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

Abstract

The invention relates to the technical field of nucleic acid extraction and purification, in particular to a DNA and RNA co-extraction reaction reagent and an extraction method, the reaction reagent comprises a nucleic acid protective agent RP, a sample lysis binding solution LB, a protein washing solution WB1, a nucleic acid rinsing solution WB2 and a nucleic acid eluent EB; the nucleic acid protective agent RP comprises DTT, mercaptoethanol, TCEP and sodium hydroxide; the sample cracking binding solution LB comprises 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 absolute ethyl alcohol; the protein washing liquid WB1 comprises Tris-HCl, benzenesulfonic acid, sodium citrate, guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, triton 100, sodium chloride, isopropanol and absolute ethyl alcohol; the nucleic acid rinsing liquid WB2 is prepared from Tris-HCl, sodium chloride, isopropanol and absolute ethyl alcohol; and the nucleic acid eluent EB is nuclease-free water. The reaction reagent can be used for rapidly and sufficiently enriching and purifying DNA and RNA of a sample at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid extraction and purification, and in particular to a DNA and RNA co-extraction reaction reagent, a kit and an extraction method. Background Art

[0002] In pathogen detection, nucleic acid extraction is a key step, which directly affects the accuracy and sensitivity of subsequent detection. The selection and use of nucleic acid extraction kits are crucial to the experimental results. The detection technologies involved in pathogen detection mainly include fluorescent quantitative PCR, isothermal amplification LAMP, high-throughput metagenomic sequencing mNGS, and high-throughput targeted sequencing. These molecular detection technologies all rely on sample nucleic acids of good quality. Therefore, in pathogen detection, it is particularly important to enrich and purify sample nucleic acids efficiently and without bias.

[0003] Pathogen detection involves a variety of sample types, including blood, plasma, sputum, oral swabs, feces, tissue, alveolar lavage fluid, cerebrospinal fluid, joint fluid, pleural effusion, etc.; and pathogens are of a wide range of types, including fungi, DNA viruses, RNA viruses, parasites, Gram-positive bacteria, Gram-negative bacteria, mycoplasma / chlamydia, etc. In particular, unknown pathogen detection such as mNGS requires that all microbial nucleic acids be enriched and purified without bias regardless of the type of sample to ensure the accuracy and sensitivity of the detection.

[0004] There are many nucleic acid extraction kits on the market. Although some of the nucleic acid extraction kits can simultaneously enrich, purify and extract DNA and RNA nucleic acids in samples, there are still shortcomings: the reaction reagents of existing kits cannot fully lyse and break the target cells when the sample components are complex, and they also require physical manual grinding of zirconium beads or glass beads and other sample pre-treatments, resulting in complicated processes. Summary of the invention

[0005] The purpose of the present invention is to avoid the shortcomings of the prior art and provide a DNA and RNA co-extraction reaction reagent, which only needs to be mixed with the sample to quickly and fully enrich and purify the DNA and RNA of the sample at the same time, and has the advantages of low cost and easy operation.

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

[0007] Provide a DNA and RNA co-extraction reaction reagent, including nucleic acid protective agent RP, sample lysis and binding solution LB, protein washing solution WB1, nucleic acid rinsing solution WB2, and nucleic acid elution solution EB;

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

[0009] The sample lysis and 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 solution EB is nuclease-free water.

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

[0014] In some embodiments, in the sample lysis and binding solution LB, the concentration of Tris-HCl is 10mM to 500mM, the concentration of benzenesulfonic acid is 5mM to 100mM, the concentration of sodium citrate is 10mM to 100mM, the concentration of guanidine hydrochloride is 0.5M to 5.5M, the concentration of guanidine isothiocyanate is 0.5M to 5.5M, the concentration of guanidine thiocyanate is 0.5 to 4M, the concentration of urea is 10mM to 1000mM, and the concentration of Triton 100 is 100mM. The concentration is 1% to 20%, the SDS concentration is 0.5% to 2%, the potassium chloride concentration is 10mM to 200mM, the sodium chloride concentration is 50mM to 500mM, the bromhexine concentration is 5mM to 50mM, the N-acetylcysteine ​​concentration is 5mM to 50mM, the ambroxol hydrochloride concentration is 5mM to 50mM, the isopropanol concentration is 10% to 40%, the anhydrous ethanol concentration is 10% to 40%, and the solution pH is 5.0 to 8.0.

