A nucleic acid extraction treatment reagent suitable for a general-purpose sample, a kit thereof, and a nucleic acid extraction method

Abstract

The present application relates to the technical field of nucleic acid detection, and in particular to a nucleic acid extraction processing reagent suitable for general samples, a kit thereof and a nucleic acid extraction method. The nucleic acid extraction processing reagent comprises a pH buffer component, a cell wall / membrane lysis component, a surfactant component, a sticky protein and polysaccharide dissolution component, a nucleic acid protection agent component and a PCR enhancer component. For different sample types, the nucleic acid extraction processing reagent selects a suitable temperature treatment mode, can crack the cell wall or cell membrane of the sample, degrade nucleic acid binding proteins, release nucleic acids in the sample into solution, prevent nucleic acid oxidation and degradation under the action of the nucleic acid protection agent, directly perform downstream molecular experiments without any purification, simplify the entire detection process, save a large amount of human and economic costs, and has the advantages of rapid detection, high sensitivity, strong specificity and good reliability.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to a nucleic acid extraction and processing reagent suitable for general-purpose samples, a kit, and a nucleic acid extraction method. Background Technology

[0002] In the field of molecular diagnostics, nucleic acid extraction is a core step in ensuring the accuracy of experimental results; however, existing nucleic acid purification technologies have many problems. Commonly used column extraction and magnetic bead extraction techniques involve multiple stages, including sample pretreatment, cell membrane lysis, nucleic acid adsorption, impurity washing, alcohol precipitation for desalting, and elution recovery, with each operation taking at least 40 minutes. While these methods are widely used in nucleic acid extraction, their cumbersome procedures make them unsuitable for high-throughput detection.

[0003] Furthermore, the diversity of sample types, coupled with the fact that most existing nucleic acid extraction or sample processing reagents are developed for specific sample types and have poor compatibility, increases the complexity and cost of molecular detection. For large-scale sample testing, time costs and reagent management difficulties increase significantly, and cumbersome operating procedures are prone to human error.

[0004] Regarding lysis efficiency, existing sample processing reagents mostly rely on a single enzyme or a single surfactant, resulting in low lysis efficiency. For example, the surfactant SDS and denaturant urea commonly used in lysis buffers, while capable of disrupting cell and nuclear membranes, are not effective for certain complex samples. Furthermore, existing reagents typically require additional purification steps to remove inhibitors, further prolonging the processing time. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a nucleic acid extraction and processing reagent suitable for general-purpose samples. This nucleic acid extraction and processing reagent suitable for general-purpose samples can be applied to various types of samples and has the advantages of high processing efficiency, low cost and easy operation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: We provide nucleic acid extraction and processing reagents suitable for general-purpose samples, comprising the following components: The pH buffer component includes one or more of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, wherein the concentration of the pH buffer component is 10mM to 100mM, and the pH buffer component adjusts the pH of the sample processing reagent to 7.5 to 8.8. Cell wall / membrane lysis components, comprising strong lysing agents and moderate lysing agents, wherein the strong lysing agent comprises one or more of guanidine isothiocyanate, guanidine hydrochloride, and urea, and the moderate lysing agent comprises one or more of SDS and CTAB, wherein the concentrations of guanidine isothiocyanate and guanidine hydrochloride are 5 mM to 100 mM, the mass-to-volume ratio of SDS and CTAB is 0.01% to 0.5%, and the concentration of urea is 10 mM to 100 mM; The surfactant component includes one or more of Triton X-100, NP40, and Tween 20, wherein the volume ratio of the surfactant component is 0.01% to 5%. The soluble components of the viscous protein and polysaccharide include one or a combination of two of acetylcysteine ​​and bromhexine hydrochloride; NaOH; PVP40, wherein the concentration of acetylcysteine ​​is 1mM~50mM, the concentration of NaOH is 0.1mM~1M, the concentration of bromhexine hydrochloride is 1mM~100mM, and the concentration of PVP40 is 0.1%~10%; The nucleic acid protective agent components include DTT, TCEP, and EDTA2Na, wherein the concentration of DTT or TCEP is 0.1mM~50mM, and the concentration of EDTA2Na is 1mM~20mM. The PCR enhancer components include BSA and / or single-stranded binding protein, KCl and / or MgCl2, PEG400~8000, betaine, trehalose and / or DMSO; wherein the concentration of BSA is 0.1 mg / mL~5 mg / mL, the concentration of single-stranded binding protein is 0.01 mg / mL~1 mg / mL, the concentration of KCl is 10 mM~200 mM, the concentration of MgCl2 is 1 mM~30 mM, the mass percentage of PEG is 0.5%~10%, the concentration of betaine is 100 mM~2 M, and the volume percentage of DMSO is 0.5%~10%.

