Rapid extraction method and kit for nucleic acid of biological sample based on magnetic bead method

Abstract

The application belongs to the technical field of gene detection, and particularly relates to a biological sample nucleic acid rapid extraction method and kit based on a magnetic bead method. The method comprises the following steps in sequence: S1, liquefying a biological sample by using a liquefying solution; the liquefying treatment is to add 1-4 times the volume of the liquefying solution to the biological sample, shake and mix, and then place at room temperature for 15-30 minutes; the liquefying solution comprises guanidine salt, potassium salt and / or sodium salt, Tris-HCl, EDTA and isopropyl alcohol; S2, lysing the biological sample; adding a lysing solution and magnetic beads to the system obtained in S1, and heating at 90-100 DEG C for 3-20 minutes to lyse the biological sample; S3, cleaning the biological sample; and S4, eluting the nucleic acid. The method does not need centrifugal separation in the operation process, and can realize automatic and efficient extraction of the nucleic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene detection, and particularly relates to a biological sample nucleic acid rapid extraction method and kit based on a magnetic bead method. BACKGROUND

[0002] Currently, common biological samples in clinical practice include sputum, whole blood, serum, plasma, lavage fluid, tissue fluid, swab eluate, urine, saliva, etc. Sputum is a liquid secreted by the respiratory tract after stimulation, also known as sputum, and its composition includes mucus, foreign matter, pathogenic microorganisms, various inflammatory cells, necrotic and exfoliated mucosal epithelial cells, etc. Pathogen detection in sputum is an important way for the diagnosis and treatment of respiratory diseases. Nucleic acid detection is one of the important methods for pathogen detection, and the identification of pathogen types through the detection of pathogen nucleic acid in sputum samples is one of the most accurate and fastest diagnostic methods.

[0003] The high accuracy of nucleic acid detection depends on the high-quality nucleic acid extraction of biological samples. However, sputum samples are complex and have high viscosity, making it difficult to directly extract nucleic acid. Therefore, the sputum sample needs to be liquefied to reduce viscosity and reduce the interference of inhibitors in the sputum on subsequent nucleic acid extraction. Currently, common sputum liquefaction methods include sodium hydroxide method, DTT method, and protease method, etc. Among them, the sodium hydroxide method is the most commonly used sputum liquefaction method. This method is relatively simple, but it needs to centrifuge to enrich pathogenic microorganisms after liquefaction, and remove sodium hydroxide to reduce the inhibition of nucleic acid extraction. The operation is complex, and incomplete centrifugation can lead to the loss of pathogenic microorganisms, reducing the sensitivity of detection. The protease method uses proteinase K to digest sputum mucin. This method is high in cost and time-consuming in enzymatic reaction, and the operation is complicated. The DTT method uses the reducing effect of sulfhydryl to break the sputum mucin. This method needs to use high-concentration DTT, and often needs to be centrifuged. Moreover, due to the poor stability of DTT, it needs to be stored at low temperature. The sputum liquefied by this method is often not uniform, and it is easy to change from a solution state to a gel state.

[0004] The traditional sputum nucleic acid extraction method is to liquefy the sputum with sodium hydroxide, centrifuge to collect the pathogenic microorganism precipitate, wash with phosphate buffer, then add lysis solution for high-temperature lysis, and then centrifuge to take the supernatant as the template nucleic acid. This method needs multiple centrifugation and washing to remove the influence of sodium hydroxide, and the operation is very complex. Moreover, a large amount of pathogenic microorganisms can be lost in the process of centrifugation to remove the supernatant, and the removal rate of impurities is low, which can easily lead to false negative detection. The commonly used silica-based adsorption column method has greatly improved the efficiency and purity of nucleic acid extraction compared with the traditional method, but it also needs to liquefy the sputum with sodium hydroxide or protease method, etc. The liquefaction process is complex and the effect is not good. At the same time, the silica-based adsorption column method also involves many centrifugation steps, and the operation is complex.

[0005] The magnetic bead method for extracting nucleic acid depends on nano magnetic beads. The magnetic beads use superparamagnetic nano microspheres as carriers and can quickly move to one side and gather together in a magnetic field. After the magnetic field is removed, the magnetic beads can quickly return to a dispersed state. A specific group is modified on the surface of the magnetic beads. The magnetic beads can quickly adsorb nucleic acid under specific conditions and release the nucleic acid under other conditions, which is used in subsequent detection steps. The magnetic bead method for extracting nucleic acid does not need to use toxic organic solvents, and the operation is safe and simple. The magnetic bead method for extracting nucleic acid can be combined with automatic equipment, which is conducive to the automation and high-throughput extraction of nucleic acid. The magnetic bead method for extracting nucleic acid is an important direction of the development of the current nucleic acid extraction method. At present, the magnetic bead nucleic acid extraction method is mainly applied to whole blood, serum, plasma, swab eluate, urine and other samples. For the extraction of pathogen nucleic acid in sputum, sodium hydroxide or protease method is still needed for liquefying sputum in advance, which can cause the loss of pathogens and increase the complexity of the operation, and is not conducive to automation.

