A rapid nucleic acid release agent without extraction for RPA constant temperature amplification and application thereof

Abstract

The application discloses a no-extraction fast nucleic acid releasing agent suitable for RPA constant temperature amplification and application thereof, and belongs to the technical field of biological detection. The no-extraction fast nucleic acid releasing agent is composed of the following components: 10 mM Tris, 20 mM sodium hydroxide, 50 mM Triton X-100, 0.01% SDS in weight ratio, 5 mM PEG4000, 1.00 g / L SE-15, 5 mM KCl and 25 mM NaCl, 10 mM guanidine hydrochloride, and the rest is DEPC water. The technical scheme can meet the technical requirement of RPA constant temperature amplification.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and more specifically, to an extraction-free rapid nucleic acid release agent suitable for RPA isothermal amplification and its application. Background Technology

[0002] Recombinase polymerase isothermal amplification (RPA) technology, as a new generation of rapid nucleic acid amplification technology, can achieve efficient nucleic acid amplification under isothermal conditions of 37℃–42℃. It does not require a thermal cycler or complex temperature control equipment and has outstanding advantages such as speed, sensitivity and portability. It is a core supporting technology for on-site POCT testing.

[0003] For RPA isothermal amplification nucleic acid release agents, the core technical requirements are: (1) rapid lysis at room temperature: complete nucleic acid release from clinical samples such as swabs and body fluids within 30 seconds at room temperature. (2) Purification-free compatibility: the lysate does not require centrifugation, washing, or magnetic bead purification; it can be directly added to the RPA system for amplification without inhibiting recombinase / polymerase activity. (3) High sensitivity and broad spectrum: adaptable to various pathogens and sample types, stable detection of low-copy samples, and strong signal on the test strip.

[0004] In response, many companies have proposed nucleic acid release agents for RPA isothermal amplification, such as: Reference 1 (CN115094118A) uses a core lysis system of surfactant, Triton X-100, and NaOH. While this nucleic acid release agent has a simple composition, it is insufficiently lysed in complex clinical samples such as swabs, secretions, and bodily fluids, resulting in incomplete release of viral / pathogen nucleic acids, leading to low detection sensitivity and an increased risk of false negatives.

[0005] Reference 2: CN112280832A requires heating at a high temperature of 42℃~72℃ for 2min~30min to operate, and cannot achieve POCT detection at room temperature; at the same time, this reference uses very complex components, which poses a potential risk of multiple inhibition of the RPA enzyme system (as shown in Table 1).

[0006]

[0007] Reference 3: CN113337500A, which consists of "Triton X-100, Tween-20, isopropanol, and ethanol". Corresponding to RPA, its amplification depends on the fine three-dimensional structure of proteins such as the recombinant enzyme UvsX and the polymerase Bsu. High concentrations of organic solvents (isopropanol, ethanol) will disrupt the protein hydration layer, leading to enzyme denaturation and precipitation, thereby completely inhibiting the RPA amplification reaction.

[0008] Reference 4, CN121344153A, uses EDTA (a metal ion chelating agent), which irreversibly chelates magnesium ions in the reaction system, leading to a sharp decrease in amplification efficiency. Therefore, the nucleic acid release agent in this reference requires continuous replenishment of magnesium ions during use. This contradicts the technical requirement of RPA (Reproductive Processing Acquisition) of "extraction-free, direct loading." Furthermore, the "preservative Proclin 300" contained in this system may have a potential inhibitory effect on the RPA enzyme system.

[0009] In summary, the research and development challenges for extraction-free rapid nucleic acid release agents suitable for RPA isothermal amplification are summarized as follows: First, lysis and amplification are incompatible.

[0010] High-concentration guanidine salts offer high lysis efficiency but strongly inhibit RPA enzymes. While single-base lysis (such as NaOH) has low inhibitory effects, it results in incomplete lysis, leading to incomplete release of viral nucleic acid and a high risk of false negatives. Therefore, achieving complete lysis and compatibility between lysis and RPA amplification remains a significant research challenge.