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

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

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

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

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

[0020] Step 1, obtaining a sample, adjusting the sample to a solid-liquid mixture containing physiological saline, and allowing the physiological saline to soak the sample as a sample to be tested;

[0021] Step 2: adding lysis binding solution RB and nucleic acid protection solution RP to the solid-liquid mixture, wherein when the sample is a sputum sample, the nucleic acid protection solution RP is added in step 1, and mixed evenly to obtain a mixture.

[0022] The mixture is heated in a water bath at 55°C to 70°C for 10 to 15 minutes, cooled to room temperature, and then the nucleic acid adsorption magnetic beads are added and vibrated to mix so that the nucleic acid in the sample is bound to the magnetic beads, and then placed in a magnetic stand to adsorb the magnetic beads, and the liquid is removed to obtain the first magnetic beads;

[0023] Step 3, adding protein washing solution WB1 to the first magnetic beads, mixing evenly until the first magnetic beads are fully suspended, then placing them in a magnetic stand to adsorb the first magnetic beads and removing the liquid to obtain second magnetic beads;

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

[0025] Step 5, centrifuging the third magnetic beads until the surface of the third magnetic beads is no longer reflective;

[0026] Step 6. Add nucleic acid elution solution EB to elute the DNA and RNA bound to the surface of the third magnetic beads. After mixing evenly, centrifuge and then place in a magnetic rack to fix the magnetic beads. 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 carboxyl- or surface hydroxy-lated magnetic beads.

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

[0029] In some embodiments, in step 1,

[0030] When the sample is a liquid sample of urine, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, or blood, the sample is taken out and equilibrated to room temperature to serve as a sample to be tested;

[0031] When the sample is a dry swab sample, the added physiological saline is immersed in the dry swab, and the mixture is shaken and mixed to form a sample to be tested;

[0032] When the sample is a sample containing a swab and a preservation solution, if the preservation solution is large, the sample is centrifuged, the supernatant is discarded, and the precipitate is resuspended with physiological saline to serve as the sample to be tested;

[0033] When the sample is a stool sample, if the stool sample does not contain a preservative solution, physiological saline is added and mixed evenly to serve as a sample to be tested; if the stool sample contains a preservative solution, the mixture is mixed evenly to serve as a sample to be tested;

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

[0035] When the sample is thick sputum, add nucleic acid protection solution RP to liquefy it and use it 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, incubated at 50° C. to 60° C., centrifuged, the upper layer solution is removed, physiological saline is added and mixed evenly, and the sample to be tested is used.

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

[0038] Beneficial effects of a DNA and RNA co-extraction reaction reagent of the present invention:

[0039] (1) The DNA and RNA co-extraction reaction reagent of the present invention comprises a nucleic acid protective agent RP, a sample lysis and binding solution LB, a protein washing solution WB1, a nucleic acid rinsing solution WB2, and a nucleic acid elution solution EB. Among the above components, Tris-HCl, benzenesulfonic acid, and sodium citrate can stabilize the pH of the reaction reagents, prevent the entire system from being affected by the pH fluctuation and the reaction effect of each reaction reagent, and maintain the binding efficiency of DNA and RNA with magnetic beads; DTT, mercaptoethanol, and TCEP are used to prevent nucleic acid oxidation and degradation, and ensure that the nucleic acid can stabilize its nucleic acid structure and quality during the reaction process; guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate, urea, Triton 100, and SDS are used to variably dissolve the protein in the sample, improve the lysis and cell wall breaking of the sample Efficiency, to ensure that DNA and RNA can be fully enriched and purified; potassium chloride and sodium chloride are used to increase the salt strength of the solution and improve the lysis and wall breaking strength; bromhexine, N-acetylcysteine, and ambroxol hydrochloride are used to reduce the viscosity of sample mucopolysaccharides or mucins, so that each component can fully act on the sample and improve the efficiency of lysis and wall breaking; PEG6000, PEG8000, spermidine, anhydrous ethanol, and isopropanol are used to modify the surface of magnetic beads and nucleic acids, so that the magnetic beads can achieve an efficiency balance value whether they adsorb DNA or RNA, and promote the binding of DNA and RNA to the magnetic beads. Therefore, there is no need to use additional physical manual grinding pre-processing samples such as zirconium beads or glass beads, which effectively improves the efficiency of enriching and purifying DNA and RNA.