[0006] Compared with the existing publicly disclosed technical solutions for extracting nucleic acid from sputum samples, the patent “Sputum sample nucleic acid extraction device and extraction method” (publication number: CN117187036A) still uses traditional sodium hydroxide to liquefy sputum, and the liquefaction effect is poor. Moreover, the device is relatively complex. The patent “Method for efficiently extracting Mycobacterium tuberculosis nucleic acid and application” (publication number: CN116426518A) also uses traditional sodium hydroxide to liquefy sputum, and needs centrifugal operation. The patent “Method for quickly extracting sputum sample genome by using magnetic bead method” (publication number: CN114717228A) combines sputum liquefaction and sample lysis into one step. The liquefaction solution / lysis solution used contains dithiothreitol. Dithiothreitol can easily lose effectiveness at room temperature and needs to be prepared on site, which increases the complexity of the operation and is not conducive to the automation of extraction. Moreover, the sample lysis temperature is 60-75°C, which is too low for difficult-to-lyse pathogens such as Mycobacterium tuberculosis. The patent “Alcohol-free cleaning solution suitable for nucleic acid extraction of various samples and nucleic acid extraction kit” (publication number: CN113462683A) is suitable for samples including sputum, but does not mention the reagent and method used for sputum liquefaction. The nucleic acid extraction in the patent needs multiple centrifugal operations, which is complicated and not conducive to automation. The patent “Kit for quickly extracting Mycobacterium tuberculosis nucleic acid in sputum” (publication number: CN112063617A) does not liquefy sputum, but directly mixes sputum with a lysis solution. However, the processing needs centrifugal operation, and hydrochloric acid solution, a hazardous chemical, is used for cleaning. Sodium hydroxide solution with a high pH is used for elution, which can affect downstream molecular experiments. SUMMARY

[0007] To solve the problems of poor sputum liquefaction effect, complex composition of liquefying agent, need for centrifugation after sputum liquefaction and inability to be combined with automatic equipment, the present application provides a biological sample nucleic acid rapid extraction method based on a magnetic bead method.

[0008] Another object of the present application is to provide a biological sample nucleic acid rapid extraction kit based on magnetic bead method.

[0009] The technical scheme adopted by the present application to solve its technical problems is:

[0010] A biological sample nucleic acid rapid extraction method based on magnetic bead method, which comprises the following steps in sequence:

[0011] S1, liquefying the biological sample by using a liquefying solution:

[0012] The liquefying treatment is to add 1-4 times the volume of the liquefying solution to the biological sample, shake and mix, and then place at room temperature for 15-30 minutes; the liquefying solution comprises guanidine salt, potassium salt and / or sodium salt, Tris-HCl, EDTA and isopropyl alcohol;

[0013] The biological sample is sputum;

[0014] S2, lysing the biological sample:

[0015] Add a lysing solution and magnetic beads to the system obtained in S1, heat at 90-100℃ for 3-20 minutes to lyse the biological sample;

[0016] The lysing solution comprises guanidine salt, potassium salt and / or sodium salt, Tris-HCl, EDTA, isopropyl alcohol and 0.5-2wt% sodium deoxycholate;

[0017] The magnetic beads are selected from one or more of silicon hydroxyl magnetic beads, carboxyl magnetic beads, amino magnetic beads and polyethyleneimine modified magnetic beads, the diameter of the magnetic beads is 500-1000nm, and the amount of the magnetic beads is 0.5-1mg / mL;

[0018] S3, washing the biological sample:

[0019] Place the system obtained in S2 on a magnetic stand, discard the liquid after the magnetic beads are adsorbed, add a washing solution, mix, adsorb the magnetic beads by the magnetic stand, discard the liquid, and repeat the washing step for several times;

[0020] S4, eluting the nucleic acid:

[0021] Add an eluting solution to the mixed solution obtained in S3, shake and mix, place on a magnetic stand, and then absorb the supernatant to obtain an eluting solution containing nucleic acid extract.

[0022] The method of the present application first liquefies and lyses the biological sample, then enriches it by using magnetic bead method, and at the same time, uses the aggregation effect of magnetic beads in the magnetic field to clean the magnetic beads, avoiding the sample loss caused by traditional centrifugal separation and avoiding the reduction of detection sensitivity. The separation of magnetic beads in the magnetic field is to place the container containing magnetic beads in the magnetic field, which can provide a magnetic force of sufficient strength to make the magnetic beads adhere to the wall in the container; then the liquid in the container is removed by a pipette, or the magnetic beads and the solution are separated by other similar methods.

[0023] The guanidine salt is added in the liquefying solution to denature the mucin in the sputum, thereby effectively liquefying the sputum, and at the same time, the guanidine salt provides the cation required for the salt bridge in the subsequent nucleic acid extraction for the combination of nucleic acid and magnetic beads, and the high concentration guanidine salt can also effectively protect the pathogen nucleic acid in the sputum from being degraded. Further, the guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, preferably guanidine hydrochloride. The concentration of the guanidine salt in the liquefying solution or the lysis solution is 2-6 mol / L; and the further optimized concentration is 2-3 mol / L.