[0011] Second, how to synergistically control anionic / nonionic surfactants.

[0012] Surfactants commonly used include SDS and Triton X-100. SDS is highly efficient at lysing membrane proteins but inhibits UvsX recombinase even in trace amounts; Triton X-100 is mild but inefficient when used alone on thick-walled samples.

[0013] Third, how to shield the cell debris and deformed proteins generated during the lysis process.

[0014] Cell debris and deformable proteins generated during lysis can non-specifically inhibit the adsorption of RPA enzymes, resulting in low amplification efficiency. Current technologies address this by adding a "purification" step. However, the absence of a "purification" step is one of the technical requirements for extraction-free rapid nucleic acid release agents suitable for isothermal RPA amplification.

[0015] In summary, there is a need to develop an extraction-free rapid nucleic acid release agent suitable for RPA isothermal amplification. Summary of the Invention

[0016] The primary objective of this application is to address the shortcomings of the prior art by providing an extraction-free rapid nucleic acid release agent suitable for RPA isothermal amplification and its application.

[0017] The technical solution of this application is: An extraction-free nucleic acid release agent suitable for RPA isothermal amplification consists of the following components: (1) Buffer solution: 10 mM Tris; (2) Alkaline pyrolysis agent: 20mM sodium hydroxide; (3) Nonionic surfactant: 50mM Triton X-100; (4) Anionic surfactant: 0.01% SDS by weight; (5) Macromolecular stabilizer: 5mM PEG4000; (6) Anti-interference agent: 1.00 g / L SE-15; (7) Ionic strength modifiers: 5 mM KCl and 25 mM NaCl; (8) Auxiliary denaturant: 10mM guanidine hydrochloride; (9) Solvent: DEPC water.

[0018] An extraction-free nucleic acid release agent suitable for RPA isothermal amplification consists of the following components: (1) Buffer solution: 10 mM Tris; (2) Alkaline lysis agent: sodium hydroxide, the pH value of the nucleic acid release agent is 10.0±0.1; (3) Nonionic surfactant: 50mM Triton X-100; (4) Anionic surfactant: 0.01% SDS by weight; (5) Macromolecular stabilizer: 5mM PEG4000; (6) Anti-interference agent: 1.00 g / L SE-15; (7) Ionic strength modifiers: 5 mM KCl and 25 mM NaCl; (8) Auxiliary denaturant: 10mM guanidine hydrochloride; (9) Solvent: DEPC water.

[0019] A method for preparing an extraction-free nucleic acid release agent suitable for RPA isothermal amplification includes the following steps: S100, take DEPC water; S200, add Tris, sodium hydroxide, Triton X-100, SDS, PEG4000, SE-15, KCl, NaCl, and guanidine hydrochloride in sequence and dissolve thoroughly; S300, stir and mix well, then adjust the pH to 10.0±0.1.

[0020] Furthermore, the concentrations of each component added during the S200 process are as follows: Tris concentration is 10 mmol / L, sodium hydroxide concentration is 20 mmol / L, Triton X-100 concentration is 50 mmol / L, SDS mass concentration is 0.01%, PEG4000 concentration is 5 mmol / L, SE-15 concentration is 1 g / L, KCl concentration is 5 mmol / L, NaCl concentration is 25 mmol / L, and guanidine hydrochloride concentration is 10 mmol / L.

[0021] Furthermore, the pH in S300 is adjusted by adding HCl or NaOH.

[0022] Furthermore, it also includes: S400, which uses a 0.22μm sterilization filter membrane for filtration and sterilization.

[0023] A nucleic acid release agent prepared using the aforementioned preparation method.

[0024] A nucleic acid amplification method, comprising the following steps: S100, Place the sample to be tested into the nucleic acid release agent; S200, at room temperature, manually shake left and right 8-10 times; S300: Directly use the lysis mixture as an amplification template and add it to the RPA amplification system; S400, isothermal amplification at 37℃-42℃.

