A nucleic acid release reagent, its preparation method and application in detecting swab samples

Abstract

The application discloses a nucleic acid releasing reagent, a preparation method thereof and application thereof in detection of swab samples, and belongs to the field of molecular biology detection. The nucleic acid releasing reagent comprises a surfactant, a buffer system, a nucleic acid coprecipitation agent, a PCR enhancer and water. The application adopts a low-inhibition surfactant complex system combined with a PCR enhancer double mechanism, so that nucleic acid can be efficiently lysed and released, and the release product can be directly added into a PCR reaction system, and the amplification reaction is not significantly inhibited. Moreover, the reagent can be stored for a long time at room temperature, and when the reagent is used for PCR reaction detection of clinical samples such as throat swabs and nose swabs, heating and centrifugation are not needed, nucleic acid release can be completed by standing at room temperature for 10 minutes, and the release product can be stably stored for more than 4 hours at room temperature, and the reagent has a good detection rate for low-load samples.

Description

Technical Field

[0001] This application relates to a nucleic acid release reagent, its preparation method, and its application in detecting swab samples, belonging to the field of molecular biology detection. Background Technology

[0002] With the rapid development of molecular diagnostic technology, nucleic acid amplification-based detection methods have become the gold standard for pathogen detection. However, the bottleneck in the entire detection process often lies in the nucleic acid extraction and purification step. Traditional methods such as magnetic bead extraction and silica membrane centrifugation column methods are complex, time-consuming (30-60 minutes), and rely on large centrifugation equipment and skilled personnel, making it difficult to meet the rapid response needs of outpatient clinics, emergency rooms, customs, and primary healthcare institutions.

[0003] To simplify the process, "direct amplification" reagents have emerged on the market. The core idea is to eliminate the purification step and add the sample directly to the amplification reaction system. For example, CN112266910A discloses a nucleic acid release agent comprising 1-10% (v / v) polyethylene glycol octylphenyl ether, 1-10% (v / v) ethylphenyl polyethylene glycol, 1-3% (v / v) sorbitan monolaurate, 0.5-500 mM tromethamine, 50-200 mM sodium chloride, 0.1-5 mg / ml proteinase K, 1-10% (v / v) water betaine, 0.1-50 mM ethylenediaminetetraacetic acid, 0.1-5 mM nuclease inhibitor, and 3-10 mM bovine serum albumin. The specific steps of the nucleic acid release method using this release agent are as follows: S1, prepare the nucleic acid release agent; S2, scrape human epidermal and mucous membrane cells and infiltrate them into the nucleic acid release agent; S3, let stand at room temperature for 15 minutes; S4, briefly centrifuge and collect the supernatant to obtain the released nucleic acid. CN115094118A discloses a rapid nucleic acid release agent, comprising the following components: surfactant, Triton X-100, and NaOH. CN113930418A discloses a nucleic acid release agent and its nucleic acid release method. The nucleic acid release agent comprises 0.5-1.5M Tris-HCl, 30-70mM EDTA, 0.5-1.5M cysteine, 10-30U / μL RRI, DEPC water, and NP-40 at a volume percentage concentration of 15-30% and PolyA at a volume percentage concentration of 3-7%, wherein the RRI is an RNase inhibitor. The nucleic acid release agent is equilibrated to room temperature and then shaken well; the shaken nucleic acid release agent is added to a PCR reaction tube; then the sample to be tested is added and mixed by pipetting to obtain the target nucleic acid, wherein the volume ratio of the nucleic acid release agent to the sample to be tested is 1:3. CN112813140A discloses a nucleic acid releasing agent and a method for releasing nucleic acid from it. The nucleic acid releasing agent is composed of 20-100 mM Tris-HCl, 0.01%-0.5% SDS (w / v), 0.04%-0.4% Triton 100 (v), 10500 mM ammonium sulfate, and 0.01%-2% betaine (w / v). The specific steps are as follows: samples from various sample sources are added to the nucleic acid releasing agent in a suitable manner, then shaken to mix, and left at room temperature for 1 min. The supernatant is then aspirated to obtain the released nucleic acid.CN113528508A discloses a nucleic acid releasing agent and a method for nucleic acid release. The nucleic acid releasing agent is composed of the following components: 50-200 mM Tris-HCl, 80-800 mM disodium magnesium ethylenediaminetetraacetate, sodium dodecyl sulfate (SDS) at a mass / volume ratio of 0.02%-2.2%, dithiothreitol (DTT) at a mass / volume ratio of 0.06%-3%, phosphate betaine at a mass / volume ratio of 0.02%-3.2%, and catechol at a mass / volume ratio of 0.08%-1.6%. CN113444771 discloses a lysis agent suitable for direct amplification of DNA or RNA viruses, comprising: Tris-HCl, a dissociative salt, a surfactant, a reducing agent, EDTA, and the RNase inhibitor Rnasin.

