extraction efficiency of the nucleic acid extraction reagent on serum and bronchoalveolar lavage fluid samples. Serum samples (numbered 1-3) from 3 patients clinically diagnosed with hepatitis C virus infection and bronchoalveolar lavage fluid samples (numbered 4-6) from 3 patients clinically diagnosed with Mycobacterium tuberculosis infection were collected. Nucleic acid extraction was performed using the nucleic acid extraction reagent described in Example 6, and the specific procedures were as follows: (1) Take 200 μL of sample, add 400 μL of lysis buffer and 100 μL of silanol magnetic bead suspension, and heat at 37°C for 2 minutes; (2) Place the centrifuge tube on the magnetic rack, let it adhere to the magnet for 30 seconds, and then discard the liquid; (3) Add 1 mL of cleaning solution to the centrifuge tube, mix well, magnetically aspirate for 30 s, and discard the liquid; (4) Add 100 μL of elution buffer, mix by pipetting or shaking, incubate at 80℃ for 3 min, magnetically aspirate for 30 s, and aspirate the liquid to obtain the nucleic acid extraction product. Use 50 μL of the extracted nucleic acid as a template and perform qPCR amplification according to the instructions of the Hepatitis C Virus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method) (National Medical Device Registration Certificate 20253400497) and the Mycobacterium tuberculosis Complex Nucleic Acid Detection Kit (Patent Application Publication No.: CN 114836554 A). The reaction program is 50℃, 2 min; 60℃, 30 min; 95℃, 2 min; 95℃, 10 s; 60℃, 22 s (45 cycles). The PCR amplification results are shown in Table 8.

[0087] Table 8

[0088] The results showed that the nucleic acid extraction reagent of Example 6 extracted serum and bronchoalveolar lavage fluid samples, and the PCR test results were positive, consistent with the clinical results. This indicates that the nucleic acid extraction reagent of the present invention can achieve nucleic acid extraction from serum and bronchoalveolar lavage fluid samples.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alcohol-free, proteinase K-free nucleic acid extraction reagent, characterized in that, The extraction reagent includes a lysis buffer, which comprises guanidine isothiocyanate at a molar concentration of 2-4 M, a mercapto reducing agent at 1%-5% v / v, sodium isoascorbate at 0.02%-0.5% w / v, and a surfactant at 1%-10% w / v. The pH of the lysis solution is 7-8.

5.

2. The extraction reagent according to claim 1, characterized in that, The lysis buffer also includes a buffer solution; Preferably, the buffer solution is a 50-200 mM Tris-HCl buffer or a Tricine buffer.

3. The extraction reagent according to claim 1, characterized in that, The thioglycolic reducing agent includes at least one of thioglycerol, hydroxylamine hydrochloride, DTE or DTT, preferably thioglycerol.

4. The extraction reagent according to claim 1, characterized in that, The surfactant includes at least one of Brij35, Triton X-100, or NP40.

5. The extraction reagent according to claim 4, characterized in that, The extraction reagent also includes washing solution, elution solution and magnetic beads; Preferably, the washing solution comprises 1%~5% w / v trisodium citrate dihydrate and 0.01%~0.05% w / v preservative; Preferably, the pH of the washing solution is 5-6; Preferably, the preservative is P-300.

6. The nucleic acid extraction reagent according to claim 5, characterized in that, The eluent comprises Tris-HCl at a molar concentration of 5-15 mM and sodium azide at a molar concentration of 0.01%-0.05% v / v; Preferably, the magnetic beads are silanol magnetic beads.

7. A rapid nucleic acid extraction method, characterized in that, The method uses the nucleic acid extraction reagent according to any one of claims 1 to 6, and includes the following steps: A. Mix the sample with the lysis buffer and magnetic bead suspension for lysis, magnetically attract the sample, discard the liquid, and add washing solution to clean the magnetic beads; B. Elute the magnetic beads obtained in step A with elution buffer, and collect the liquid to obtain the nucleic acid extraction product.

8. The rapid nucleic acid extraction method according to claim 7, characterized in that, The amount of lysis buffer used is 1.5 to 2 times the sample volume; Preferably, the concentration of the magnetic bead suspension is 5~20 mg / ml; Preferably, the amount of the magnetic bead suspension is 25~150μl, more preferably 100μl; Preferably, the pyrolysis includes pyrolysis at 20~50℃ for 2~5 minutes, more preferably pyrolysis at 37℃ for 2 minutes; Preferably, the magnetic attraction time in step A is 20~60s, more preferably 30s; Preferably, the step of adding washing liquid to clean the magnetic beads includes adding washing liquid, mixing evenly, magnetically attracting the beads, and then discarding the liquid; Preferably, the amount of the washing solution used is 0.6~1.2ml, more preferably 1ml; Preferably, the time for magnetic attraction after uniform mixing is 20-60 seconds, and more preferably 30 seconds.

9. The rapid nucleic acid extraction method according to claim 7, characterized in that, Step B includes adding elution buffer, mixing thoroughly, incubating for elution, magnetically aspirating, and removing the liquid. Preferably, the amount of the eluent is 50~150 μl; Preferably, the temperature for the incubation and elution is 50~80℃; Preferably, the incubation and elution time is 1-5 minutes, more preferably 3 minutes; Preferably, the magnetic attraction time in step B is 20~60s, more preferably 30s.

10. The rapid nucleic acid extraction method according to any one of claims 7 to 9, characterized in that, The sample includes at least one of swab washing fluid, serum, plasma, or bronchoalveolar lavage fluid.

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