A DNA and RNA co-extraction reaction reagent, kit and extraction method

Abstract

The present application relates to the technical field of nucleic acid extraction and purification, and particularly relates to a DNA and RNA co-extraction reaction reagent and an extraction method, the reaction reagent comprising a nucleic acid protective agent RP, a sample lysis and binding liquid LB, a protein washing liquid WB1, a nucleic acid rinsing liquid WB2, and a nucleic acid elution liquid EB; the nucleic acid protective agent RP comprising DTT, mercaptoethanol, TCEP, and sodium hydroxide; the sample lysis and binding liquid LB comprising Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, triton X-100, SDS, potassium chloride, sodium chloride, bromhexine, N-acetylcysteine, ambroxol hydrochloride, isopropyl alcohol, and anhydrous ethanol; the protein washing liquid WB1 comprising Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, triton X-100, sodium chloride, isopropyl alcohol, and anhydrous ethanol; the nucleic acid rinsing liquid WB2 comprising Tris-HCl, sodium chloride, isopropyl alcohol, and anhydrous ethanol; and the nucleic acid elution liquid EB being nuclease-free water; the reagent can rapidly and fully enrich and purify DNA and RNA of a sample simultaneously.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction and purification technology, specifically to a DNA and RNA co-extraction reaction reagent, kit, and extraction method. Background Technology

[0002] Nucleic acid extraction is a crucial step in pathogen detection, directly impacting the accuracy and sensitivity of subsequent tests. The selection and use of nucleic acid extraction kits are critical to experimental results. Pathogen detection technologies primarily include quantitative real-time PCR, isothermal amplification (LAMP), high-throughput metagenomic sequencing (mNGS), and high-throughput targeted sequencing. These molecular detection techniques all rely on high-quality nucleic acid samples. Therefore, efficient and unbiased enrichment and purification of sample nucleic acids are particularly important in pathogen detection.

[0003] Pathogen detection involves a wide variety of sample types, including blood, plasma, sputum, oral swabs, feces, tissue, bronchoalveolar lavage fluid, cerebrospinal fluid, synovial fluid, and pleural and peritoneal fluid. The types of pathogens are extensive, including fungi, DNA viruses, RNA viruses, parasites, Gram-positive bacteria, Gram-negative bacteria, and mycoplasma / chlamydia. Especially for the detection of unknown pathogens, such as mNGS, it is required that all microbial nucleic acids be enriched and purified without deviation from the sample type to ensure the accuracy and sensitivity of the detection.

[0004] There are many nucleic acid extraction kits on the market. Although some nucleic acid extraction kits can enrich, purify and extract DNA and RNA nucleic acids from samples at the same time, they still have shortcomings: the reaction reagents of existing kits cannot fully lyse and disrupt target cells when the sample composition is complex. They also require sample pretreatment such as physical manual grinding with zirconium beads or glass beads, which leads to complicated procedures. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art by providing a DNA and RNA co-extraction reaction reagent. This DNA and RNA co-extraction reaction reagent can quickly and fully enrich and purify the DNA and RNA of the sample simultaneously by simply mixing it with the sample. It has the advantages of low cost and ease of operation.

[0006] To achieve one of the above objectives, the present invention provides the following technical solution:

[0007] A DNA and RNA co-extraction reaction reagent is provided, including nucleic acid protectant RP, sample lysis binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB;

[0008] The nucleic acid protective agent RP includes DTT, mercaptoethanol, TCEP, and sodium hydroxide;

[0009] The sample lysis binding solution LB includes Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, Triton 100, SDS, potassium chloride, sodium chloride, bromhexine, N-acetylcysteine, ambroxol hydrochloride, isopropanol, and anhydrous ethanol.

[0010] The protein washing solution WB1 includes Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, Triton 100, sodium chloride, isopropanol, and anhydrous ethanol.

[0011] The nucleic acid washing solution WB2 includes Tris-HCl, sodium chloride, isopropanol, and anhydrous ethanol;

[0012] The nucleic acid elution buffer EB is nuclease-free water.

[0013] In some embodiments, the nucleic acid protectant RP contains DTT at a concentration of 0.1 mM to 20 mM, mercaptoethanol at a concentration of 0.1% to 10%, TCEP at a concentration of 0.1 mM to 30 mM, sodium hydroxide at a concentration of 10 mM to 200 mM, and a solution pH of 4.5 to 8.0.

[0014] In some embodiments, the sample lysis binding buffer LB contains Tris-HCl at a concentration of 10 mM to 500 mM, benzenesulfonic acid at a concentration of 5 mM to 100 mM, sodium citrate at a concentration of 10 mM to 100 mM, guanidine hydrochloride at a concentration of 0.5 M to 5.5 M, guanidine isothiocyanate at a concentration of 0.5 M to 5.5 M, guanidine thiocyanate at a concentration of 0.5 M to 4 M, urea at a concentration of 10 mM to 1000 mM, and Triton 100 at a concentration of [missing information]. The concentrations of the following components are as follows: pH 1%–20%, SDS concentration 0.5%–2%, potassium chloride concentration 10mM–200mM, sodium chloride concentration 50mM–500mM, bromhexine concentration 5mM–50mM, N-acetylcysteine ​​concentration 5mM–50mM, ambroxol hydrochloride concentration 5mM–50mM, isopropanol concentration 10%–40%, anhydrous ethanol concentration 10%–40%, and solution pH 5.0–8.0.