[0007] In some embodiments, the molecular weight of the PEG is any one or more of 400, 800, 1000, 4000, 6000, and 8000.

[0008] Nucleic acid extraction kits are also provided, which contain the above-mentioned nucleic acid extraction and processing reagents suitable for general-purpose samples.

[0009] A nucleic acid extraction method is also provided, characterized by comprising the following steps: The sample containing the nucleic acid to be extracted is prepared into a sample solution. The sample includes one or more of the following: animal and plant tissues, blood, body fluids, sputum, and microbial cultures. Add the above-mentioned nucleic acid extraction reagent suitable for general samples to the sample solution, vortex to mix, and then treat at room temperature to 100℃ for 5 min to 20 min, followed by centrifugation for 1 min to 2 min. Collect the supernatant after centrifugation. The supernatant was used as a nucleic acid template for molecular detection experiments.

[0010] In some embodiments, the molecular detection experiments include nucleic acid amplification experiments, viral load monitoring, rapid identification of microorganisms, detection of plant and animal polymorphisms, rapid gene cloning, rapid detection of plant and animal pathogens, and plant and animal quarantine and prevention.

[0011] In some implementations, the nucleic acid amplification experiments include qRT-PCR, LAMP, and CRISPR-Cas12a.

[0012] The beneficial effects of this invention's nucleic acid extraction and processing reagent suitable for general-purpose samples are as follows: (1) The nucleic acid extraction and processing reagent of the present invention, suitable for general-purpose samples, can efficiently lyse and release nucleic acids from different sample types (including animal and plant tissues, blood, body fluids, sputum, and microbial cultures, etc.). Through the dynamic synergistic effect of multi-dimensional technical modules, it overcomes the limitations of traditional sample processing reagents, such as single sample type, narrow lysis range, large amount of inhibitor residue, and poor PCR compatibility. It achieves compatibility with multiple sample types, high nucleic acid release efficiency, and minimizes the inhibitory effect of subsequent molecular experiments such as PCR. Specifically, it is reflected in the following synergistic mechanism: Its cell wall / membrane lysis component consists of a gradient lysis group, in which guanidine isothiocyanate, guanidine hydrochloride, and urea act as strong denaturants; SDS and CTAB act as moderate lysis agents; and the surfactants X-100, NP40, and Tween 20 act as mild solubilizers. Through the dynamic combination of lysis reagents of varying strengths, it enables cell lysis and nucleic acid release from samples with diverse cell wall structures, including plant and fungal cell walls. It is also applicable to various samples with membrane structures but no cell wall, such as blood leukocytes, cultured cells, and bacteria; and is equally suitable for viral samples with intact cell structures. Through gradient lysis adaptation, it achieves full-type sample coverage from viral capsids to plant cell walls, enabling cell lysis of various samples without the need for additional reagents.

[0013] The surfactants include nonionic detergents X-100, Tween 20, and NP40. These nonionic detergents can intercalate into the cell membrane lipid bilayer, thereby reducing cell membrane tension. Furthermore, X-100, Tween 20, and NP40, combined with potassium and magnesium ions in the aqueous solution and PEG and DMSO in the PCR enhancer components, can form cationic polymers. These cationic polymers enhance the efficiency of nucleic acid release from the cell nucleus. In addition, the use of nonionic detergents X-100, Tween 20, and NP40 in the PCR system can antagonize the strong denaturants in the cell wall / membrane lysis components, counteracting the damaging effects of these components on the PCR enzyme. At lower temperatures, such as room temperature, X-100, Tween 20, and NP40 can shield the effects of strong denaturants, reducing their destructive effects on nucleic acids and dynamically balancing lysis capacity, achieving a dynamic synergistic effect of gradient lysis.

[0014] Its viscous protein and polysaccharide-dissolving components include acetylcysteine, NaOH, bromhexine hydrochloride, and PVP40, which can decompose the complex polysaccharide structure in bacterial capsules and fungal cell walls, as well as decompose viscous proteins in sputum, ensuring the effective processing of difficult-to-lyse samples such as Gram-negative bacteria, or the liquefaction and nucleic acid release of samples such as sputum, further improving the processing capability of viscous samples and realizing the applicability to spectral sample types.