[0004] However, existing technologies generally face the following key challenges: First, PCR inhibition: Existing technologies and formulations using ionic detergents and strongly alkaline environments significantly inhibit PCR reactions, requiring post-processing of the lysed products or limiting the amount of lysed products to reduce PCR inhibition. Second, insufficient room temperature stability: Most lysis release reagents need to be stored at low temperatures, and the released nucleic acids are easily degraded by residual nucleases at room temperature, affecting sample preservation and transportation. Third, limited detection rate for low-load samples: Without purification, trace pathogen nucleic acids are difficult to release from a large amount of background material and be effectively captured, resulting in insufficient detection limits.

[0005] Therefore, there is an urgent need to develop a nucleic acid release reagent that can rapidly release nucleic acids at room temperature, maintain nucleic acid stability, and be directly added to the PCR reaction system without affecting subsequent PCR reactions. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a nucleic acid release reagent that is simple to operate, rapid, efficient, and has no inhibitory effect. The nucleic acid release reagent comprises a surfactant, a buffer system, a nucleic acid co-precipitant, a PCR enhancer, and water. This nucleic acid release reagent can be directly applied to swab samples, achieving the release and stabilization of pathogen nucleic acids within minutes. The released solution can be directly used for subsequent nucleic acid amplification and detection without any heating, centrifugation, or purification procedures.

[0007] According to a first aspect of this application, a nucleic acid release reagent is provided.

[0008] A nucleic acid release reagent, the nucleic acid release reagent comprising a surfactant, a buffer system, a nucleic acid coprecipitant, a PCR enhancer, and water.

[0009] Optionally, the surfactant is nonionic, and the surfactant includes polysorbate 20, polyoxyethylene lauryl ether, octylphenol polyoxyethylene ether, and nonylphenol polyoxyethylene ether.

[0010] Surfactants are the core components for cell or virus lysis. They include at least four nonionic surfactants, have no inhibitory effect on subsequent PCR reactions, have a synergistic effect with multiple detergents, enhance the effect, are stable in room temperature solutions, and can be stored effectively.

[0011] Optionally, the buffer system is weakly alkaline and includes tris(hydroxymethyl)aminomethane hydrochloric acid and 4-hydroxyethylpiperazine ethanesulfonic acid.

[0012] The buffer system provides longer-term buffering capacity, ensuring long-term reagent preservation without increasing osmotic pressure or affecting the amount added to the PCR system.

[0013] Optionally, the nucleic acid coprecipitant includes linear acrylamide.

[0014] Nucleic acid coprecipitants compete with nucleases and negatively charged surfaces that may exist in the sample nucleic acid during the initial stage of lysis and release, protecting low-copy-number target nucleic acids from degradation or adsorption loss.

[0015] Optionally, the PCR enhancer includes betaine.

[0016] Betaine can play multiple roles in PCR or RT-PCR reactions, enhancing the effects of RT-PCR and PCR, and neutralizing the effects of certain PCR inhibitors.

[0017] Optionally, the nucleic acid release reagent further includes a protease.

[0018] Optionally, the protease includes proteinase K.

[0019] Proteinase K can be used for the detection of fungal samples, lysis of small tissue samples, and whole blood samples.

[0020] Optionally, the mass ratio of the surfactant, the buffer system, the nucleic acid coprecipitant, the PCR enhancer, and the water is 2~5:1~5:0.1~0.5:5~15:80~95.