[0015] In some embodiments, the protein washing solution WB1 contains Tris-HCl at a concentration of 10 mM to 500 mM, benzenesulfonic acid at a concentration of 5 mM to 100 mM, sodium citrate at a concentration of 10 mM to 100 mM, guanidine hydrochloride at a concentration of 0.5 M to 5.5 M, guanidine isothiocyanate at a concentration of 0.5 M to 5.5 M, guanidine thiocyanate at a concentration of 0.5 M to 4 M, urea at a concentration of 10 mM to 1000 mM, Triton 100 at a concentration of 1% to 20%, sodium chloride at a concentration of 50 mM to 500 mM, isopropanol at a concentration of 10% to 40%, anhydrous ethanol at a concentration of 10% to 40%, and a solution pH of 5.0 to 8.0.

[0016] In some embodiments, the nucleic acid washing solution WB2 contains Tris-HCl at a concentration of 10 mM to 500 mM, sodium chloride at a concentration of 50 mM to 500 mM, isopropanol at a concentration of 50% to 85%, anhydrous ethanol at a concentration of 50% to 85%, and a solution pH of 6.5 to 8.0.

[0017] A kit for co-extraction of DNA and RNA is also provided, comprising the aforementioned DNA and RNA co-extraction reaction reagents.

[0018] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0019] A method for co-extracting DNA and RNA is provided, comprising the following steps, using the aforementioned DNA and RNA co-extraction reaction reagent;

[0020] Step 1: Obtain the sample and adjust it into a solid-liquid mixture containing physiological saline. Soak the sample in the physiological saline to obtain the sample to be tested.

[0021] Step 2: Add lysis binding solution RB and nucleic acid protection solution RP to the solid-liquid mixture. If the sample is a sputum sample, add the nucleic acid protection solution RP in step 1, mix thoroughly, and obtain the mixture.

[0022] The mixture was heated in a water bath at 55℃~70℃ for 10min~15min, cooled to room temperature, and then nucleic acid adsorption magnetic beads were added and shaken to mix, so that the nucleic acid in the sample could bind to the magnetic beads. Then the mixture was placed in a magnetic rack to adsorb the magnetic beads, and the liquid was removed to obtain the first magnetic beads.

[0023] Step 3: Add protein washing solution WB1 to the first magnetic bead, mix well until the first magnetic bead is fully suspended, then place it in a magnetic rack to adsorb the first magnetic bead and remove the liquid to obtain the second magnetic bead;

[0024] Step 4: Add nucleic acid washing solution WB2 to the second magnetic bead, mix well until the second magnetic bead is fully suspended, then place it in a magnetic rack to adsorb the second magnetic bead and remove the liquid to obtain the third magnetic bead;

[0025] Step 5: Centrifuge the third magnetic bead until its surface no longer reflects light;

[0026] Step 6: Add nucleic acid elution buffer EB to elute the DNA and RNA bound to the surface of the third magnetic bead. After mixing thoroughly, centrifuge and then place the magnetic beads in a magnetic rack to fix them. The remaining liquid is a mixed nucleic acid solution for enriching and purifying DNA and RNA.

[0027] In some embodiments, the magnetic beads are surface carboxylated magnetic beads or surface hydroxylated magnetic beads.

[0028] In some embodiments, the magnetic bead particle size is 100nm to 1000nm.

[0029] In some implementations, in step one,

[0030] When the sample is a liquid sample such as urine, pleural effusion, peritoneal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, or blood, the sample should be removed and equilibrated to room temperature before being used as the sample to be tested.

[0031] When the sample is a dry swab sample, add physiological saline to submerge the dry swab, shake to mix, and use it as the sample to be tested;

[0032] When the sample contains a swab and a preservation solution, if there is a large amount of preservation solution, the sample should be centrifuged, the supernatant discarded, and the precipitate resuspended in physiological saline as the sample to be tested.

[0033] When the sample is a fecal sample, if the fecal sample does not contain a preservation solution, add physiological saline and mix well to use as the test sample; if the fecal sample contains a preservation solution, mix well to use as the test sample.

[0034] When the sample is a tissue sample, add an appropriate amount of physiological saline as the sample to be tested;

[0035] When the sample is viscous sputum, it is liquefied by adding nucleic acid protection solution RP and used as the sample to be tested;

[0036] When the sample is a paraffin specimen, the paraffin specimen is scraped off and a dewaxing agent is added. It is then incubated at 50℃~60℃, centrifuged, the supernatant is removed, and physiological saline is added and mixed evenly to obtain the sample to be tested.

[0037] In some embodiments, the magnetic frame adsorbs the magnetic beads for 1 to 2 minutes.