[0024] The sodium salt or potassium salt is added in the liquefying solution to further increase the ionic strength, which is helpful for the liquefaction of sputum and the subsequent nucleic acid extraction. Further, the sodium salt is sodium chloride or sodium iodide, and the potassium salt is potassium chloride or potassium iodide, and the concentration is 1-2 mol / L. Tris-HCl and EDTA are added in the liquefying solution to enhance the stability of nucleic acid in the sputum sample. Isopropyl alcohol is added in the liquefying solution to further denature the mucin in the sputum and enhance the liquefaction effect, and the isopropyl alcohol also has a protective effect on nucleic acid.

[0025] The liquefying solution provided by the present application has similar components as the lysis solution, and the only difference is that the lysis solution further contains sodium deoxycholate, which can reduce the complexity of reagents and is conducive to the subsequent separation and purification steps. Sodium deoxycholate is an ionic detergent, which can be used for cell lysis and protein dissolution, especially for dissolving some water-insoluble proteins, which is helpful for lysis of pathogens and treatment of impurities in the sample. As a further optimization of the present application, the lysis solution contains 1 wt% of sodium deoxycholate.

[0026] As a preferred embodiment, the guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, and the concentration of the guanidine salt in the liquefying solution or the lysis solution is 2-6 mol / L; the sodium salt is sodium chloride or sodium iodide, and the potassium salt is potassium chloride or potassium iodide.

[0027] As a preferred embodiment, the liquefying solution contains 2-6 mol / L guanidine salt, 1-2 mol / L potassium salt and / or sodium salt, 10-20 mM Tris-HCl, 5-10 mM EDTA and 20-50 vol% isopropyl alcohol;

[0028] The lysis solution comprises 2-6 mol / L guanidine salt, 1-2 mol / L potassium salt and / or sodium salt, 10-20 mM Tris-HCl, 5-10 mM EDTA, 20-50 vol% isopropanol and 0.5-2 wt% sodium deoxycholate.

[0029] Preferably, the washing solution comprises 25-75 vol% ethanol, 0.5-2 mol / L sodium chloride or potassium chloride; and the elution solution is nuclease-free water or TE buffer.

[0030] Preferably, the lysis solution comprises 1 wt% sodium deoxycholate; and the washing solution comprises 75 vol% ethanol and 0.5 mol / L sodium chloride.

[0031] A biological sample nucleic acid rapid extraction kit, comprising a liquefaction solution, a lysis solution, a washing solution, an elution solution and magnetic beads,

[0032] The liquefaction solution comprises 2-6 mol / L guanidine salt, 1-2 mol / L potassium salt and / or sodium salt, 10-20 mM Tris-HCl, 5-10 mM EDTA and 20-50 vol% isopropanol.

[0033] The lysis solution comprises 2-6 mol / L guanidine salt, 1-2 mol / L potassium salt and / or sodium salt, 10-20 mM Tris-HCl, 5-10 mM EDTA, 20-50 vol% isopropanol and 0.5-2 wt% sodium deoxycholate.

[0034] The washing solution comprises 25-75 vol% ethanol, 0.5-2 mol / L sodium chloride or potassium chloride.

[0035] The elution solution is nuclease-free water or TE buffer.

[0036] The magnetic beads are selected from one or more of a silicon hydroxyl magnetic bead, a carboxyl magnetic bead, an amino magnetic bead and a polyethyleneimine-modified magnetic bead, the magnetic beads have a diameter of 500-1000 nm, and the magnetic beads are used in an amount of 0.5-1 mg / mL.

[0037] Preferably, the biological sample nucleic acid rapid extraction kit comprises:

[0038] The liquefaction solution comprises 3 mol / L guanidine salt, 2 mol / L potassium salt and / or sodium salt, 20 mM Tris-HCl, 10 mM EDTA and 20 vol% isopropanol, and has a pH of 7.0.

[0039] The lysis solution comprises 3 mol / L guanidine salt, 2 mol / L potassium salt and / or sodium salt, 20 mM Tris-HCl, 10 mM EDTA, 20 vol% isopropanol and 1 wt% sodium deoxycholate, pH 7.0;

[0040] The washing solution comprises 75 vol% ethanol and 0.5 mol / L sodium chloride;

[0041] The elution solution is TE buffer.

[0042] The application of the biological sample nucleic acid rapid extraction kit in the extraction of sputum nucleic acid.

[0043] The application has the following advantages:

[0044] The biological sample nucleic acid rapid extraction kit can separate the magnetic beads directly through a magnetic field without the need of large equipment such as a centrifuge, and thus the nucleic acid in the biological sample can be extracted rapidly.