[0015] Its pH buffer components include Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, while its nucleic acid protective agent components include DTT, TCEP, and EDTA2Na. These nucleic acid protective agent components work synergistically with the pH buffer components. Since TCEP is a weakly acidic organic compound with an aqueous solution pH of approximately 2.5, TCEP forms a pH buffer system with the weakly basic substance Tris or Tris-HCl. The mechanism of action is to respond to pH changes in the environment through protonation / deprotonation. TCEP's temperature resistance and hydrolysis resistance further enhance the pH buffering capacity, achieving dynamic pH stability and environmental adaptation for nucleic acid protection within the process control. Specifically, under storage conditions of 4-100℃, pH fluctuations can be controlled within the range of 0.2, avoiding nucleic acid degradation during low-temperature transportation or handling. During the lysis phase (typically maintained at 30-100℃), the pH buffer components counteract the pH decrease caused by temperature increases through proton buffering, ensuring that the pH of the subsequent PCR system is not altered during enzymatic reactions, thus guaranteeing thermal stability.

[0016] Its PCR enhancer components include BSA, single-stranded binding protein, KCl, MgCl2, PEG400~8000, betaine, and DMSO. These PCR enhancer components can regulate polymerase activity, maintain the amplification efficiency at the original level, and prevent the sample processing solution from affecting the amplification mechanism.

[0017] Furthermore, the surfactant encapsulates phenolic compounds, guanidine salts, and heme molecules through steric hindrance, while DTT or TCEP acts as a reducing agent to decompose DNA polymerase inhibitors (such as SDS derivatives). Trehalose, Tween 20, X-100, or BSA in the formulation act as antagonists for PCR inhibitors (such as guanidine salts, SDS, PVP40, and proteins in the sample), enabling simultaneous inhibitor neutralization during the lysis phase without the need for additional purification steps. Therefore, this invention achieves a synergistic effect across the entire chain of universal lysis, nucleic acid protection, and amplification protection through the above components, enabling simple processing and direct rapid detection of various sample types. This gives the reagent a significant competitive advantage in fields such as clinical diagnosis, environmental microbial detection, and genetically modified crop screening, and is particularly suitable for the rapid processing needs of various sample types in resource-constrained scenarios. Attached Figure Description

[0018] Figure 1 This is a schematic diagram showing the successful amplification of target genes of different lengths in rice and maize in Experiment Example 1.

[0019] Figure 2 This is a graph showing the rapid pathogen detection results of the nucleic acid extraction and processing reagent for the general-purpose sample in Experiment Example 2 in the animal and aquaculture industry.

[0020] Figure 3 This is the qPCR amplification curve and melting curve of sweet potato leaf curl virus in Experiment Example 2.

[0021] Figure 4 This is the amplification curve and melting curve of the sweet potato internal reference gene in Experiment Example 2.

[0022] Figure 5 This is the amplification curve from Experiment Example 5.

[0023] Figure 6 This is the effect of treating the sputum in Experiment Example 6 with the universal sample processing reagent of this invention.

[0024] Figure 7 This is the amplification curve of isothermal amplification detection in Experiment Example 6.

[0025] Figure 8 This is a diagram showing the amplification results of the negative control before the amplification reaction and the positive control reaction in Experiment Example 7.

[0026] Figure 9 This is a visualization of the LAMP detection results from Experiment Example 8. Detailed Implementation

[0027] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0028] Abbreviated definition:

[0029] Example 1

[0030] This embodiment discloses a nucleic acid extraction and processing reagent suitable for general-purpose samples, comprising the following components: The pH buffer component includes one or more of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid. The concentration of the pH buffer component is 10mM to 100mM. The pH buffer component is used to adjust the pH of the sample processing reagent to 7.5 to 8.8. The specific parameters can be selected according to actual conditions.

[0031] The pH buffer components include Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid. The nucleic acid protective agent components include DTT, TCEP, and EDTA2Na. These components work synergistically with the pH buffer components. Since TCEP is a weakly acidic organic compound with an aqueous solution pH of approximately 2.5, TCEP forms a pH buffer system with the weakly basic Tris or Tris-HCl. Its mechanism of action involves protonation / deprotonation to respond to pH changes in the environment. TCEP's temperature and hydrolysis resistance further enhance the pH buffering capacity, achieving dynamic pH stability and environmental adaptation for nucleic acid protection within the process control. Specifically, under storage conditions of 4-100℃, pH fluctuations can be controlled within 0.2, preventing nucleic acid degradation during low-temperature transportation or handling. During the lysis phase (typically maintained at 30-100℃), the pH buffer components counteract the pH decrease caused by temperature increases through proton buffering, ensuring that the pH of the subsequent PCR system is not altered during enzymatic reactions, thus guaranteeing thermal stability.