[0038] The beneficial effects of the DNA and RNA co-extraction reaction reagent of the present invention are as follows:

[0039] (1) The DNA and RNA co-extraction reaction reagent of the present invention includes nucleic acid protectant RP, sample lysis binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB. Among the above components, Tris-HCl, benzenesulfonic acid, and sodium citrate can stabilize the pH of the reaction reagent, prevent the reaction effect of each reagent from being affected by pH fluctuation, and maintain the binding efficiency of DNA and RNA with magnetic beads; DTT, mercaptoethanol, and TCEP are used to prevent nucleic acid oxidative degradation and ensure that the nucleic acid can maintain its structure and quality during the reaction; guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, Triton 100, and SDS are used to dissolve proteins in the sample and improve the lysis and cell wall disruption of the sample. Efficiency is maintained to ensure sufficient enrichment and purification of DNA and RNA; potassium chloride and sodium chloride are used to increase the salt strength of the solution, improving the lysis and cell disruption strength; bromhexine, N-acetylcysteine, and ambroxol hydrochloride are used to reduce the viscosity of sample mucopolysaccharides or mucins, allowing each component to act fully on the sample and improving the efficiency of lysis and cell disruption; PEG6000, PEG8000, spermidine, anhydrous ethanol, and isopropanol modify the surface of magnetic beads and nucleic acids, so that the magnetic beads can achieve a balance in efficiency whether adsorbing DNA or RNA, promoting the binding of DNA and RNA to the magnetic beads. Therefore, there is no need to use zirconium beads or glass beads for physical manual grinding of the sample pretreatment, effectively improving the efficiency of enrichment and purification of DNA and RNA.

[0040] The beneficial effects of the DNA and RNA co-extraction method of the present invention are as follows:

[0041] (1) The present invention provides a method for co-extraction of DNA and RNA. By using the above-mentioned reaction reagents, the sample can be fully lysed and cell walls broken without the need for physical grinding conditions such as zirconium beads and glass beads, thereby efficiently enriching and purifying DNA and RNA at the same time, realizing one-step extraction of DNA and RNA, improving extraction efficiency, and suitable for large-scale production and application.

[0042] (2) The present invention provides a method for co-extraction of DNA and RNA, which eliminates the need for sample pretreatment by grinding, effectively avoids sample loss, saves sample volume, and helps improve detection accuracy.

[0043] (3) The present invention provides a method for co-extraction of DNA and RNA. Due to the use of the above-mentioned reaction reagents, it can fully remove substances that affect nucleic acid detection. It is suitable for a variety of complex samples and has the flexibility of use. For various types of clinical samples, DNA and RNA can be co-extracted by manual extraction, magnetic rod automatic extraction, or pipetting automatic extraction according to the actual situation. It can better adapt to different application scenarios and clinical needs and effectively expand the detection range. Attached Figure Description

[0044] Figure 1 These are agarose gel electrophoresis images of nucleic acids co-extracted from DNA and RNA of different clinical samples. Detailed Implementation

[0045] 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.

[0046] Example 1

[0047] The DNA and RNA co-extraction reaction reagents disclosed in this embodiment include nucleic acid protectant RP, sample lysis binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB.

[0048] The amounts of the above-mentioned nucleic acid protectant RP, sample lysis binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB can be selected according to actual needs, as long as the sample can be fully mixed with each component separately, and the above-mentioned nucleic acid protectant RP, sample lysis binding buffer LB, protein washing buffer WB1, nucleic acid rinsing buffer WB2, and nucleic acid elution buffer EB are stored independently before use and are not mixed together.

[0049] The nucleic acid protectant RP includes DTT (dithiothreitol), mercaptoethanol, TCEP (tris(2-carboxyethyl)phosphine), and sodium hydroxide. The amount of each component in the nucleic acid protectant RP can be selected according to actual needs.

[0050] The sample lysis binding solution LB includes Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, Triton 100, SDS, potassium chloride, sodium chloride, bromhexine, N-acetylcysteine, ambroxol hydrochloride, isopropanol, and anhydrous ethanol; the amount of each component of the lysis binding solution LB can be selected according to actual needs.

[0051] The protein washing solution WB1 includes Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, Triton 100, sodium chloride, isopropanol, and anhydrous ethanol; the amount of each component in the protein washing solution WB1 can be selected according to actual needs.

[0052] The nucleic acid washing solution WB2 includes Tris-HCl, sodium chloride, isopropanol, and anhydrous ethanol; the amounts of each component in the nucleic acid protectant RP can be selected according to actual needs.

[0053] The nucleic acid elution buffer EB is nuclease-free water.

[0054] In the nucleic acid protective agent RP, the final concentration of DTT is 0.1mM to 20mM, mercaptoethanol is 0.1% to 10%, TCEP is 0.1mM to 30mM, sodium hydroxide is 10mM to 200mM, and the solution pH is 4.5 to 8.0.