[0045] Among many biological samples, the nucleic acid sample of sputum is preferably extracted. The liquefying solution provided in the kit is low in price and short in liquefying time, and can effectively protect the nucleic acid sample from degradation. The liquefying solution is similar to the lysis solution in design, reduces the complexity of the system, and is suitable for the environment required for the adsorption of nucleic acid by the magnetic beads, and thus the operation of removing the liquefying solution by centrifugation is not needed. The sodium deoxycholate is additionally added in the lysis solution, which is helpful for the extraction of nucleic acid of difficult-to-lyse pathogens such as Mycobacterium tuberculosis.

[0046] The method of the application adopts the traditional heating lysis mode, which is simple and easy to operate. The components of the washing solution and the elution solution are simple and low in price, the pH of the elution solution is 8.0, which is close to the pH of the PCR reaction buffer, reduces the inhibition to the downstream PCR reaction, and the operation process does not need centrifugal separation; the extraction efficiency of the nucleic acid in the sample by using the kit is also significantly higher than that of the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 It is a comparison chart of the liquefying effect of sputum by using the liquefying solution in the method of the application or 4% NaOH;

[0048] Figure 2 It is a fluorescence curve chart of real-time fluorescent quantitative PCR (qPCR) amplification after the extraction of Mycobacterium tuberculosis nucleic acid according to the Mycobacterium tuberculosis nucleic acid detection kit (PCR-fluorescent probe method) instruction after the liquefaction of sputum by using the liquefying solution in the method of the application or 4% NaOH and the storage of the sputum at 4 degrees for 0 day, 3 days, 5 days and 7 days;

[0049] Figure 3is a fluorescence curve chart of real-time fluorescent quantitative PCR (qPCR) amplification after liquefied sputum is liquefied using the liquefied liquid in the method of the application, and after adding or not adding sodium deoxycholate, SDS, Tween-100 to extract the nucleic acid of Mycobacterium tuberculosis in the cracking liquid;

[0050] Figure 4 is a fluorescence curve chart of real-time fluorescent quantitative PCR (qPCR) amplification using the nucleic acid of Mycobacterium tuberculosis in the sputum sample extracted by the method of the application and the comparative kit as a template;

[0051] Figure 5 is a fluorescence curve chart of real-time fluorescent quantitative PCR (qPCR) amplification using the nucleic acid of human parvovirus B19 in the plasma extracted by the method of the application and the comparative kit as a template. DETAILED DESCRIPTION

[0052] The technical solutions of the application are further specifically described below through specific examples. It should be understood that the implementation of the application is not limited to the following examples, and any form of variation and / or change of the application will fall within the protection scope of the application.

[0053] In the application, all the parts and percentages are weight units, and the equipment and raw materials used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional methods in the art, unless otherwise specified.

[0054] The reagents used in the following examples can be purchased from conventional biochemical reagent stores, unless otherwise specified.

[0055] The TE buffer described in the application is a buffer for dissolving nucleic acid prepared from Tris and EDTA, which is a conventional reagent in the biochemical field.

[0056] The solvents used for the preparation of the liquefied liquid, cracking liquid, washing liquid and other reagents in the application are all water.

[0057] Example 1

[0058] A biological sample nucleic acid extraction kit, the kit comprising:

[0059] 1. Silica hydroxyl magnetic beads with a particle size of 500 nm, purchased from Suzhou Nanmi Technology Co., Ltd.

[0060] 2. Liquefied liquid: guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0.

[0061] 3. Lysis solution: guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, 1 wt% sodium deoxycholate, pH 7.0.

[0062] 4. Washing solution: 75% (v / v) ethanol, 0.5 mol / L sodium chloride.

[0063] 5. Elution solution: TE buffer.

[0064] The rapid extraction method of sputum nucleic acid based on the magnetic bead method realized by the above-mentioned kit specifically comprises the following steps:

[0065] (1) Take 1 mL of sputum, add 3 mL of liquefaction solution, and liquefy at room temperature for 15 minutes;

[0066] (2) Add the liquefied sputum to a 2 mL centrifuge tube, add 1 mL of lysis solution and 40 μL of silanol magnetic beads (magnetic bead particle size 500 nm, concentration 25 mg / mL), heat and lyse at 95°C for 10 minutes;

[0067] (3) Place the centrifuge tube in a magnetic stand, and after the magnetic beads are adsorbed to the wall of the tube, discard the liquid;

[0068] (4) Add 1 mL of washing solution to the centrifuge tube, invert and mix for 15 seconds, and discard the liquid;

[0069] (5) Repeat step (4);

[0070] (6) Take the centrifuge tube out of the magnetic stand, add 100 μL of elution solution, shake and elute for 60 seconds, and collect the supernatant to obtain the elution solution containing the nucleic acid extract.

[0071] Example 2

[0072] The sputum clinically determined to be negative for Mycobacterium tuberculosis is evenly divided into multiple portions by scissors, and is treated according to the test group and the control group respectively. After shaking and liquefying at room temperature for 15 minutes, the clarity of the sputum is observed, and the OD value is measured by a UV spectrophotometer to judge the liquefaction effect of the sputum.