[0032] The cell wall / membrane lysis component includes a strong lysing agent and a moderate lysing agent. The strong lysing agent includes one or more of guanidine isothiocyanate, guanidine hydrochloride, and urea. The moderate lysing agent includes one or more of SDS and CTAB. The concentrations of guanidine isothiocyanate and guanidine hydrochloride are 5 mM to 100 mM. The mass-to-volume ratio of SDS and CTAB is 0.01% to 0.5%. The concentration of urea is 10 mM to 100 mM. The volume parameters can be selected according to actual conditions.

[0033] Among the aforementioned cell wall / membrane lysis components, guanidine isothiocyanate, guanidine hydrochloride, and urea act as strong denaturants; SDS and CTAB act as moderate lysis agents; and the surfactants X-100, NP40, and Tween 20 act as mild solubilizers. Through the dynamic combination of lysis reagents of varying strengths, this allows for cell lysis and nucleic acid release from samples with diverse cell wall structures, including plant and fungal cell walls. It is also applicable to various samples without cell walls but possessing membrane structures, such as blood leukocytes, cultured cells, and bacteria; and is equally suitable for viral samples with intact cell structures. Through gradient lysis adaptation, it achieves full-type sample coverage from viral capsids to plant cell walls, enabling cell lysis of various samples without the need for additional reagents.

[0034] The surfactant component includes one or more of Triton X-100, NP40, and Tween 20, wherein the volume ratio of the surfactant component is 0.01% to 5%. The surfactant components mentioned above include nonionic detergents X-100, Tween 20, and NP40. These nonionic detergents can insert into the lipid bilayer of the cell membrane, thereby reducing cell membrane tension. Furthermore, X-100, Tween 20, and NP40, combined with potassium and magnesium ions in the aqueous solution and PEG and DMSO in the PCR enhancer components, can form cationic polymers. These cationic polymers enhance the release efficiency of nucleic acids from the cell nucleus. In addition, the use of nonionic detergents X-100, Tween 20, and NP40 in the PCR system of this invention can antagonize the strong denaturants in the cell wall / membrane lysis components, counteracting the damaging effects of these components on PCR enzymes. At lower temperatures, such as room temperature, X-100, Tween 20, and NP40 can shield the effects of strong denaturants, reducing their destructive effects on nucleic acids and dynamically balancing lysis capacity, achieving a dynamic synergistic effect of gradient lysis.

[0035] The soluble components of the viscous protein and polysaccharide include one or a combination of two of acetylcysteine ​​and bromhexine hydrochloride; NaOH; PVP40, wherein the concentration of acetylcysteine ​​is 1mM~50mM, the concentration of NaOH is 0.1mM~1M, the concentration of bromhexine hydrochloride is 1mM~100mM, and the concentration of PVP40 is 0.1%~10%; The aforementioned viscous protein and polysaccharide dissolving components include acetylcysteine, NaOH, bromhexine hydrochloride, and PVP40, which can decompose the complex polysaccharide structure in bacterial capsules and fungal cell walls, as well as decompose viscous proteins in sputum, ensuring the effective processing of difficult-to-lyse samples such as Gram-negative bacteria, or the liquefaction and nucleic acid release of samples such as sputum, further improving the processing capability for viscous samples and achieving applicability to spectral sample types.

[0036] The nucleic acid protective agent components include DTT, TCEP, and EDTA2Na, wherein the concentration of DTT or TCEP is 0.1mM~50mM, and the concentration of EDTA2Na is 1mM~20mM. The PCR enhancer components include one or a combination of two of BSA and single-stranded binding protein; one or a combination of two of KCl and MgCl2; PEG 400~8000; and one or a combination of two of trehalose or DMSO, wherein the concentration of BSA is 0.1 mg / mL~5 mg / mL, the concentration of single-stranded binding protein is 0.01 mg / mL~1 mg / mL, the concentration of KCl is 10 mM~200 mM, the concentration of MgCl2 is 1 mM~30 mM; the mass ratio of PEG is 0.5%~10%; the concentration of betaine is 100 mM~2 M; and the concentration of DMSO is 0.5%~10%.

[0037] The PCR enhancer components mentioned above include BSA, single-stranded binding protein, KCl, MgCl2, PEG400~8000, betaine, and DMSO. These PCR enhancer components can regulate polymerase activity, maintain the amplification efficiency at the original level, and prevent the sample processing solution from affecting the amplification mechanism.

[0038] The molecular weight of the PEG is any one or more of 400, 800, 1000, 4000, 6000, and 8000.