[0055] In this embodiment, the sample lysis binding buffer LB contains the following concentrations: Tris-HCl (10 mM–500 mM), benzenesulfonic acid (5 mM–100 mM), sodium citrate (10 mM–100 mM), guanidine hydrochloride (0.5 M–5.5 M), guanidine isothiocyanate (0.5 M–5.5 M), guanidine thiocyanate (0.5 M–4 M), urea (10 mM–1000 mM), and Triton 100 (…). The concentrations of the following components are as follows: 1%–20%, SDS concentration 0.5%–2%, potassium chloride concentration 10mM–200mM, sodium chloride concentration 50mM–500mM, bromhexine concentration 5mM–50mM, N-acetylcysteine ​​concentration 5mM–50mM, ambroxol hydrochloride concentration 5mM–50mM, isopropanol concentration 10%–40%, anhydrous ethanol concentration 10%–40%, and solution pH 5.0–8.0.

[0056] In this embodiment, the protein washing solution WB1 contains Tris-HCl at a concentration of 10mM to 500mM, benzenesulfonic acid at a concentration of 5mM to 100mM, sodium citrate at a concentration of 10mM to 100mM, guanidine hydrochloride at a concentration of 0.5M to 5.5M, guanidine isothiocyanate at a concentration of 0.5M to 5.5M, guanidine thiocyanate at a concentration of 0.5M to 4M, urea at a concentration of 10mM to 1000mM, Triton 100 at a concentration of 1% to 20%, sodium chloride at a concentration of 50mM to 500mM, isopropanol at a concentration of 10% to 40%, anhydrous ethanol at a concentration of 10% to 40%, and a solution pH of 5.0 to 8.0.

[0057] In this embodiment, the nucleic acid washing solution WB2 has a Tris-HCl concentration of 10mM to 500mM, a sodium chloride concentration of 50mM to 500mM, an isopropanol concentration of 50% to 85%, an anhydrous ethanol concentration of 50% to 85%, and a solution pH of 6.5 to 8.0.

[0058] The appropriate concentration ratio of the above components can achieve sufficient lysis and cell wall disruption of the sample to obtain nucleic acid.

[0059] Prepare the above-mentioned DNA and RNA co-extraction reaction reagents, and implement the DNA and RNA co-extraction method, including the following steps:

[0060] Step 1: Obtain the sample and adjust it into a solid-liquid mixture containing physiological saline. Soak the sample in the physiological saline to prepare it as the sample to be tested. Specifically, when the sample itself contains biological fluid or the biological fluid is insufficient, physiological saline can be added to adjust the sample to a solid-liquid mixture state, which is convenient for subsequent testing.

[0061] Specifically, the samples are clinical samples, which may include: urine, pleural effusion, peritoneal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, blood, and other bodily fluid samples; swab samples; stool samples; tissue samples; sputum; and tissue sections. They may also be other clinical samples, and in practical applications are not limited to the types of samples listed above.

[0062] Step 2: Add lysis binding solution RB and nucleic acid protection solution RP to the solid-liquid mixture to lyse the cells in the sample and protect the lysed nucleic acids.

[0063] When the sample is a sputum sample, in step one, nucleic acid protection solution RP is added to liquefy the sputum, and the mixture is mixed evenly to obtain a mixture.

[0064] The mixture is heated in a water bath at 55-70°C for 10-15 minutes and then cooled to room temperature. Magnetic beads for nucleic acid adsorption are then added and the mixture is shaken to mix, so that the nucleic acid in the sample binds to the magnetic beads. The mixture is then placed in a magnetic rack to adsorb the magnetic beads and remove the liquid to obtain the first magnetic beads. The first magnetic beads with nucleic acid bound are fixed by the adsorption of the magnetic rack, thus achieving the first purification of nucleic acid.

[0065] Step 3: Add protein washing solution WB1 to the first magnetic bead to wash the protein on the nucleic acid adsorbed on the first magnetic bead. Mix evenly until the first magnetic bead is fully suspended. Then place it in a magnetic rack to adsorb the first magnetic bead and remove the liquid to obtain the second magnetic bead. The nucleic acid on the second magnetic bead has been deproteinized.

[0066] Step 4: Add nucleic acid washing solution WB2 to the second magnetic bead, mix well until the second magnetic bead is fully suspended, then place it in a magnetic rack to adsorb the second magnetic bead and remove the liquid to obtain the third magnetic bead. The nucleic acid on the third magnetic bead is further purified.

[0067] Step 5: Centrifuge the third magnetic bead until its surface is no longer reflective. This allows the nucleic acid to adhere further to the surface of the magnetic bead, maximizing the collection of nucleic acid.

[0068] Step Six: Add nucleic acid elution buffer EB to elute the DNA and RNA bound to the surface of the third magnetic bead. After mixing thoroughly, centrifuge and then fix the magnetic beads in a magnetic rack. The remaining liquid is a mixed nucleic acid solution enriched and purified from DNA and RNA. At this point, elute the purified nucleic acids from the magnetic beads into the solution and collect them.

[0069] The magnetic beads are surface carboxylated or surface hydroxylated magnetic beads, with a particle size of 100nm to 1000nm. The magnetic beads have excellent DNA and RNA adsorption capacity.

[0070] In step one, when the sample is a liquid sample such as urine, pleural effusion, peritoneal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, or blood, the sample is taken out and equilibrated to room temperature before being used as the sample to be tested.