[0021] Optionally, in the surfactant, the volume ratio of polysorbate 20, polyoxyethylene lauryl ether, octylphenol polyoxyethylene ether, and nonylphenol polyoxyethylene ether is 0.1~1:0.1~0.5:0.5~2:0.2~1.

[0022] Optionally, in the nucleic acid release reagent, the concentration of tris(hydroxymethyl)aminomethane-hydrochloric acid is 20-100 mM, and the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 10-50 mM.

[0023] Optionally, the concentration of the protease is 0.05~1 mg / ml.

[0024] Optionally, the pH value of the nucleic acid release reagent is 7.4 to 8.2.

[0025] The nucleic acid release reagent in this application has the following characteristics: First, it employs a compound formulation of multiple nonionic surfactants. This synergistic effect of multiple nonionic surfactants maintains lysis efficiency while significantly reducing the potential inhibitory risk to subsequent enzyme activity from a single high-concentration surfactant. The formulation contains no ionic surfactants and does not inhibit PCR, unlike other existing patent formulations. Second, a dual buffer system creates a mild and stable lysis and release environment. By precisely controlling the pH and ionic strength, it ensures complete lysis of viruses or cells while maximally protecting the released nucleic acid molecules from degradation, and maintains extremely low ion concentrations to avoid affecting subsequent PCR reactions. Third, it integrates nucleic acid protectants and PCR enhancers to ensure detection efficiency for low-load samples. Fourth, all components of the formulation are stable at room temperature and in solution, facilitating long-term storage at room temperature, and do not contain any known PCR inhibitors. The synergistic effect of these components enables this reagent to achieve rapid and uninhibited nucleic acid release at room temperature.

[0026] According to a second aspect of this application, a method for preparing the nucleic acid release reagent described above is provided.

[0027] A method for preparing the above-described nucleic acid release reagent, the method comprising: The nucleic acid release reagent is obtained by dissolving a mixture containing surfactant, buffer system, nucleic acid coprecipitant and PCR enhancer in water.

[0028] Optionally, the preparation method further includes dissolving the protease in water.

[0029] According to a third aspect of this application, an application of a nucleic acid release reagent in the detection of swab samples is provided.

[0030] The application of a nucleic acid release reagent in the detection of swab samples includes the following steps: S1. Dissolve the collected swab samples in a centrifuge tube containing nucleic acid release reagent and shake for I. S2, incubate statically, then shake II; S3. Take the supernatant and add it to the reaction system for detection; The nucleic acid release reagent is selected from at least one of the nucleic acid release reagents described above and the nucleic acid release reagents prepared by the method described above.

[0031] Optionally, the volume of the centrifuge tube in step S1 is 100~200μL, and the shaking time of step I is 10~15 seconds.

[0032] Optionally, the static incubation temperature in step S2 is 15~30°C and the time is 5~10 minutes.

[0033] Optionally, the reaction system in step S3 includes PCR or isothermal amplification, and the reaction system is made up using the supernatant.

[0034] This nucleic acid release reagent can be directly mixed with swab samples, disrupting the viral envelope or cell membrane through a gentle yet efficient lysis process to release nucleic acids. The reagent contains no PCR inhibitors and has extremely low ion concentrations, allowing it to be added to the PCR reaction system in any proportion without affecting PCR efficiency. The reagent components are stable in solution and contain no potentially inactivated or easily degraded components, allowing for storage at room temperature for at least 6 months without affecting usability. After sample lysis, incubation at room temperature for 2 hours does not affect PCR results. For testing swab samples with fungal infections, or for testing thick sputum, whole blood, or tissue samples, additional proteinase K can be added, followed by incubation before PCR.

[0035] The beneficial effects that this application can produce include: 1. High efficiency and speed: This nucleic acid release reagent can release viral / bacterial nucleic acid from swab samples within 15 minutes. From sample processing to obtaining a template that can be used for amplification, the entire process takes no more than 10 minutes, which greatly shortens the detection time window.