[0039] Nucleic acid extraction is performed using the above-mentioned nucleic acid extraction reagents suitable for general-purpose samples, including the following steps: The sample containing the nucleic acid to be extracted is prepared into a sample solution. The sample includes one or more of the following: animal and plant tissues, blood, body fluids, sputum, and microbial cultures. Add nucleic acid extraction reagent to the sample solution, vortex to mix, and then treat at room temperature to 100℃ for 5-20 minutes. Centrifuge for 1-2 minutes and collect the supernatant. The supernatant was used as a nucleic acid template for molecular detection experiments; The components whose English abbreviations are mentioned above are listed in Table 1. Table 1

[0040] Effect verification

[0041] To further illustrate the effectiveness of the nucleic acid extraction and processing reagent of the present invention for general-purpose samples, the following experimental verification was conducted: The reagent formulations described in Table 2 were used in all the following experiments.

[0042] Table 2

[0043] Experimental Example 1

[0044] In this experimental example, PCR amplification was performed on rice leaves and corn ears after pretreatment. The pretreatment method is as follows: Take about 0.5g of rice leaves and corn cobs, and cut them into 1.5mL centrifuge tubes. The rice leaves should be about 0.5cm square and the corn cobs should be about 0.5cm long.

[0045] Add 200 μL of the general sample processing reagent from Table 2 to each centrifuge tube, place the centrifuge tube in a constant temperature water bath, and treat at 100°C for 10-15 minutes.

[0046] Remove the centrifuge tubes from the constant temperature water bath, vortex mix for 10-30 seconds, then centrifuge for 10 seconds using a handheld centrifuge to obtain the supernatant.

[0047] Housekeeping gene primers for rice and maize with different amplicon lengths were designed. Using commercially available PCR amplification reagents, 5 μL of the supernatant was taken for PCR amplification. Finally, the results were detected by agarose gel electrophoresis.

[0048] Test results as follows Figure 1 As shown, the results indicate that after simple treatment with the universal sample processing reagent of the present invention, the supernatant can be obtained for PCR amplification, and target genes of different lengths can be successfully amplified, indicating that the universal sample processing reagent of the present invention can process different samples and achieve good results.

[0049] Experiment Example 2

[0050] The following are the steps for rapid identification of sweet potato leaf curl virus (SPLCV) infection in sweet potato leaves using the qPCR probe method in this experimental example: Collect three sweet potato leaves that have shown initial symptoms of infection with sweet potato leaf curl virus. Use sterile scissors to cut the infected parts of the leaves into small pieces of about 0.5 cm and put them into 1.5 mL centrifuge tubes. Add the general sample processing reagents in Table 2 to the centrifuge tubes and place the centrifuge tubes in a constant temperature water bath and treat them at 100℃ for 10-15 min.

[0051] Remove the centrifuge tubes from the constant temperature water bath, vortex mix for 10-30 seconds, then centrifuge for 10 seconds using a handheld centrifuge to obtain the supernatant. We designed sweet potato leaf curl virus-specific primer pairs and probes, and used commercially available quantitative real-time PCR reagents to amplify the supernatant.

[0052] The experimental results are as follows: Figure 3 This is a qPCR amplification curve for sweet potato leaf curl virus. Figure 4 The image shows the amplification curve of the sweet potato internal reference gene. The results indicate that the universal sample processing reagent of this invention can quickly detect sweet potato leaf curl virus infection without nucleic acid extraction and purification, providing a rapid pathogen screening guarantee for the large-scale planting and promotion of sweet potato industry.

[0053] Experimental Example 3

[0054] This experimental example demonstrates the application of the universal sample processing reagent of this invention in the rapid detection of pathogens in the animal and aquaculture industries. The experimental procedure is as follows: The experiment focused on detecting liver parasites in shrimp, specifically the rapid detection of Enterocytozoon hepatopenaei (EHP).

[0055] Purchased shrimp enterocytozoon nucleic acid detection kit: Sample processing: A. Place small shrimp larvae, small fish larvae, or other small animal tissues into a homogenizing bag and thoroughly crush the bag with a glass jar to grind the tissues; or place the tissues into a 1.5mL centrifuge tube and repeatedly cut and shred them with sterile small scissors (which can be rinsed with clean water and then cauterized with an open flame, or wiped with 75% alcohol). Transfer 10mg (rice grain size) to 20mg (mung bean size) of the shredded tissues into a new centrifuge tube.

[0056] B, Aquaculture water sample: Filter more than 500 mL of aquaculture water using a water-based filter membrane with a pore size of 0.22 μm. After removing the filter membrane, cut it into small pieces and put it into centrifuge tubes.

[0057] C. Add 3 times the volume (10mg tissue is counted as 100uL) of the general sample processing reagent in Table 2 to the centrifuge tube containing the tissue fragments or filter membrane. Place the tube in a constant temperature water bath or metal bath and incubate at 85-95℃ for 10-15 minutes. Then remove the centrifuge tube, vortex to mix, and centrifuge at the maximum speed for 1 minute. Take 5-10uL of the supernatant for qPCR experiments.