[0040] Beneficial effects of the method for co-extraction of DNA and RNA of the present invention:

[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 broken without the use of physical grinding conditions such as zirconium beads and glass beads, and then the DNA and RNA can be efficiently enriched and purified at the same time, thereby realizing the one-step extraction of DNA and RNA, and improving the extraction efficiency, which is suitable for large-scale production and application.

[0042] (2) The method for co-extraction of DNA and RNA of the present invention does not require the use of grinding means for sample pre-treatment, which effectively avoids sample loss, can save sample volume, and is conducive to improving the accuracy of detection.

[0043] (3) The method for co-extraction of DNA and RNA of the present invention can fully remove substances that affect nucleic acid detection by using the above-mentioned reaction reagents, is applicable to a variety of complex samples, and has flexibility in use. For various types of clinical samples, manual extraction, magnetic rod automatic extraction, pipetting automatic extraction, etc. can be used for co-extraction of DNA and RNA according to actual conditions. It can better adapt to different application scenarios and clinical needs and effectively expand the detection range. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the agarose gel electrophoresis diagram of nucleic acid co-extracted from DNA and RNA of different clinical samples. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the preferred embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present 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 and binding solution LB, protein washing solution WB1, nucleic acid rinsing solution WB2, and nucleic acid elution solution EB;

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

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

[0050] The sample lysis and 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 and 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 amount of each component in the nucleic acid protecting agent RP can be selected according to actual needs.

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

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

[0055] In this embodiment, in the sample lysis and binding solution LB, the concentration of Tris-HCl is 10mM to 500mM, the concentration of benzenesulfonic acid is 5mM to 100mM, the concentration of sodium citrate is 10mM to 100mM, the concentration of guanidine hydrochloride is 0.5M to 5.5M, the concentration of guanidine isothiocyanate is 0.5M to 5.5M, the concentration of guanidine thiocyanate is 0.5 to 4M, the concentration of urea is 10mM to 1000mM, and the concentration of Triton 100 is 1%~20%, SDS concentration is 0.5%~2%, potassium chloride concentration is 10mM~200mM, sodium chloride concentration is 50mM~500mM, bromhexine concentration is 5mM~50mM, N-acetylcysteine ​​concentration is 5mM~50mM, ambroxol hydrochloride concentration is 5mM~50mM, isopropanol concentration is 10%~40%, anhydrous ethanol concentration is 10%~40%, and the solution pH is 5.0~8.0.

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

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

[0058] The reasonable concentration ratio of the above components can fully lyse and break the sample to obtain nucleic acid.

[0059] The above-mentioned DNA and RNA co-extraction reaction reagents are prepared and the method for implementing DNA and RNA co-extraction comprises the following steps:

[0060] Step 1: Obtain a sample, adjust the sample to a solid-liquid mixture containing physiological saline, and soak the sample in the physiological saline to serve as a 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 so that the sample is in a solid-liquid mixture state, which is convenient for subsequent detection.

[0061] Specifically, the sample is a clinical sample, which can be: urine, pleural effusion, cerebrospinal fluid, alveolar lavage fluid, saliva, blood and other body fluid samples; swab samples; stool samples; tissue samples; sputum; slices. It can also be other clinical samples, and is not limited to the sample types listed above in actual applications.

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

[0063] Wherein, when the sample is a sputum sample, in step 1, a 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 to 70° C. for 10 to 15 minutes and then cooled to room temperature, and then magnetic beads for nucleic acid adsorption are added and vibrated to mix, so that the nucleic acid in the sample is bound to the magnetic beads, and then placed in a magnetic frame to adsorb the magnetic beads and remove the liquid to obtain first magnetic beads, and the first magnetic beads bound to the nucleic acid are fixed by the adsorption action of the magnetic frame to achieve the first purification of the nucleic acid;

[0065] Step 3: Add protein washing solution WB1 to the first magnetic beads to wash the proteins on the nucleic acid adsorbed on the first magnetic beads, mix evenly until the first magnetic beads are fully suspended, and then place them in a magnetic stand to adsorb the first magnetic beads and remove the liquid to obtain second magnetic beads, from which the proteins on the nucleic acid have been removed;

[0066] Step 4: Add nucleic acid washing solution WB2 to the second magnetic beads, mix evenly until the second magnetic beads are fully suspended, then place them in a magnetic rack to adsorb the second magnetic beads and remove the liquid to obtain third magnetic beads, and the nucleic acid on the third magnetic beads is further purified;

[0067] Step 5: Centrifuge the third magnetic beads until the surface of the third magnetic beads does not reflect light, so that the nucleic acid is further attached to the surface of the magnetic beads, thereby maximizing the collection of nucleic acid.