[0036] 2. Highly compatible, no PCR inhibition: Comparative tests showed that the cycle threshold (Ct value) of nucleic acid templates released using this nucleic acid release reagent for quantitative PCR differed from that of nucleic acid templates purified by standard column extraction by ≤0.5, indicating that the reagent essentially eliminated the inhibition of the amplification reaction, ensuring detection sensitivity and accuracy.

[0037] 3. Excellent room temperature stability: It can be stored in a sealed container at room temperature for at least 6 months. The released nucleic acid can be stably stored in a reagent environment at 25°C for at least 2 hours, with no significant drift in Ct value (change <1). This facilitates on-site sample processing, temporary storage, and batch processing, reducing immediate reliance on cold chain transportation.

[0038] 4. Excellent detection capability for low viral load: For simulated samples with low viral load (e.g., 100 copies / mL), this reagent, when used in conjunction with commercial PCR kits, can achieve a positive detection rate of over 95%, meeting the clinical need for high-sensitivity detection.

[0039] 5. Extremely simple operation: The entire release process does not require heating modules, centrifuges or other equipment, only vortex oscillation and static incubation, which greatly reduces the technical requirements for operators and hardware costs, making it particularly suitable for resource-limited environments and on-site rapid testing scenarios. Detailed Implementation

[0040] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0041] Unless otherwise specified, the raw materials and reagents used in the embodiments of this application were all purchased commercially.

[0042] Unless otherwise specified, all test methods are standard and all instrument settings are those recommended by the manufacturer.

[0043] Example 1 Take 50 ml of 1M tris(hydroxymethyl)aminomethane-hydrochloric acid solution (pH 8.8) and 50 ml of 1M 4-hydroxyethylpiperazine ethanesulfonic acid solution (pH 7.5), add them to 600 ml of deionized water, add 117 g of molecularly pure betaine to the above solution, stir until completely dissolved, then add 2.5 ml of polysorbate 20 (final concentration 0.25%, v / v), 1 ml of polyoxyethylene lauryl ether (final concentration 0.1%, v / v), 5 ml of octylphenol polyoxyethylene ether (final concentration 0.5%, v / v), 10 ml of nonylphenol polyoxyethylene ether (final concentration 1%, v / v) and 2 ml of linear acrylamide solution (5 mg / ml, final concentration 10 μg / ml), adjust the pH to 7.5 with 1M concentrated hydrochloric acid, and bring the volume to 1 L to obtain the nucleic acid release reagent, which is designated as sample 1#.

[0044] Test Example 1: Comparison of Release Efficiency and Inhibition between Sample 1# and Commercially Available Products Twenty pharyngeal swab samples with known positive (influenza A virus) results were collected. Each sample was divided into two portions. One portion was prepared by dissolving the collected pharyngeal swab sample in a centrifuge tube containing sample #1, shaking, incubating for 5 minutes, gently shaking, and then collecting the supernatant as template A. The other portion was used to extract and purify nucleic acid using a column-based nucleic acid extraction kit purchased from Qiager (Germany), which was used as template B. Parallel detection of template A and template B was performed using the same batch of influenza A virus real-time PCR kit with the same primers and probes. The results showed that all 20 samples were positive. The mean Ct value for template A was 24.3 ± 1.2, and the mean Ct value for template B was 24.1 ± 1.1. There was no statistically significant difference in Ct values ​​between the two groups (P > 0.05, paired t-test), with a mean difference of only 0.2.

[0045] Test Example 2: Room Temperature Stability Test of Sample 1# Ten nasal swab samples that tested positive for SARS-CoV-2 nucleic acid (ORF1ab gene Ct value between 25-30) were dissolved in a centrifuge tube containing sample #1, vortexed, and incubated for 5 minutes with gentle vortexing. The supernatant was then collected. Each supernatant was divided into two tubes: one tube was immediately used for qPCR testing (0-hour group), and the other tube was stored in a 25°C incubator. Samples were retrieved for qPCR testing at 0.5, 1, 2, and 3 hours after storage. The results showed that within 2 hours, the Ct value changes of all samples were within ±1; by 3 hours, the Ct value of two samples was delayed by more than 1.5, indicating that the nucleic acid released by this reagent is stable at 25°C for at least 2 hours.