[0073] Test group 1: one portion is added with 3 times the volume of the sputum of the liquefaction solution of the present application: guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0.

[0074] Test group 2: one portion is added with 3 times the volume of the sputum of the liquefaction solution: sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0.

[0075] Test group 3: one portion of liquefied sputum was added with 3 times the volume of the liquefied solution: guanidine hydrochloride 3 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0.

[0076] Test group 4: one portion of liquefied sputum was added with 3 times the volume of the liquefied solution: guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, pH 7.0.

[0077] Control group: one portion of liquefied sputum was added with 3 times the volume of 4% NaOH.

[0078] The OD of the liquefied sputum in the test groups and the control group was measured by ultraviolet spectrophotometry, and the measurement results are shown in Table 1.

[0079] Table 1

[0080]

[0081] According to the data in Table 1, the OD value of the liquefied sputum in test group 1 is smaller than that in other test groups, indicating that the guanidine salt, sodium chloride and isopropanol in the liquefied solution of the application are helpful for the liquefaction of sputum; the OD value of the liquefied sputum in test group 1 is significantly smaller than that in the control group, and as shown in Table 1, the liquefied sputum in test group 1 has higher clarity and fewer particulate matters than the control group, indicating that the effect of liquefying sputum in test group 1 is better than that in the control group, and the impurities in the sputum are less. Figure 1

[0082] Example 3

[0083] The sputum of a patient clinically determined to be positive for Mycobacterium tuberculosis infection was taken in two portions, each 2 mL, and treated according to the test group and the control group, respectively.

[0084] Test group: one portion of liquefied sputum was added with 3 times the volume of the liquefied solution of the application: guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0.

[0085] Control group: one portion of liquefied sputum was added with 3 times the volume of 4% NaOH.

[0086] ​After vortexing and mixing, the mixture was liquefied at room temperature for 15 minutes. After storage at 4°C for 0, 3, 5, and 7 days, Mycobacterium tuberculosis nucleic acid was extracted according to the instructions of the Mycobacterium tuberculosis Nucleic Acid Detection Kit (PCR-fluorescent probe method, purchased from Sansure Biotech, National Medical Device Registration Certificate 20173400166): The liquefied liquid and the sputum liquefied with 4% NaOH were centrifuged at 12000 rpm for 3 minutes, and the supernatant was removed; the precipitate was resuspended in 1 mL of sterile physiological saline, centrifuged at 12000 rpm for 3 minutes, the supernatant was removed, 50 μL of the nucleic acid release agent provided in the kit was added, heated at 100°C for 10 minutes, centrifuged at 12000 rpm for 3 minutes, and the supernatant was used as the nucleic acid to be tested. Nucleic acid was amplified by qPCR according to the instructions of the Mycobacterium tuberculosis nucleic acid detection kit (PCR-fluorescent probe method): the reaction system consisted of 37 μL of reaction solution per person, 2 μL of enzyme mixture per person, 1 μL of internal standard per person, and 10 μL of template per person; the reaction program was 50℃, 2 minutes, 1 cycle, 94℃, 5 minutes, 1 cycle, 94℃, 15 seconds, 57℃, 30 seconds, 45 cycles.

[0087] The fluorescence curve of PCR amplification is shown in the figure. Figure 2 As shown in Table 2, the red line represents the amplification curve of liquefied sputum using the liquefaction solution of this invention, and the blue line represents the amplification curve of liquefied sputum using 4% NaOH. The Ct values ​​of PCR amplification are shown in Table 2.

[0088] Table 2

[0089]

[0090] according to Figure 2 According to the results in Table 2, the extraction of nucleic acid from sputum liquefied with the liquefaction solution of this invention is better than that from sputum liquefied with 4% NaOH; the difference between the PCR Ct value after 7 days of storage at 4℃ and the Ct value at 0 days is significantly smaller than that after using 4% NaOH, indicating that the liquefaction solution of this invention has a protective effect on nucleic acids in sputum.

[0091] Example 4

[0092] Sputum samples were collected from patients clinically diagnosed with Mycobacterium tuberculosis infection and the following tests were performed:

[0093] (1) Take 2 mL of positive sputum and liquefy it at room temperature for 15 minutes with 6 mL of the liquefaction solution (3 mol / L guanidine hydrochloride, 2 mol / L sodium chloride, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0) in the method of this invention.