[0071] When the sample is a dry swab sample, add physiological saline to submerge the dry swab, shake to mix, and use it as the sample to be tested;

[0072] When the sample contains a swab and a preservation solution, if there is a large amount of preservation solution, the sample should be centrifuged, the supernatant discarded, and the precipitate resuspended in physiological saline as the sample to be tested.

[0073] When the sample is a fecal sample, if the fecal sample does not contain a preservation solution, add physiological saline and mix well to use as the test sample; if the fecal sample contains a preservation solution, mix well to use as the test sample.

[0074] When the sample is a tissue sample, add an appropriate amount of physiological saline as the sample to be tested;

[0075] When the sample is viscous sputum, it is liquefied by adding nucleic acid protection solution RP and used as the sample to be tested;

[0076] When the sample is a paraffin specimen, the paraffin specimen is scraped off and a dewaxing agent is added. It is then incubated at 50-60°C, centrifuged, the supernatant is removed, and physiological saline is added and mixed evenly to obtain the sample to be tested.

[0077] The magnetic frame adsorbs the magnetic beads for 1 to 2 minutes, and the specific time can be adjusted according to the actual situation.

[0078] The above reagents and methods can complete the enrichment and purification of all nucleic acids, including DNA and RNA. The extracted DNA & RNA mixed nucleic acid can be applied to the simultaneous single-tube detection of all pathogenic microorganisms, including fungi, bacteria, DNA viruses and RNA viruses, as well as the simultaneous detection of DNA and RNA of tumor fusion genes. This not only saves precious clinical sample volume, but also saves repetitive personnel operation time and manpower requirements, meeting the current needs of molecular experiments.

[0079] Based on the method of this invention, a DNA & RNA co-extraction kit using magnetic beads has been developed. The DNA & RNA co-extraction method and kit of this invention do not rely on physical grinding conditions such as zirconium beads or glass beads during sample processing, exhibiting high operational and sample compatibility. For various types of clinical samples, DNA & RNA co-extraction can be performed using manual extraction, automated magnetic rod extraction, or automated pipetting extraction, adapting to different application scenarios and clinical needs.

[0080] Example 2

[0081] To illustrate the method for co-extracting DNA and RNA of the present invention in more detail, the DNA and RNA co-extraction reaction reagent of Example 1 is used. This example discloses the following steps:

[0082] Step 1: Sample pretreatment, including

[0083] If the sample is a body fluid such as urine, pleural effusion, peritoneal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, or blood, remove the sample and allow it to equilibrate to room temperature before proceeding to step two.

[0084] If the sample is a swab sample:

[0085] For dry swab samples, add a certain volume (enough to submerge the swab) of physiological saline, vortex to mix, and then use. For samples containing swabs and preservation solution, vortex to mix, and then take 300 μL for step 2. If the sample volume is >400 μL, transfer the sample to a 1.5 mL nuclease-free centrifuge tube, centrifuge at 12000 rpm (13800 × g) for 5 min, discard the supernatant, resuspend the precipitate with 350 μL of physiological saline, and take 300 μL for step 2.

[0086] If the sample is a fecal sample:

[0087] For samples without preservation solution, add 5 times the volume of physiological saline, vortex to mix, and proceed to the next step. For samples containing preservation solution, vortex to mix, and then take 300 μL for step two.

[0088] If the sample is a tissue sample:

[0089] Take a tissue block, add an appropriate amount of physiological saline, and then proceed to step two.

[0090] If the sample is sputum

[0091] If the sputum is viscous, add about 1 / 6 of the sputum volume of nucleic acid protection solution RP in advance to liquefy it, and then take 300ul volume for step two.

[0092] If the sample is a slice

[0093] Take paraffin-coated specimens from 8-10 glass slides, scrape them all off with a disposable blade, transfer them to an EP tube, add 320 μL of dewaxing agent, incubate at 56°C for 3 min, centrifuge at 12000 rpm for 1 min to remove the upper layer of dewaxing agent. Add 300 μL of physiological saline to the centrifuge tube and proceed to step two.

[0094] Step 3: After processing the above samples, add 300 μL of sample to a 1.5 mL centrifuge tube, along with 400 μL of lysis binding buffer RB, 50 μL of nucleic acid protection buffer RP (if it is a sputum sample, RP was already added during pretreatment, so it does not need to be added again in this step), and 20 μL of Proteinase K. Tightly cap the tube. Incubate the centrifuge tube on a metal bath at 65°C for 10-15 minutes.

[0095] Step 4: Remove the centrifuge tube from the metal bath and allow it to cool to room temperature. Add 20 μL of nucleic acid adsorption magnetic beads, vortex to mix for 30 seconds, and let stand for 10 minutes. Mix once every 1-2 minutes during the process.

[0096] Step 5: Place the centrifuge tube on the magnetic rack for 2 minutes. Once the magnetic beads are fully adsorbed, carefully remove the liquid with a pipette.

[0097] Step 6: Add 750 μL of protein washing buffer WB1 and vortex for 2 minutes to fully suspend the magnetic beads.