[0025] A kit containing the aforementioned extraction-free nucleic acid release agent suitable for RPA isothermal amplification.

[0026] The application of one of the aforementioned nucleic acid release agents in the preparation of a rapid RPA detection kit for respiratory pathogens, enteroviruses, or genitourinary pathogens.

[0027] The beneficial effects of this application are as follows: First, the nucleic acid release agent of this application has the following advantages: (1) Rapid lysis at room temperature, greatly shortening the detection time. The entire process is carried out at room temperature, with lysis within 30 seconds. Combined with rapid amplification by RPA, the overall detection time is shortened to within 20 minutes, significantly improving detection efficiency.

[0028] (2) Strong lysis ability and more thorough nucleic acid release. The synergistic lysis system of strong alkali + compound surfactant + low concentration guanidine salt is adopted to efficiently break the viral envelope, capsid and host cell structure. The nucleic acid release rate is much higher than that of single alkali lysis solution, ensuring stable detection of low copy samples.

[0029] (3) Highly compatible with RPA enzyme system, amplification is more stable. The formula has been precisely optimized, with no redundant inhibitory components, does not interfere with the activity of recombinase and polymerase, does not affect the specific binding of primers and probes, and the test strip has strong signal and good repeatability.

[0030] (4) The operation is extremely simple and requires little equipment and personnel. No centrifuges, purification columns, or cold chain equipment are needed. It is a single-tube operation with few steps, and even non-professionals can quickly get started. It is suitable for grassroots and field use.

[0031] (5) It can be stored and transported at room temperature, significantly reducing costs. It can be stored stably for a long time at 2–30℃. Room temperature transportation does not require a cold chain, saving the investment in purification consumables and equipment. The cost per person is lower, making it suitable for large-scale universal screening.

[0032] (6) It is applicable to a wide range of samples and has high sensitivity. It is suitable for various samples such as nasopharyngeal swabs, genital secretions, and body fluids. It can stably detect low concentrations of pathogen nucleic acid, and its sensitivity meets the requirements for clinical diagnosis and screening.

[0033] Second, the key point of the nucleic acid release agent in this application is: (1) How to lyse. This application adopts a lysis scheme dominated by "alkaline environment of pH 10.0 + Triton X-100 (50mM)" to achieve a membrane rupture effect of 30s at room temperature without the need for heating and centrifugation. At the same time, the lysis is assisted by "SDS (0.01%) + 10mM guanidine hydrochloride" without triggering RAP enzyme inhibition.

[0034] (2) Active anti-interference. This application uses “SE-15 (1g / L)” to adsorb / shield lysate fragments and denatured proteins, eliminating the non-specific inhibition of RPA recombinase by the extract-free product.

[0035] Third, this invention is compatible with various sample types, including respiratory tract swabs, genitourinary tract swabs, saliva, and urine, and is suitable for respiratory viruses (influenza A virus, influenza B virus, respiratory syncytial virus, Chlamydia pneumoniae, rhinovirus, adenovirus, enterovirus EV71, enterovirus CA16, and general enteroviruses) as well as genitourinary tract pathogens (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and high-risk HPV types 16 / 18 / 52). Attached Figure Description

[0036] The present application will be further described in detail below with reference to the embodiments in the accompanying drawings, but this does not constitute any limitation on the present application.

[0037] Figure 1 This is a graph showing the experimental results of this application (room temperature pyrolysis).

[0038] Figure 2 This is a graph showing the experimental results of Comparative Example 1.

[0039] Figure 3 This is a graph showing the experimental results of Comparative Example 2.

[0040] Figure 4 This is a graph showing the experimental results of Comparative Example 3.