[0046] Test Example 3: Clinical sample validation results of Sample 1# Pharyngeal swab samples were collected from 100 patients suspected of having respiratory infections. Two parallel processing methods were employed: a direct release method (the collected pharyngeal swab samples were dissolved in a centrifuge tube containing Sample 1#, vortexed, incubated for 5 minutes, gently vortexed, and the supernatant was collected) and a conventional column extraction method. Detection was then performed using a multiplex respiratory pathogen PCR detection panel. Using the column extraction method as a reference standard, Sample 1# showed detection sensitivities of 97.1%, 96.3%, 95.8%, and 100% for influenza A virus, influenza B virus, respiratory syncytial virus, and adenovirus, respectively, with a specificity of 100% for all. Even for low-load samples (Ct value > 30 in the reference method), the detection concordance rate of Sample 1# remained at 93.5%. This demonstrates that Sample 1# exhibits good detection performance in real-world clinical scenarios.

[0047] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. A nucleic acid release reagent, characterized in that, The nucleic acid release reagent includes a surfactant, a buffer system, a nucleic acid coprecipitant, a PCR enhancer, and water.

2. The nucleic acid release reagent according to claim 1, characterized in that, The surfactant is nonionic and includes polysorbate 20, polyoxyethylene lauryl ether, octylphenol polyoxyethylene ether, and nonylphenol polyoxyethylene ether. Preferably, the buffer system is weakly alkaline, and the buffer system comprises tris(hydroxymethyl)aminomethane-hydrochloric acid and 4-hydroxyethylpiperazine ethanesulfonic acid; Preferably, the nucleic acid coprecipitant comprises linear acrylamide; Preferably, the PCR enhancer includes betaine.

3. The nucleic acid release reagent according to claim 1, characterized in that, The nucleic acid release reagent also includes protease; Preferably, the protease includes proteinase K.

4. The nucleic acid release reagent according to claim 1, characterized in that, The mass ratio of the surfactant, the buffer system, the nucleic acid coprecipitant, the PCR enhancer, and the water is 2~5:1~5:0.1~0.5:5~15:80~95; Preferably, in the surfactant, the volume ratio of polysorbate 20, polyoxyethylene lauryl ether, octylphenol polyoxyethylene ether, and nonylphenol polyoxyethylene ether is 0.1~1:0.1~0.5:0.5~2:0.2~1; Preferably, in the nucleic acid release reagent, the concentration of tris(hydroxymethyl)aminomethane-hydrochloric acid is 20-100 mM, and the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 10-50 mM.

5. The nucleic acid release reagent according to claim 1, characterized in that, The concentration of the protease is 0.05~1 mg / ml.

6. The nucleic acid release reagent according to claim 1, characterized in that, The pH value of the nucleic acid release reagent is 7.4~8.

2.

7. A method for preparing the nucleic acid release reagent according to any one of claims 1 to 6, characterized in that, The preparation method includes: The nucleic acid release reagent is obtained by dissolving a mixture containing surfactant, buffer system, nucleic acid coprecipitant and PCR enhancer in water.

8. The preparation method according to claim 7, characterized in that, The preparation method also includes dissolving the protease in water.

9. The application of a nucleic acid release reagent in detecting swab samples, characterized in that, Includes the following steps: S1. Dissolve the collected swab samples in a centrifuge tube containing nucleic acid release reagent and shake for I. S2, incubate statically, then shake II; S3. Take the supernatant and add it to the reaction system for detection; The nucleic acid release reagent is selected from at least one of the nucleic acid release reagents according to any one of claims 1 to 6, or the nucleic acid release reagents prepared by the method according to claim 7 or 8.

10. The application according to claim 9, characterized in that, The volume of the centrifuge tube in step S1 is 100~200μL, and the shaking time of step I is 10~15 seconds; Preferably, the static incubation temperature in step S2 is 15~30°C and the time is 5~10 minutes; Preferably, the reaction system in step S3 includes PCR or isothermal amplification, and the reaction system is made up using the supernatant.