[0098] Step 7: Place the centrifuge tube on the magnetic rack for 1 minute. Once the magnetic beads are fully adsorbed, carefully remove the liquid with a pipette.

[0099] Step 8: Add 750 μL of nucleic acid washing buffer WB2 and vortex for 2 minutes to fully suspend the magnetic beads.

[0100] Step 9: Place the centrifuge tube on the magnetic rack for 1 minute. Once the magnetic beads are fully adsorbed, carefully remove the liquid with a pipette.

[0101] Step 10: After a brief centrifugation, place the tube on a magnetic rack to remove any residual droplets at the bottom. Allow it to air dry at room temperature for 3-5 minutes until the surface of the magnetic beads is no longer reflective.

[0102] Step 11: Add 50-100 μL of nucleic acid elution buffer EB, vortex to mix for 5 min, and then briefly centrifuge.

[0103] Step 12: Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed, carefully transfer the nucleic acid solution to a new EP tube to obtain a mixed nucleic acid of DNA and RNA, and store it at -20℃.

[0104] Test case verification

[0105] Experimental Example 1

[0106] Experiment 1 used different clinical samples: blood, bronchoalveolar lavage fluid, sputum, cerebrospinal fluid, swabs, and paraffin-embedded tissue sections, with each sample repeated twice. 1000 copies of a SARS-CoV-2 pseudovirus standard were added to each experiment to verify the RNA extraction efficiency.

[0107] Following the procedure in Example 2, DNA and RNA were co-extracted manually. Finally, the nucleic acid concentration and purity were detected using a spectrophotometer, and nucleic acid band analysis was performed using agarose gel electrophoresis. The results are shown in Table 1. Figure 1 Agarose gel electrophoresis images of co-extracted nucleic acids (DNA and RNA) from different clinical samples.

[0108] Finally, qPCR was performed using primers and probes for the SARS-CoV-2 pseudovirus, primers and probes for the human internal reference gene GAPDH, and mRNA primers and probes for the human internal reference gene GAPDH, respectively, to determine the DNA and RNA extraction status. The results are shown in Table 2: qPCR detection results for SARS-CoV-2 pseudovirus, DNA internal reference, and RNA internal reference.

[0109] Table 1. Results of co-extraction of DNA and RNA from different clinical samples

[0110] Serial Number sample repeat Nucleic acid concentration ng / uL A260 / A280 1 blood 1 60.686 2.03 2 blood 2 63.615 2.05 3 bronchoalveolar lavage fluid 1 43.913 1.93 4 bronchoalveolar lavage fluid 2 36.352 1.88 5 sputum 1 28.467 1.91 6 sputum 2 35.282 2.06 7 swab 1 23.957 1.72 8 swab 2 26.742 1.81 9 Cerebrospinal fluid 1 14.155 1.75 10 Cerebrospinal fluid 2 13.483 1.81 11 Paraffin sections 1 26.892 1.95 12 Paraffin sections 2 23.730 2.03

[0111] Table 2. qPCR detection results of SARS-CoV-2 pseudovirus, DNA internal control, and RNA internal control.

[0112]

[0113] The results above show that the present invention can simultaneously enrich and purify DNA and RNA from a variety of clinical samples, including all nucleic acids, including viral RNA. It is applicable to various sample types and suitable for large-scale production and application.

[0114] Experimental Example 2

[0115] To demonstrate that this invention can perform unbiased enrichment and purification of all microorganisms in clinical samples, Experiment 2 was conducted. Specifically, using the extraction method of Example 2, 500 copies of standard strains of Cryptococcus neoformans (fungus), Mycobacterium tuberculosis (special bacteria), Escherichia coli (Gram-negative bacteria), Staphylococcus aureus (Gram-positive bacteria), and Influenza A (RNA virus) were added to 300 μL of blood for co-extraction of DNA and RNA. Each sample was repeated twice. The nucleic acid concentration and purity were detected using a spectrophotometer. Finally, qPCR was performed using the corresponding microbial primers and probes for verification. The results are shown in Table 3. Results of co-extraction of DNA and RNA for different microbial types.

[0116] Table 3 Results of co-extraction of DNA and RNA from different microbial types

[0117]

[0118]

[0119] As shown in Table 3, the present invention can simultaneously extract and purify all microbial DNA and RNA nucleic acids, including fungi, bacteria, and viruses, from clinical samples, meeting the needs of various downstream clinical applications such as qPCR pathogen diagnosis.

[0120] Experimental Example 3

[0121] Automated co-extraction of DNA and RNA was performed using the magnetic rod method. The same sample protocol and extraction kit as in Experiment 1 were used, and a common automated magnetic rod nucleic acid extractor was employed. The following procedures were performed:

[0122] (1) Add 400 μL of lysis binding buffer LB to well A1 of a 96-well plate, add 20 μL of nucleic acid adsorption magnetic bead solution (50 mg / mL) to well A2, add 750 μL of protein washing buffer WB1 to well A3, add 750 μL of nucleic acid rinsing buffer WB2 to wells A4 and A5 respectively, and add 50-100 μL of nucleic acid elution buffer EB buffer to well A6.