[0068] Step 6: Add nucleic acid elution solution EB to elute the DNA and RNA bound to the surface of the third magnetic bead, mix evenly, centrifuge, and then place in a magnetic frame to fix the magnetic beads. The remaining liquid is a mixed nucleic acid solution enriched with purified DNA and RNA. At this time, the purified nucleic acid on the magnetic beads is eluted into the solution to collect it.

[0069] The magnetic beads are surface carboxylation magnetic beads or surface hydroxylation magnetic beads, the particle size of the magnetic beads is 100nm-1000nm, and the magnetic beads have good DNA and RNA adsorption capabilities.

[0070] In step 1, when the sample is a liquid sample of urine, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, or blood, the sample is taken out and equilibrated to room temperature to serve as a sample to be tested;

[0071] When the sample is a dry swab sample, the added physiological saline is immersed in the dry swab, and the mixture is shaken and mixed to form a sample to be tested;

[0072] When the sample is a sample containing a swab and a preservation solution, if the preservation solution is large, the sample is centrifuged, the supernatant is discarded, and the precipitate is resuspended with physiological saline as the sample to be tested;

[0073] When the sample is a stool sample, if the stool sample does not contain a preservative solution, physiological saline is added and mixed evenly to serve as a sample to be tested; if the stool sample contains a preservative solution, the mixture is mixed evenly to serve as a sample to be tested;

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

[0075] When the sample is thick sputum, add nucleic acid protection solution RP to liquefy it and use it 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, incubated at 50-60° C., centrifuged, the upper layer solution is removed, physiological saline is added and mixed evenly, and the sample to be tested is used.

[0077] The time for the magnetic frame to adsorb the magnetic beads is 1 min to 2 min, and the specific time is adjusted according to actual conditions.

[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 acids 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, etc., which not only saves precious clinical samples, but also saves repeated personnel operation time and manpower requirements, and meets the current needs of molecular experiments.

[0079] Based on the method of the present invention, a DNA & RNA co-extraction kit using a magnetic bead method has been developed. The DNA & RNA nucleic acid co-extraction method and kit of the present invention do not rely on physical grinding conditions such as zirconium beads and glass beads when processing samples, have great operational compatibility and sample compatibility, and can be used for DNA & RNA co-extraction by manual extraction, automatic extraction using a magnetic rod method, automatic extraction using a pipetting method, etc., according to actual conditions for various types of clinical samples, to adapt to different application scenarios and clinical needs.

[0080] Example 2

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

[0082] Step 1: Sample pretreatment, including

[0083] If the sample is a body fluid sample such as urine, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, blood, etc., take out the sample and equilibrate it to room temperature, and proceed to step 2;

[0084] If the sample is a swab sample:

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

[0086] If the sample is a stool sample:

[0087] If the sample does not contain preservation solution, add 5 times the volume of normal saline, vortex and mix, and then proceed to the next step. If the sample contains preservation solution, vortex and mix, then take 300ul and proceed to step 2;

[0088] If the sample is a tissue sample:

[0089] Take the tissue block and add an appropriate amount of saline, then proceed to step 2.

[0090] If the sample is sputum

[0091] If the sputum is thick, add about 1 / 6 of the sputum volume of nucleic acid protection solution RP in advance to liquefy it, and then take 300ul of the volume to proceed to step 2.

[0092] If the sample is a slice

[0093] Take 8 to 10 paraffin specimens from the slides, scrape them off with a disposable blade and transfer them to an EP tube, add 320ul of dewaxing agent, incubate at 56℃ for 3min, centrifuge at 12000rpm for 1min, and remove the upper layer of dewaxing agent. Add 300uL of physiological saline to the centrifuge tube and proceed to step 2.

[0094] Step 3: After the above samples are processed, take 300ul of the sample and add it to a 1.5mL centrifuge tube, add 400ul of lysis binding solution RB and 50ul of nucleic acid protection solution RP (if it is a sputum sample, the protection solution RP has been added during the pretreatment, and no need to add it again in this step) and 20ul of Proteinase K, and cover the tube tightly. Place the centrifuge tube in a metal bath and incubate at 65℃ for 10-15min.