[0094] (2) Prepare seven 2mL tubes. Take 1mL of liquefied sputum into each 2mL tube, and then add 1mL of different lysis buffers:

[0095] Control lysis solution: guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0;

[0096] Test group lysis solution 1: control lysis solution plus 1 wt% sodium deoxycholate;

[0097] Test group lysis solution 2: control lysis solution plus 2 wt% sodium deoxycholate;

[0098] Test group lysis solution 3: control lysis solution plus 1 wt% SDS;

[0099] Test group lysis solution 4: control lysis solution plus 2 wt% SDS;

[0100] Test group lysis solution 5: control lysis solution plus 1% (v / v) Tween-100;

[0101] Test group lysis solution 6: control lysis solution plus 2% (v / v) Tween-100;

[0102] 40 μL of silicon-hydroxyl magnetic beads (magnetic bead particle size 500 nm, concentration 25 mg / mL) were added to the lysis solution, and heated at 95°C for 10 minutes;

[0103] (3) The 2 mL tube was placed in a magnetic stand, and after the magnetic beads were adsorbed to the tube wall, the liquid was discarded;

[0104] (4) 1 mL of washing solution (75% (v / v) ethanol, 0.5 mol / L sodium chloride) was added to the 2 mL tube, inverted and mixed for 15 seconds, and the liquid was discarded;

[0105] (5) Step (4) was repeated;

[0106] (6) The 2 mL tube was removed from the magnetic stand, 100 μL of elution solution was added, and the elution solution was eluted by shaking for 60 seconds, and the elution solution was the extracted nucleic acid;

[0107] (7) 10 μL of the extracted nucleic acid was taken as a template to perform qPCR amplification according to the Mycobacterium tuberculosis nucleic acid detection kit (PCR-fluorescent probe method, purchased from Shengxiang Biology, national drug registration 20173400166): the reaction system was 37 μL of reaction solution per person, 2 μL of enzyme mixture per person, 1 μL of internal standard per person, and 10 μL of template per person; the reaction program was 50°C for 2 minutes, 1 cycle, 94°C for 5 minutes, 1 cycle, 94°C for 15 seconds, 57°C for 30 seconds, 45 cycles.

[0108] The fluorescence curve of PCR amplification is shown in Figure 3As shown, the Ct values of the amplification curves of the nucleic acids extracted from the lysis solution 1 and 2 in the test group as templates are basically consistent, and are about 1.9 smaller than the Ct values of the amplification curves of the nucleic acids extracted from the lysis solution in the control group as templates, indicating that the addition of sodium deoxycholate in the lysis solution helps to improve the nucleic acid extraction efficiency of Mycobacterium tuberculosis. The Ct values of the amplification curves of the nucleic acids extracted from the lysis solution 3 and 4 in the test group as templates are basically consistent, and are larger than the Ct values of the amplification curves of the nucleic acids extracted from the lysis solution in the control group as templates, indicating that the addition of SDS in the lysis solution affects the nucleic acid extraction. The Ct values of the amplification curves of the nucleic acids extracted from the lysis solution 5 and 6 in the test group as templates are basically consistent, and are basically consistent with the Ct values of the amplification curves of the nucleic acids extracted from the lysis solution in the control group as templates, indicating that the addition of Tween-100 in the lysis solution cannot increase the nucleic acid extraction efficiency.

[0109] Example 5

[0110] In this test example, the kit provided in Example 1 and the existing kit (Microbiome DNA Isolation Kit, Nanjing Nuowezan, Item No.: DC502) are used to extract the nucleic acids of Mycobacterium tuberculosis in a sputum sample, respectively, and then real-time fluorescence quantitative PCR (qPCR) amplification is performed. The specific steps are as follows: Microbiome DNA Isolation Kit, Nanjing Nuowezan, Item No.: DC502) are used to extract the nucleic acids of Mycobacterium tuberculosis in a sputum sample, respectively, and then real-time fluorescence quantitative PCR (qPCR) amplification is performed. The specific steps are as follows:

[0111] (1) Take 1 mL of sputum of a patient clinically determined to be positive for Mycobacterium tuberculosis infection, and liquefy 3 mL of the liquefaction solution (3 mol / L guanidine hydrochloride, 2 mol / L sodium chloride, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, pH 7.0) in the method of the present application at room temperature for 15 minutes;

[0112] (2) Take 1 mL of the liquefied sputum in a 2 mL tube, add 1 mL of the lysis solution (3 mol / L guanidine hydrochloride, 2 mol / L sodium chloride, 20 mM Tris-HCl, 10 mM EDTA, 20% (v / v) isopropanol, 1 wt% sodium deoxycholate, pH 7.0) in the method of the present application and 40 μL of silanol magnetic beads (magnetic bead particle size 500 nm, concentration 25 mg / mL), and heat at 95°C for 10 minutes;

[0113] (3) Place the 2 mL tube in a magnetic stand, and after the magnetic beads are adsorbed to the wall of the tube, discard the liquid;

[0114] (4) Add 1 mL of the washing solution (75% (v / v) ethanol, 0.5 mol / L sodium chloride) to the 2 mL tube, invert and mix for 15 seconds, and discard the liquid;

[0115] (5) Repeat step (4);

[0116] (6) Take the 2 mL tube out of the magnetic stand, add 100 μL eluent, shake and elute for 60 seconds, and the eluent is the extracted nucleic acid;

[0117] (7) 10 μL of the extracted nucleic acid was taken as a template for qPCR amplification according to the instructions of the Mycobacterium tuberculosis nucleic acid detection kit (PCR-fluorescent probe method, purchased from Shengxiang Biology, national equipment approval 20173400166): the reaction system was 37 μL of reaction solution per person, 2 μL of enzyme mixture per person, 1 μL of internal standard per person, and 10 μL of template per person; the reaction program was 50°C for 2 minutes, 1 cycle, 94°C for 5 minutes, 1 cycle, 94°C for 15 seconds, 57°C for 30 seconds, and 45 cycles.