[0123] (2) The program settings are as follows: ① The heating temperature of hole A1 is 65℃, the heating time is 20min, the vertical vibration intensity of the robotic arm is set to level 4, and the vibration amplitude is set to 0%~90%; ② The magnetic rod in hole A2 is set to magnetic attraction time of 30s, then transferred to hole A1, the magnetic rod descends to the bottom of the liquid, the magnetic attraction time is 0 (releasing the magnetic bead), the vertical vibration intensity of the robotic arm is set to level 6, the amplitude is set to 0%~100%, and the duration is set to 15min; ③ The magnetic rod in hole A1 is set to magnetic attraction time of 1min, then transferred to the bottom of the liquid in hole A3, the magnetic bead is released, the vertical vibration intensity of the robotic arm is set to level 9, the vibration time is 10min, then the magnetic attraction time is set to 1min, and transferred to hole A4; ④ The program and parameters for holes A4 and A5 are the same as those for hole A3; ⑤ The parameters for hole A6 are: the magnetic rod stays in the air for 3min, then descends to the bottom of the eluent, the magnetic bead is released, the vertical vibration intensity of the robotic arm is set to level 10, and the duration is set to 5min.

[0124] (3) After the instrument program is set, the clinical sample is pretreated according to the sample pretreatment scheme in Example 2. Then, 300uL of sample is mixed with 50uL of nucleic acid protectant RP and 20uL of proteinase K and added to the A1 well of the 96-well plate. Then, the 96-well plate is placed in the designated position of the instrument and the instrument is started to begin automatic DNA and RNA co-extraction.

[0125] (4) After the instrument runs out, transfer the DNA and RNA solutions in well A6 to a new nuclease-free centrifuge tube to obtain a mixed DNA and RNA solution. Use a spectrophotometer to detect the concentration and purity. The results are shown in Table 4.

[0126] Table 4 Results of automated DNA and RNA co-extraction using the magnetic rod method

[0127]

[0128] As shown in Table 4, the DNA and RNA co-extraction reaction reagents provided by this invention can be directly adapted from manual extraction schemes to automated magnetic rod extraction schemes without any additional steps beyond the manual scheme. It is simple to operate, has wide adaptability, and the concentration and purity of the extracted nucleic acids are not significantly different from those of the manual extraction scheme.

[0129] Test Example 4

[0130] Example 4 of this study provides a clinical application of using mixed nucleic acids of DNA and RNA obtained from the reaction reagents for metagenomic mNGS detection of pathogenic microorganisms.

[0131] Nucleic acid was extracted from the sample in Experiment 3. The DNA and RNA co-construction library kit from Shanghai Yisheng Biotechnology Co., Ltd. was used to construct the library according to the kit instructions. The constructed library was sent to a metagenomic sequencing company for high-throughput sequencing and bioinformatics analysis. The results are shown in Table 5.

[0132] Table 5. Results of mNGS high-throughput sequencing of nucleic acids extracted from both DNA and RNA.

[0133]

[0134]

[0135] In Table 5, the microorganisms with green backgrounds are clearly positive microorganisms in the samples, all of which were detected. The microbial types covered fungi, bacteria, RNA viruses, etc., while the remaining microorganisms were colonizing bacteria in some samples or background bacteria from environmental contamination. The results show that the DNA and RNA co-extraction reaction reagent provided by this invention can extract mixed nucleic acids that, in addition to being applicable to qPCR experiments (Example 3), can also be applied to high-throughput sequencing (NGS). A single nucleic acid extraction can complete the simultaneous detection of DNA and RNA, greatly simplifying the downstream molecular detection process, saving clinical sample volume, and reducing manpower and economic costs.