[0095] Step 4: Remove the centrifuge tube from the metal bath, lower the temperature to room temperature, add 20ul of nucleic acid adsorption magnetic beads, vortex and mix for 30 seconds, let stand for 10 minutes, and mix every 1 to 2 minutes.

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

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

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

[0099] Step 8. Add 750ul 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. When the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette.

[0101] Step 10. Centrifuge briefly again and place on a magnetic rack to remove any remaining droplets at the bottom of the tube. Dry at room temperature for 3-5 minutes until the surface of the magnetic beads is no longer reflective.

[0102] Step 11. Add 50-100ul of nucleic acid elution buffer EB, vortex mix for 5 minutes, and briefly centrifuge.

[0103] Step 12. Place the centrifuge tube on the 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°C.

[0104] Test case verification

[0105] Test Example 1

[0106] Experimental Example 1 used specific different clinical samples: blood, alveolar lavage fluid, sputum, cerebrospinal fluid, swabs, paraffin sections, and each sample was repeated twice. 1000 copies of the new coronavirus pseudovirus standard were added to each experimental example to verify the RNA nucleic acid extraction effect.

[0107] According to the operation flow of Example 2, DNA and RNA were co-extracted manually, and finally the nucleic acid concentration and purity were detected by spectrophotometer, and nucleic acid band analysis was performed by agarose gel electrophoresis. The results are shown in Table 1. DNA and RNA co-extraction results of different clinical samples and Figure 1 Agarose gel electrophoresis of nucleic acid co-extracted DNA and RNA from different clinical samples.

[0108] Finally, qPCR detection was performed using the novel coronavirus pseudovirus primer probe, the human internal reference gene GAPDH primer probe, and the human internal reference gene GAPDH mRNA primer probe to determine the DNA and RNA extraction conditions. The results are shown in Table 2. The novel coronavirus pseudovirus, DNA internal reference, and RNA internal reference qPCR detection results.

[0109] Table 1 DNA and RNA co-extraction results of 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 test results of SARS-CoV-2 pseudovirus, DNA internal reference, and RNA internal reference

[0112]

[0113] From the above results, it can be seen that the present invention can simultaneously enrich and purify DNA and RNA of various clinical samples, including all nucleic acids including viral RNA, is applicable to various sample types, and is suitable for large-scale production and application.

[0114] Test Example 2

[0115] In order to illustrate that the present invention can be used for unbiased enrichment and purification of all microorganisms in clinical samples, Experimental Example 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 300uL blood, and DNA and RNA were co-extracted. Each sample was repeated twice, and the nucleic acid concentration and purity were detected by a spectrophotometer. Finally, qPCR verification was performed using the corresponding microbial primer probes. The results are shown in Table 3. DNA and RNA co-extraction results of different microbial types.

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

[0117]

[0118]

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

[0120] Test Example 3

[0121] The magnetic rod method was used for automated co-extraction of DNA and RNA. The same sample scheme as in Experiment 1 and the extraction kit of Example 1 were used. A common magnetic rod method automated nucleic acid extraction instrument was used to perform the following operations:

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

[0123] (2) The program settings are as follows: ① The heating temperature of well A1 is 65℃, the heating time is 20min, the mechanical arm's up and down vibration intensity is selected to level 4, and the vibration amplitude is selected to be 0% to 90%; ② The magnetic rod attraction time of well A2 is set to 30s, and then it is transferred to well A1. The magnetic rod is lowered to the bottom of the liquid, the attraction time is 0 (releasing the magnetic beads), the mechanical arm's up and down vibration intensity is selected to level 6, and the amplitude is selected to be 0% to 100%; the duration is set to 15min; ③ The magnetic rod attraction time of well A1 is set to 1min, and then it is transferred to the bottom of the liquid in well A3 to release the magnetic beads, and the mechanical arm's up and down vibration intensity is set to level 9, the vibration time is 10min, and then the attraction time is set to 1min, and then it is transferred to well A4; ④ The setting procedures and parameters of wells A4 and A5 are the same as those of well A3; ⑤ The setting parameters of well A6 are that the magnetic rod stays in the air for 3min, then descends to the bottom of the eluent to release the magnetic beads, the mechanical arm's up and down vibration intensity is set to level 10, and the duration is set to 5min.