[0118] Microbiome DNA Isolation Kit method:

[0119] (1) 1 mL of the above positive sputum was taken, 3 mL of 4% NaOH was added and liquefied at room temperature for 15 minutes, then 1 mL of the liquefied sputum was taken, centrifuged at 12000 rpm for 5 minutes, and the supernatant was removed. The bacterial pellet was washed once with 1 mL of phosphate buffer, and the washed pellet was used to extract nucleic acid with the comparative kit. Finally, elution was performed in 100 μL of eluent provided by the kit;

[0120] (2) 10 μL of the extracted nucleic acid was taken as a template for qPCR amplification according to the instructions of the Mycobacterium tuberculosis nucleic acid detection kit (PCR-fluorescent probe method): the reaction system was 37 μL of reaction solution per person, 2 μL of enzyme mixture per person, 1 μL of internal standard per person, and 10 μL of template per person; the reaction program was 50°C for 2 minutes, 1 cycle, 94°C for 5 minutes, 1 cycle, 94°C for 15 seconds, 57°C for 30 seconds, and 45 cycles.

[0121] The fluorescence curve of PCR amplification is shown in Figure 4 , the red line is the amplification curve using the nucleic acid extracted by the comparative kit as the template, and the blue line is the amplification curve using the nucleic acid extracted by the method provided by the present application as the template. The Ct value of the blue amplification curve is about 2.9 less than that of the red amplification curve, indicating that the efficiency of extracting Mycobacterium tuberculosis nucleic acid from sputum using the method provided by the present application is higher than that of the comparative kit.

[0122] Example 6

[0123] After extracting the human parvovirus B19 nucleic acid from the human plasma sample using the method of the present application and the comparative kit (viral genomic DNA / RNA extraction kit, purchased from Beijing Tiangu, item number: DP315), real-time fluorescent quantitative PCR (qPCR) amplification was performed, and the specific steps were as follows:

[0124] (1) Take 0.5 mL of plasma from a patient confirmed to be positive for human parvovirus B19 infection, and directly mix it with 1.5 mL of lysate (3M guanidine hydrochloride, 2M sodium chloride, 20 mM Tris-HCl, 10 mM EDTA, 20% isopropyl alcohol, 1% sodium deoxycholate, pH 7.0) and 40 μL of silicon hydroxyl magnetic beads (magnetic bead particle size 500 nm, concentration 25 mg / mL) in a 2 mL tube (no need for a liquefaction step for plasma, whole blood, serum, lavage fluid, tissue fluid, swab eluate, urine, and saliva samples). Heat at 95 degrees for 10 minutes;

[0125] (2) Place the 2 mL tube in a magnetic stand, and discard the liquid after the magnetic beads are adsorbed to the tube wall;

[0126] (3) Add 1 mL of washing solution (75% (v / v) ethanol, 0.5 mol / L sodium chloride) to the 2 mL tube, mix well by inverting for 15 seconds, and discard the liquid;

[0127] (4) Repeat step (3);

[0128] (5) Take the 2 mL tube out of the magnetic stand, add 100 μL of elution solution, and shake for 60 seconds to elute the nucleic acid, which is the extracted nucleic acid;

[0129] (6) Take 4 μL of the extracted nucleic acid as a template and perform qPCR amplification according to the instructions of the human parvovirus B19 fluorescent PCR kit (purchased from Beijing Bai'ao Leibo, item number: SYA774): the reaction system is 20 μL of reaction solution per person, 1 μL of enzyme mixture per person, and 4 μL of template per person; the reaction program is 94°C for 2 minutes, 1 cycle, 94°C for 15 seconds, 55°C for 30 seconds, and 40 cycles.

[0130] Virus genomic DNA / RNA extraction kit method:

[0131] (1) Take 0.5 mL of the above positive plasma, and extract the nucleic acid using the comparative kit (purchased from Beijing Tian'gen, item number: DP315), and finally elute it in 100 μL of elution solution provided by the kit;

[0132] (2) Take 4 μL of the nucleic acid extracted by the comparative kit as a template, and perform qPCR amplification according to the instructions of the human parvovirus B19 fluorescent PCR kit (purchased from Beijing Bai'ao Leibo, item number: SYA774): the reaction system is 20 μL of reaction solution per person, 1 μL of enzyme mixture per person, and 4 μL of template per person; the reaction program is 94°C for 2 minutes, 1 cycle, 94°C for 15 seconds, 55°C for 30 seconds, and 40 cycles.