[0058] qPCR detection: The reaction system was prepared according to the instructions of the purchased shrimp enterocytozoon nucleic acid detection kit. 5 μL of the supernatant after step C was used as a template for qPCR detection.

[0059] The test results are as follows: Figure 2 ,Depend on Figure 2 It is known that the release agent of the present invention can rapidly detect pathogens in animal and aquatic industries.

[0060] Experiment Example 4

[0061] The universal sample processing reagent of this invention is also applicable to the field of POCT rapid detection, especially to the rapid diagnosis of key pet pathogens in POCT, such as feline herpesvirus, feline coronavirus, canine coronavirus, feline panleukopenia virus, canine distemper virus, rabies virus, etc.

[0062] The testing procedure is as follows: Take 100uL of the general sample processing reagent from Table 2 and place the reagent into the sample well or sample processing reagent well of the POCT instrument's sample processing cartridge. Then, take 100uL of a pet's nasal or pharyngeal swab sample and add it to the sample well of the cartridge. Start the POCR instrument's testing program, and in the sample processing step of the program, adjust the heating temperature and time to 55℃ for 5-10 minutes. Keep other program parameters unchanged. After the program finishes running, the result report will be available.

[0063] Experimental Example 5

[0064] This experimental example provides a rapid sample processing method for rapid detection of respiratory infection viruses. Take 100 μL of a COVID-19 positive oral swab sample (verified by a third party), add 100 μL of the universal sample processing reagent listed in Table 2, vortex to mix, and incubate at room temperature for 10-15 min. Then, add 5-10 μL of the sample processing solution to the PCR reaction system and perform qPCR detection according to the COVID-19 nucleic acid detection kit instructions. The results are as follows: Figure 5 As shown, the amplification curves, from left to right, are the amplification curves of the internal reference gene, the N region, and the O region, demonstrating that the universal sample processing reagent of this invention has a good pretreatment effect on various types of samples.

[0065] Experimental Example 6

[0066] Isothermal amplification detection of sputum-bound mycobacteria

[0067] This experimental example provides an application of direct processing and amplification of viscous clinical samples such as sputum. Specifically, a clinically validated sputum sample positive for Mycobacterium tuberculosis is taken. The volume of the sputum sample is estimated, or 100-500 μL of sputum sample is aspirated into a centrifuge tube using a 1000 μL pipette tip (without the tip removed). An equal volume of the general sample processing reagent listed in Table 2 is added, and the mixture is vortexed for 1-3 minutes to ensure complete liquefaction of the sputum. The tube is then placed in a constant temperature water bath at 100°C for 10-15 minutes. Finally, the centrifuge tube is removed, and the tube is centrifuged at its maximum speed using a handheld centrifuge or at 6000 rpm using a high-speed centrifuge for 2 minutes. 5 μL of the supernatant is taken as a template and added to the Mycobacterium tuberculosis isothermal amplification detection (fluorescent probe method) reaction system. The fluorescence quantitative PCR amplification program is set according to the instructions of the isothermal amplification detection kit, and the isothermal amplification detection is started.

[0068] the following Figure 6 The effect of treating sputum with the universal sample processing reagent of this invention; Figure 7 The amplification curve is obtained by taking 5 μL of supernatant and adding it to the isothermal amplification system for isothermal amplification detection. The red line is the amplification curve of Mycobacterium tuberculosis, and the green line is the amplification curve of positive control.

[0069] Experimental Example 7

[0070] Color visual isothermal amplification detection can be used for pathogen detection and screening, transgenic identification, species identification, etc.

[0071] The universal sample processing reagent of this invention is also applicable to sample pretreatment related to rapid isothermal amplification detection using colorimetric visual methods. Specifically, according to different sample types, add an equal volume of the universal sample processing reagent in Table 1, perform sample pretreatment according to different sample types, and release nucleic acids into the liquid.

[0072] Sample pretreatment: Appropriate pretreatment methods are applied to different samples to release their nucleic acids into a solution for use in downstream experiments. These downstream experiments are related to gene and nucleic acid experiments, which will not be elaborated here. The main purpose of this invention is to determine how to pretreat samples, with different processing methods for different sample types. Specifically: Pretreatment method for plant leaves: Cut the leaves into small square pieces of about 0.5cm with sterile scissors, put them into 1.5mL centrifuge tubes, add 100uL-200uL (the liquid should completely cover the leaf tissue by about one-fold), place the tubes on a constant temperature water bath, incubate at 95℃-100℃ for 10min-20min, then vortex to mix, and briefly centrifuge for 10s with a handheld centrifuge. Take 5-10uL of the supernatant for downstream experiments.