[0124] (3) After the instrument program is set, the clinical sample is pre-treated according to the sample pre-treatment scheme of Example 2, and then 300uL of the sample is mixed with 50uL of nucleic acid protectant RP and 20uL of proteinase K, and then added to the A1 well position of the 96-deep well plate. Then, the 96-deep well plate is placed in the designated position of the instrument, and the instrument is started to automatically extract DNA and RNA;

[0125] (4) After the instrument operation is completed, the DNA and RNA solutions in well A6 are transferred to a new nuclease-free centrifuge tube to obtain a mixed solution of DNA and RNA. The concentration and purity are detected by a spectrophotometer. The results are shown in Table 4.

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

[0127]

[0128] From the results in Table 4, it can be seen that the DNA and RNA co-extraction reaction reagent provided by the present invention can be directly adapted from the manual extraction scheme to the magnetic rod method automated extraction scheme without any additional step adjustment beyond the manual scheme. It is simple and easy to operate, has a wide adaptability, and the extracted nucleic acid concentration and purity are not significantly different from those of the manual extraction scheme.

[0129] Test Example 4

[0130] This Experimental Example 4 provides a clinical application of mNGS detection of pathogenic microorganism metagenomics using a mixed nucleic acid of DNA and RNA obtained using reaction reagents.

[0131] The samples of Experimental Example 3 were used to extract nucleic acids, and the DNA and RNA co-library construction kit of Shanghai Yisheng Biotechnology Co., Ltd. was used to construct the library according to the instructions of the kit. 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 DNA and RNA co-extraction nucleic acid mNGS high-throughput sequencing results

[0133]

[0134]

[0135] In the results of Table 5, the green shading microorganisms are the microorganisms with clear positive samples, all of which are detected, and the types of microorganisms cover fungi, bacteria, RNA viruses, etc. The remaining microorganisms are colonized bacteria of some samples or environmental pollution background bacteria. It can be seen from the results that the DNA and RNA co-extraction reaction reagents provided by the present invention, the extracted mixed nucleic acids, in addition to being applicable to the qPCR test example (Test Example 3), can also be applied to high-throughput sequencing (NGS), and one nucleic acid extraction can complete the simultaneous detection of DNA and RNA, greatly simplifying the downstream molecular detection process, saving clinical sample usage, and saving manpower and economic costs.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A DNA and RNA co-extraction reaction reagent, characterized in that: Including nucleic acid protective agent RP, sample lysis and binding solution LB, protein washing solution WB1, nucleic acid rinsing solution WB2, nucleic acid elution solution EB; The nucleic acid protective agent RP includes DTT, mercaptoethanol, TCEP, and sodium hydroxide; The sample lysis and 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 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 nucleic acid washing solution WB2 includes Tris-HCl, sodium chloride, isopropanol, and anhydrous ethanol; The nucleic acid elution solution EB is nuclease-free water.

2. The DNA and RNA co-extraction reaction reagent according to claim 1, characterized in that: In the nucleic acid protective 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, the concentration of sodium hydroxide is 10mM-200mM, and the pH concentration of the solution is 4.5-8.

0.

3. The DNA and RNA co-extraction reaction reagent according to claim 1, characterized in that: In the sample lysis and binding solution LB, the concentration of Tris-HCl is 10mM to 500mM, the concentration of benzenesulfonic acid is 5mM to 100mM, the concentration of sodium citrate is 10mM to 100mM, the concentration of guanidine hydrochloride is 0.5M to 5.5M, the concentration of guanidine isothiocyanate is 0.5M to 5.5M, the concentration of guanidine thiocyanate is 0.5 to 4M, the concentration of urea is 10mM to 1000mM, and the concentration of Triton 100 is 1% to 20%. 20%, SDS concentration is 0.5% ~ 2%, potassium chloride concentration is 10mM ~ 200mM, sodium chloride concentration is 50mM ~ 500mM, bromhexine concentration is 5mM ~ 50mM, N-acetylcysteine ​​concentration is 5mM ~ 50mM, ambroxol hydrochloride concentration is 5mM ~ 50mM, isopropanol concentration is 10% ~ 40%, anhydrous ethanol concentration is 10% ~ 40%, and the solution pH is 5.0 ~ 8.

0.