[0133] The fluorescence curve of PCR amplification is as follows: Figure 5As shown, the red line is the amplification curve using the nucleic acid extracted by the comparative kit as a template, and the blue line is the amplification curve using the nucleic acid extracted by the method provided by the application as a template. The Ct value of the blue amplification curve is about 1.2 less than that of the red amplification curve, indicating that the efficiency of extracting human parvovirus B19 nucleic acid from plasma by the method provided by the application is higher than that of the comparative kit.

[0134] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between various embodiments can be referred to each other. For the method disclosed by the embodiments, since it corresponds to the method disclosed by the embodiments, the description is relatively simple, and the relevant part can be referred to the method part.

[0135] The above describes the biological sample nucleic acid rapid extraction method and kit based on the magnetic bead method provided by the application in detail. The principle and implementation mode of the application are described by applying specific examples. The above embodiment is only used to help understand the method and core idea of the application. It should be pointed out that for ordinary skilled in the art, without departing from the principle of the application, the application can be improved and modified. These improvements and modifications also fall within the protection scope of the claims of the application.

Claims

1. A method for rapid extraction of nucleic acid from sputum sample based on magnetic bead method, characterized in that The method comprises the following steps in sequence: S1, liquefying the sputum sample by using a liquefying solution: The liquefying treatment is to add 1-4 volumes of the liquefying solution to the sputum sample, shake and mix, and then place at room temperature for 15-30 minutes; the liquefying solution is composed of guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, and 20 vol% isopropyl alcohol, and has a pH of 7.0; S2, lysis of the sputum sample: Add a lysis solution and magnetic beads to the system obtained in S1, and heat at 90-100°C for 3-20 minutes to lyse the sputum sample; The lysis solution is composed of 2-6 mol / L guanidine salt, 1-2 mol / L potassium salt and / or sodium salt, 10-20 mM Tris-HCl, 5-10 mM EDTA, 20-50 vol% isopropyl alcohol, and 1-2 wt% sodium deoxycholate; the guanidine salt is guanidine hydrochloride or guanidine isothiocyanate, the sodium salt is sodium chloride or sodium iodide, and the potassium salt is potassium chloride or potassium iodide; The magnetic beads are selected from one or more of a silicon hydroxyl magnetic bead, a carboxyl magnetic bead, and an amino magnetic bead, the diameter of the magnetic beads is 500-1000 nm, and the amount of the magnetic beads is 0.5-1 mg / mL; S3, washing of the sputum sample: Place the system obtained in S2 on a magnetic stand, discard the liquid after the magnetic beads are adsorbed, add a washing solution, mix, adsorb the magnetic beads by the magnetic stand, discard the liquid, and repeat the washing step for several times; the washing solution comprises 25-75 vol% ethanol and 0.5-2 mol / L sodium chloride or potassium chloride; S4, elution of nucleic acids: Add an elution solution to the mixed solution obtained in S3, shake and mix, place on a magnetic stand, and then suck the supernatant to obtain an elution solution containing a nucleic acid extract; The method for rapidly extracting nucleic acids from a sputum sample based on a magnetic bead method does not need centrifugation and can achieve nucleic acid extraction.

2. The method for rapidly extracting nucleic acids from a sputum sample based on a magnetic bead method according to claim 1, characterized in that: The elution solution is nuclease-free water or a TE buffer.

3. A rapid extraction kit for nucleic acid from sputum sample, characterized by The kit comprises a liquefying solution, a lysis solution, a washing solution, an elution solution, and magnetic beads, The liquefying solution is composed of guanidine hydrochloride 3 mol / L, sodium chloride 2 mol / L, 20 mM Tris-HCl, 10 mM EDTA, and 20 vol% isopropyl alcohol, and has a pH of 7.0; The lysis solution is composed of 2-6 mol / L guanidine salt, 1-2 mol / L potassium salt and / or sodium salt, 10-20 mM Tris-HCl, 5-10 mM EDTA, 20-50 vol% isopropyl alcohol, and 1-2 wt% sodium deoxycholate; The washing solution comprises 25-75 vol% ethanol and 0.5-2 mol / L sodium chloride or potassium chloride; The elution solution is nuclease-free water or a TE buffer; The magnetic beads are selected from one or more of a silicon hydroxyl magnetic bead, a carboxyl magnetic bead, and an amino magnetic bead, the diameter of the magnetic beads is 500-1000 nm, and the amount of the magnetic beads is 0.5-1 mg / mL. 4.The sputum sample nucleic acid rapid extraction kit of claim 3, characterized in that: the composition of the lysis solution is: 3 mol / L guanidine salt, 2 mol / L potassium salt and / or sodium salt, 20 mM Tris-HCl, 10 mM EDTA, 20 vol% isopropyl alcohol and 1 wt% sodium deoxycholate, pH 7.0; the washing solution comprises 75 vol% ethanol and 0.5 mol / L sodium chloride; the elution solution is TE buffer. 5.The use of the sputum sample nucleic acid rapid extraction kit of claim 3 in sputum nucleic acid extraction.

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