[0073] Pretreatment method for plant roots, stems and fruits: First, crush the roots, stems and fruits into pieces using a mortar and pestle or equivalent tool. Take about 0.1g-0.5g and put it into a centrifuge tube. Add 3 times the volume of the universal sample processing reagent of this invention. Place the tube on a constant temperature water bath and incubate at 95℃-100℃ for 10min-20min. Then, vortex to mix and briefly centrifuge for 10s using a handheld centrifuge. Take 5uL-10uL of the supernatant for downstream experiments.

[0074] Pretreatment method for liquids such as blood, saliva, and swab preservation solution: Take 10uL-100uL of sample into a centrifuge tube, add 1-4 times the volume of the universal sample processing reagent of this invention, vortex mix well, place it on a constant temperature water bath, incubate at 65℃~85℃ for 10min-20min, then vortex mix well, briefly centrifuge for 10s with a handheld centrifuge, and take 5-10uL of supernatant for downstream experiments.

[0075] Pretreatment method for viscous samples such as sputum: Cut off a small section of the pipette tip with scissors to enlarge the tip opening. Then, using a large-capacity pipette, aspirate approximately 100-500 μL of sample into a centrifuge tube. Add 1.5-2 times the volume of the universal sample processing reagent of this invention, vortex to mix for 1-3 minutes, and then place the tube in a constant temperature water bath. Incubate at 65-95°C for 10-20 minutes. After vortexing, briefly centrifuge for 10 seconds using a handheld centrifuge. Take 5-10 μL of the supernatant for downstream experiments.

[0076] Pretreatment method for microbial samples such as bacterial suspension and bacterial cells: Place the sample into a 1.5mL centrifuge tube, add 100uL-200uL (the liquid should completely cover the leaf tissue by about one-fold), place it on a constant temperature water bath, incubate at 95℃-100℃ for 10min-20min, then vortex to mix, briefly centrifuge for 10s with a handheld centrifuge, and take 5-10uL of the supernatant for downstream experiments.

[0077] For other sample types, adjust the volume of the sample processing reagents, incubation temperature, and time as appropriate.

[0078] Here is a specific example: A clinically validated oral swab sample of H1N1 influenza A virus infection was selected. 100 μL of the swab sample was transferred to a 1.5 mL centrifuge tube, and the general sample processing reagents listed in Table 1 were added. The sample was processed according to the above-described sample type processing method. 5 μL of the processed sample supernatant was added to a colorimetric visual isothermal amplification detection reagent (purchased externally). A negative control and a positive control (a nucleic acid sample extracted from this swab sample using a traditional magnetic bead method) were set up. The amplification reaction was repeated with the two samples. The sample was stabilized in a constant temperature metal bath according to the instructions and amplified for 60 minutes. The results are as follows: Figure 8As shown in the results, the negative control (pink reaction tube on the left) maintained its pink color before the amplification reaction, while the positive control (second reaction on the left) and the two replicate reactions all showed a bright yellow color change, indicating successful amplification and a positive sample test. This further demonstrates that the universal sample processing reagent of this invention has good pretreatment effects on various types of samples.

[0079] Experimental Example 8

[0080] This invention is also applicable to isothermal amplification detection of related gene variations such as SNPs using color visual inspection or ion indicator methods, and can be used for species identification, pathogen resistance, and genotype confirmation in precision medicine.

[0081] Two samples of botrytis cinerea infected with corn were collected, and two samples each of those showing resistance to fungicides and those showing no resistance were selected. Nucleic acid extraction and first-generation sequencing were performed to verify the N230I variant site.

[0082] Then, the present invention was used to perform nucleic acid release treatment on these four samples. The supernatant was directly added to the N230I variant detection visualization LAMP assay reagent and reacted at 65°C for 40 min. The results are shown in [Figure 1]. Figure 9 .

[0083] Depend on Figure 9 Therefore, this invention is applicable to SNP detection requirements with higher demands. Correspondingly, it is also applicable to qPCR or RT-qPCR detection systems with stronger inhibitor tolerance, which will not be elaborated further here.

[0084] This invention enables pre-processing of samples suitable for nucleic acid amplification systems such as qRT-PCR, LAMP, and CRISPR-Cas12a, and is particularly suitable for viral load monitoring (e.g., HCV RNA quantification), rapid microbial identification, plant and animal polymorphism detection, rapid gene cloning, rapid detection of plant and animal pathogens, and plant and animal quarantine and prevention. By directly lysing whole blood samples, it achieves a "sample in - result out" POCT testing mode, shortening the conventional 40-minute molecular testing process to within 15 minutes, meeting the time-sensitive needs of emergency departments and ICUs. Eliminating purification steps such as magnetic bead adsorption and alcohol precipitation reduces the cost per test by approximately 40% and avoids the risk of sample loss due to nucleic acid purification.