[0041] Figure 5 This is a diagram showing the experimental results of this application (pyrolysis at 95°C). Detailed Implementation The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0042] <Example 1> I. Components An extraction-free rapid nucleic acid release agent suitable for RPA isothermal amplification, each L of the nucleic acid release agent consists of the following components: 10 mM Tris(hydroxymethyl)aminomethane (Tris), 20 mM sodium hydroxide (NaOH), 50 mM Triton X-100, sodium dodecyl sulfate (SDS) at a weight ratio of 0.1‰, 5 mM polyethylene glycol 4000 (PEG4000), 1.00 g SE-15, 5 mM potassium chloride (KCl), 25 mM sodium chloride (NaCl), 10 mM guanidine hydrochloride, and the balance being DEPC water.

[0043] The pH value of the nucleic acid releasing agent is 10.0±0.1.

[0044] The functions of each component are shown in Table 2.

[0045]

[0046] II. Preparation Method The preparation method of the above-mentioned nucleic acid release agent is as follows: S100, measure 800mL of DEPC water into a beaker, add a magnetic stir bar, and turn on the stirrer; S200: Weigh each component accurately according to the formula, add them in batches and stir thoroughly until completely dissolved; After all components of S300 have dissolved, continue stirring and mixing for 30 minutes to ensure the system is homogeneous and stable. S400: Use an acid-base adjuster to precisely correct the pH to 10.0 ± 0.1 (the solution obtained in step S200 has a pH around 10; in S400, add a trace amount of HCl or NaOH to adjust the pH to 10). S500, bring the volume to 1L with DEPC water, and retest the pH to ensure it meets the requirements; The S600 uses a 0.22μm sterilization filter membrane for filtration and sterilization. S700, dispensed in 1mL / 2mL / 3mL sizes, sealed and stored at room temperature (2~30℃).

[0047] III. Storage and Transportation Conditions Storage temperature: 2℃–30℃.

[0048] Valid for 24 months.

[0049] Transportation conditions: Normal temperature transportation, no cold chain required.

[0050] IV. Extraction-free nucleic acid release and RPA detection method A method for releasing nucleic acids, comprising the following steps: S100: After rinsing the collected swab samples in the nucleic acid release agent for 15-30 seconds, remove them (if it is a liquid sample, release it at a 1:1 ratio). S200, at room temperature, manually shake left and right 8-10 times; S300: Directly use the lysis mixture as an amplification template and add it to the RPA amplification system; S400, isothermal amplification at 37℃-42℃, detection using test strip method, result interpretation completed in 5-20 minutes. S500 is suitable for detecting respiratory pathogens, enteroviruses, genitourinary pathogens, etc.

[0051] <Comparative Experiment: Different Nucleic Acid Release Agents> Comparative Example 1: Contains only 200mM sodium hydroxide (NaOH).

[0052] Comparative Example 2: Guanidine hydrochloride and SE-15 were removed from Example 1, while other components and their contents remained unchanged.

[0053] Comparative Example 3: The concentration of guanidine hydrochloride in Example 1 was increased to 4M, while the other components and their contents remained unchanged.

[0054] from Figure 1 , Figure 2 , Figure 3 , Figure 4 It can be seen that the nucleic acid release agent gradient of this application is good and the signal is strong; Comparative Example 1 has a poor gradient and a weak signal; Comparative Example 2 has a poor gradient and a weak signal; Comparative Example 3 has no effective amplification and the enzyme system is significantly inhibited. Figures 1-4 (The lysis temperature of all samples is room temperature). Therefore, it can be concluded that the nucleic acid release agent developed in this application is highly compatible with the RPA enzyme system and has no inhibitory effect.

[0055] <Comparative Experiment: Pyrolysis at Different Temperatures> Figure 5 The results illustrate the detection effect of the nucleic acid release agent of this application during thermal pyrolysis at 95℃.

[0056] contrast Figure 1 and Figure 5 It can be seen that room temperature pyrolysis exhibits a good gradient and strong signal over 30 seconds; thermal pyrolysis at 95℃ also exhibits a good gradient and strong signal over 30 seconds. Therefore, it can be concluded that efficient pyrolysis can be achieved at room temperature, with results comparable to thermal pyrolysis.