4. The DNA and RNA co-extraction reaction reagent according to claim 1, characterized in that: In the protein washing solution WB1, the concentration of Tris-HCl is 10mM to 500mM, the concentration of benzenesulfonic acid is 5mM to 100mM, the concentration of sodium citrate is 10mM to 100mM, the concentration of guanidine hydrochloride is 0.5M to 5.5M, the concentration of guanidine isothiocyanate is 0.5M to 5.5M, the concentration of guanidine thiocyanate is 0.5 to 4M, the concentration of urea is 10mM to 1000mM, the concentration of Triton 100 is 1% to 20%, the concentration of sodium chloride is 50mM to 500mM, the concentration of isopropanol is 10% to 40%, the concentration of anhydrous ethanol is 10% to 40%, and the pH value of the solution is 5.0 to 8.

0.

5. The DNA and RNA co-extraction reaction reagent according to claim 1, characterized in that: In the nucleic acid washing solution WB2, the concentration of Tris-HCl is 10mM to 500mM, the concentration of sodium chloride is 50mM to 500mM, the concentration of isopropanol is 50% to 85%, the concentration of anhydrous ethanol is 50% to 85%, and the pH value of the solution is 6.5 to 8.

0.

6. A kit for co-extraction of DNA and RNA, characterized in that: The invention comprises the DNA and RNA co-extraction reaction reagent according to any one of claims 1 to 5.

7. A method for co-extraction of DNA and RNA, characterized in that: The method comprises the following steps: using the DNA and RNA co-extraction reaction reagent according to any one of claims 1 to 5; Step 1, obtaining a sample, adjusting the sample to a solid-liquid mixture containing physiological saline, and allowing the physiological saline to soak the sample as a sample to be tested; Step 2: adding lysis binding solution RB and nucleic acid protection solution RP to the solid-liquid mixture, wherein when the sample is a sputum sample, the nucleic acid protection solution RP is added in step 1, and mixed evenly to obtain a mixture. The mixture is heated in a water bath at 55°C to 70°C for 10 to 15 minutes, cooled to room temperature, and then the nucleic acid adsorption magnetic beads are added and vibrated to mix so that the nucleic acid in the sample is bound to the magnetic beads, and then placed in a magnetic stand to adsorb the magnetic beads, and the liquid is removed to obtain the first magnetic beads; Step 3, adding protein washing solution WB1 to the first magnetic beads, mixing evenly until the first magnetic beads are fully suspended, then placing them in a magnetic stand to adsorb the first magnetic beads and removing the liquid to obtain second magnetic beads; Step 4: Add nucleic acid washing solution WB2 to the second magnetic beads, mix evenly until the second magnetic beads are fully suspended, and then place them in a magnetic rack to adsorb the second magnetic beads and remove the liquid to obtain third magnetic beads; Step 5, centrifuging the third magnetic beads until the surface of the third magnetic beads is no longer reflective; Step 6. Add nucleic acid elution solution EB to elute the DNA and RNA bound to the surface of the third magnetic beads. After mixing evenly, centrifuge and then place in a magnetic rack to fix the magnetic beads. The remaining liquid is a mixed nucleic acid solution for enriching and purifying DNA and RNA.

8. The method for co-extraction of DNA and RNA according to claim 7, characterized in that: The magnetic beads are surface carboxylation magnetic beads or surface hydroxylation magnetic beads.

9. The method for co-extraction of DNA and RNA according to claim 7, characterized in that: In step one, When the sample is a liquid sample of urine, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, or blood, the sample is taken out and equilibrated to room temperature to serve as a sample to be tested; When the sample is a dry swab sample, the added physiological saline is immersed in the dry swab, and the mixture is shaken and mixed to form a sample to be tested; When the sample is a sample containing a swab and a preservation solution, if the preservation solution is large, the sample is centrifuged, the supernatant is discarded, and the precipitate is resuspended with physiological saline to serve as the sample to be tested; When the sample is a stool sample, if the stool sample does not contain a preservative solution, physiological saline is added and mixed evenly to serve as a sample to be tested; if the stool sample contains a preservative solution, the mixture is mixed evenly to serve as a sample to be tested; When the sample is a tissue sample, add an appropriate amount of physiological saline to serve as a sample to be tested; When the sample is thick sputum, add nucleic acid protection solution RP to liquefy it and use it as the sample to be tested; When the sample is a paraffin specimen, the paraffin specimen is scraped off and a dewaxing agent is added, incubated at 50° C. to 60° C., centrifuged, the upper layer solution is removed, physiological saline is added and mixed evenly, and the sample to be tested is used.

10. The method for co-extraction of DNA and RNA according to claim 7, characterized in that: The time for the magnetic frame to adsorb the magnetic beads is 1 min to 2 min.

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