[0085] This sample processing reagent, through the combination of surfactants, anionic detergents, and mucin-dissolving reagents, selects appropriate temperature treatment methods for different sample types. It can lyse the cell walls or cell membranes of the samples, degrade nucleic acid-binding proteins, and release the nucleic acids in the samples into the solution. Under the action of nucleic acid protectants, it prevents the nucleic acids from being oxidized and degraded. It can directly perform downstream molecular experiments such as PCR, qPCR, and isothermal amplification LAMP without any purification, simplifying the entire detection process and saving a lot of manpower and economic costs. It has the advantages of rapid detection, high sensitivity, strong specificity, and good reliability.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nucleic acid extraction and processing reagent suitable for general-purpose samples, characterized in that, Includes the following components: The pH buffer component includes one or more of Tris-HCl, sodium acetate-acetic acid, and sodium citrate-citric acid, wherein the concentration of the pH buffer component is 10mM to 100mM, and the pH buffer component adjusts the pH of the sample processing reagent to 7.5 to 8.

8. The surfactant component includes one or more of Triton X-100, NP40, and Tween 20, wherein the volume ratio of the surfactant component is 0.01% to 5%. Cell wall / membrane lysis components, comprising strong lysing agents and moderate lysing agents, wherein the strong lysing agent comprises one or more of guanidine isothiocyanate, guanidine hydrochloride, and urea, and the moderate lysing agent comprises one or more of SDS and CTAB, wherein the concentrations of guanidine isothiocyanate and guanidine hydrochloride are 5 mM to 100 mM, the mass-to-volume ratio of SDS and CTAB is 0.01% to 0.5%, and the concentration of urea is 10 mM to 100 mM; The soluble components of the viscous protein and polysaccharide include one or a combination of two of acetylcysteine ​​and bromhexine hydrochloride; NaOH; PVP40, wherein the concentration of acetylcysteine ​​is 1mM~50mM, the concentration of NaOH is 0.1mM~1M, the concentration of bromhexine hydrochloride is 1mM~100mM, and the concentration of PVP40 is 0.1%~10%; The nucleic acid protective agent components include DTT, TCEP, and EDTA2Na, wherein the concentration of DTT or TCEP is 0.1mM~50mM, and the concentration of EDTA2Na is 1mM~20mM. The PCR enhancer components include BSA and / or single-stranded binding protein, KCl and / or MgCl2, PEG400~8000, betaine, trehalose and / or DMSO; wherein the concentration of BSA is 0.1 mg / mL~5 mg / mL, the concentration of single-stranded binding protein is 0.01 mg / mL~1 mg / mL, the concentration of KCl is 10 mM~200 mM, the concentration of MgCl2 is 1 mM~30 mM, the mass percentage of PEG is 0.5%~10%, the concentration of betaine is 100 mM~2 M, and the volume percentage of DMSO is 0.5%~10%.

2. The nucleic acid extraction and processing reagent suitable for general-purpose samples according to claim 1, characterized in that, The molecular weight of the PEG is any one or more of 400, 800, 1000, 4000, 6000, and 8000.

3. A nucleic acid extraction kit, characterized in that, The reagent comprises the nucleic acid extraction and processing reagent suitable for general-purpose samples as described in claim 1 or 2.

4. A method for nucleic acid extraction, characterized in that, Includes the following steps: The sample containing the nucleic acid to be extracted is prepared into a sample solution. The sample includes one or more of the following: animal and plant tissues, blood, body fluids, sputum, and microbial cultures. Add the nucleic acid extraction reagent suitable for general-purpose samples as described in claim 1 or 2 to the sample solution, vortex to mix, then treat at room temperature to 100°C for 5 min to 20 min, centrifuge for 1 min to 2 min, and collect the supernatant after centrifugation. Claims The supernatant was used as a nucleic acid template for molecular detection experiments.

5. The nucleic acid extraction method according to claim 4, characterized in that, The molecular detection experiments include nucleic acid amplification experiments, viral load monitoring, rapid identification of microorganisms, detection of plant and animal polymorphisms, rapid gene cloning, rapid detection of plant and animal pathogens, and plant and animal quarantine and prevention.

6. The nucleic acid extraction method according to claim 5, characterized in that, The nucleic acid amplification experiments include qRT-PCR, LAMP, and CRISPR-Cas12a.

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