[0136] 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 DNA and RNA co-extraction reaction reagent, characterized by: The nucleic acid protection agent RP, sample lysis binding liquid LB, protein washing liquid WB1, nucleic acid rinsing liquid WB2 and nucleic acid elution liquid EB are composed of DTT, mercaptoethanol, TCEP and sodium hydroxide; The nucleic acid protection agent RP is composed of DTT, mercaptoethanol, TCEP and sodium hydroxide; The sample lysis binding liquid LB is composed of TrisHCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate and urea, triton 100, SDS, potassium chloride, sodium chloride, bromhexine, N-acetylcysteine, ambroxol hydrochloride, isopropyl alcohol and anhydrous ethanol; The protein washing liquid WB1 is composed of TrisHCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, triton 100, sodium chloride, isopropyl alcohol and anhydrous ethanol; The nucleic acid rinsing liquid WB2 is composed of TrisHCl, sodium chloride, isopropyl alcohol and anhydrous ethanol; The nucleic acid elution liquid EB is nuclease-free water; In the nucleic acid protection agent RP, the concentration of DTT is 0.1mM-20mM, the concentration of mercaptoethanol is 0.1%-10%, the concentration of TCEP is 0.1mM-30mM, and the concentration of sodium hydroxide is 10mM-200mM, and the pH of the solution is 4.5-8.0; In the sample lysis binding liquid LB, the concentration of TrisHCl is 10mM-500mM, the concentration of benzenesulfonic acid is 5mM-100mM, the concentration of sodium citrate is 10mM-100mM, the concentration of guanidine hydrochloride is 0.5M-5.5M, the concentration of guanidine isothiocyanate is 0.5M-5.5M, the concentration of guanidine thiocyanate is 0.5-4M, the concentration of urea is 10mM-1000mM, the concentration of triton 100 is 1%-20%, the concentration of SDS is 0.5%-2%, the concentration of potassium chloride is 10mM-200mM, the concentration of sodium chloride is 50mM-500mM, the concentration of bromhexine is 5mM-50mM, the concentration of N-acetylcysteine is 5mM-50mM, the concentration of ambroxol hydrochloride is 5mM-50mM, the concentration of isopropyl alcohol is 10%-40%, the concentration of anhydrous ethanol is 10%-40%, and the pH of the solution is 5.0-8.0; In the protein washing liquid WB1, the concentration of TrisHCl is 10mM-500mM, the concentration of benzenesulfonic acid is 5mM-100mM, the concentration of sodium citrate is 10mM-100mM, the concentration of guanidine hydrochloride is 0.5M-5.5M, the concentration of guanidine isothiocyanate is 0.5M-5.5M, the concentration of guanidine thiocyanate is 0.5-4M, the concentration of urea is 10mM-1000mM, the concentration of triton 100 is 1%-20%, the concentration of sodium chloride is 50mM-500mM, the concentration of isopropyl alcohol is 10%-40%, the concentration of anhydrous ethanol is 10%-40%, and the pH of the solution is 5.0-8.0; In the nucleic acid rinsing liquid WB2, the concentration of TrisHCl is 10mM-500mM, the concentration of sodium chloride is 50mM-500mM, the concentration of isopropyl alcohol is 50%-85%, the concentration of anhydrous ethanol is 50%-85%, and the pH of the solution is 6.5-8.

0.

2. A kit for DNA and RNA co-extraction, characterized by: The DNA and RNA co-extraction reaction reagent of claim 1.

3. A method for DNA and RNA co-extraction, characterized by: The DNA and RNA co-extraction reaction reagent of claim 1 is used to perform the following steps: Step one, obtaining a sample, adjusting the sample to a mixture containing physiological saline, soaking the sample with the physiological saline as a sample to be tested; the sample is a body fluid sample, a swab sample, a fecal sample, a tissue sample, and a viscous sputum sample, wherein the body fluid sample is urine, pleural effusion, cerebrospinal fluid, alveolar lavage fluid, saliva, and blood; Step two, adding a lysis and binding solution RB and a nucleic acid protection agent RP to the mixture to obtain a mixture two, wherein when the sample is a sputum sample, the nucleic acid protection agent RP is added in step one and mixed uniformly; The mixture two is heated at 55-70 DEG C for 10-15 min, cooled to room temperature, then nucleic acid adsorption magnetic beads are added and mixed uniformly, the nucleic acid in the sample is combined with the magnetic beads, then the magnetic beads are adsorbed in a magnetic stand to remove the liquid to obtain first magnetic beads; Step three, adding a protein washing solution WB1 to the first magnetic beads, mixing uniformly until the first magnetic beads are fully suspended, then adsorbing the first magnetic beads in a magnetic stand to remove the liquid to obtain second magnetic beads; Step four, adding a nucleic acid rinsing solution WB2 to the second magnetic beads, mixing uniformly until the second magnetic beads are fully suspended, then adsorbing the second magnetic beads in a magnetic stand to remove the liquid to obtain third magnetic beads; Step five, centrifuging the third magnetic beads until the surface of the third magnetic beads is not reflective; Step six, adding a nucleic acid elution solution EB to elute the DNA and RNA combined on the surface of the third magnetic beads, mixing uniformly, then centrifuging, then fixing the magnetic beads in a magnetic stand, and the remaining liquid is a mixed nucleic acid solution enriched and purified DNA and RNA.

4. The method of co-extraction of DNA and RNA according to claim 3, characterized in that: The magnetic beads are carboxylated magnetic beads or hydroxylated magnetic beads.

5. The method of co-extraction of DNA and RNA according to claim 3, characterized in that: In step one, When the sample is a body fluid sample, the sample is taken out and balanced to room temperature as a sample to be tested; When the sample is a dry swab sample, the physiological saline is added to immerse the dry swab and mixed uniformly as a sample to be tested; When the sample is a sample containing a swab and a preservative, if the preservative is too much, the sample is centrifuged, the supernatant is discarded, and the precipitate is resuspended with physiological saline as a sample to be tested; When the sample is a fecal sample, if the fecal sample does not contain a preservative, physiological saline is added and mixed uniformly as a sample to be tested, and if the fecal sample contains a preservative, it is mixed uniformly as a sample to be tested; When the sample is a tissue sample, an appropriate amount of physiological saline is added as a sample to be tested; When the sample is viscous sputum, a nucleic acid protection agent RP is added for liquefaction as a sample to be tested.

6. The method of co-extraction of DNA and RNA according to claim 3, characterized in that: The time for the magnetic stand to adsorb the magnetic beads is 1-2 min.

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