[0057] The above-described embodiments are preferred embodiments of this application and are only used to facilitate the illustration of this application. They are not intended to limit this application in any way. Any person with ordinary knowledge in the art can make equivalent embodiments by making partial modifications or alterations to the technical content disclosed in this application without departing from the scope of the technical features of this application. Such equivalent embodiments are still within the scope of the technical features of this application.

Claims

1. An extraction-free nucleic acid release agent suitable for RPA isothermal amplification, characterized in that, It consists of the following components: (1) Buffer solution: 10 mM Tris; (2) Alkaline pyrolysis agent: 20mM sodium hydroxide; (3) Nonionic surfactant: 50mM Triton X-100; (4) Anionic surfactant: 0.01% SDS by weight; (5) Macromolecular stabilizer: 5mM PEG4000; (6) Anti-interference agent: 1.00 g / L SE-15; (7) Ionic strength modifiers: 5 mM KCl and 25 mM NaCl; (8) Auxiliary denaturant: 10mM guanidine hydrochloride; (9) Solvent: DEPC water.

2. An extraction-free nucleic acid release agent suitable for RPA isothermal amplification, characterized in that, It consists of the following components: (1) Buffer solution: 10 mM Tris; (2) Alkaline lysis agent: sodium hydroxide, the pH value of the nucleic acid releasing agent is 10.0 ± 0.1; (3) Nonionic surfactant: 50mM Triton X-100; (4) Anionic surfactant: 0.01% SDS by weight; (5) Macromolecular stabilizer: 5mM PEG4000; (6) Anti-interference agent: 1.00 g / L SE-15; (7) Ionic strength modifiers: 5 mM KCl and 25 mM NaCl; (8) Auxiliary denaturant: 10mM guanidine hydrochloride; (9) Solvent: DEPC water.

3. A method for preparing an extraction-free nucleic acid release agent suitable for RPA isothermal amplification, characterized in that, Includes the following steps: S100, take DEPC water; S200, add Tris, sodium hydroxide, Triton X-100, SDS, PEG4000, SE-15, KCl, NaCl, and guanidine hydrochloride in sequence and dissolve thoroughly; S300, stir and mix well, then adjust the pH to 10.0±0.

1.

4. A method for preparing an extraction-free nucleic acid release agent suitable for RPA isothermal amplification as described in claim 3, characterized in that, The concentrations of each component added during the S200 process are as follows: Tris concentration is 10 mmol / L, sodium hydroxide concentration is 20 mmol / L, Triton X-100 concentration is 50 mmol / L, SDS mass concentration is 0.01%, PEG4000 concentration is 5 mmol / L, SE-15 concentration is 1 g / L, KCl concentration is 5 mmol / L, NaCl concentration is 25 mmol / L, and guanidine hydrochloride concentration is 10 mmol / L.

5. The method for preparing an extraction-free nucleic acid release agent suitable for RPA isothermal amplification as described in claim 4, characterized in that, The method to adjust the pH in S300 is to add HCl or NaOH.

6. The method for preparing an extraction-free nucleic acid release agent suitable for RPA isothermal amplification as described in claim 3, characterized in that, Also includes: The S400 uses a 0.22μm sterilization filter membrane for filtration and sterilization.

7. A nucleic acid release agent prepared by the preparation method according to any one of claims 3 to 6.

8. A method for nucleic acid amplification, characterized in that, Includes the following steps: S100, the sample to be tested is placed in the nucleic acid releasing agent as described in claim 1, 2 or 7; S200, at room temperature, manually shake left and right 8-10 times; S300: Directly use the lysis mixture as an amplification template and add it to the RPA amplification system; S400, isothermal amplification at 37℃-42℃.

9. A reagent kit, characterized in that, It contains an extraction-free nucleic acid release agent suitable for RPA isothermal amplification as described in claim 1, 2, or 7.

10. The use of a nucleic acid releasing agent as described in claim 1, 2 or 7 in the preparation of a rapid RPA detection kit for respiratory pathogens, enteroviruses or genitourinary pathogens.

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