A virus DNA, RNA co-extraction kit and extraction method

Abstract

The present application relates to a kit and method for the co-extraction of viral DNA and RNA, and belongs to the field of biotechnology. The present invention can directly extract viral nucleic acids (DNA and RNA) from cell-free body fluids of humans and animals, such as serum, plasma, and centrifugal supernatants of tissue grinding fluid, by combining solutions such as lysate and rinse solution with extraction methods. The method and kit of the present invention can simultaneously extract DNA and RNA, and are simple to operate, especially for small virus nucleic acid extraction with high sensitivity and better extraction effect.

Description

Technical Field

[0001] The present application relates to a viral DNA and RNA co-extraction kit and an extraction method, and belongs to the field of biotechnology. Background Art

[0002] Viruses are divided into DNA viruses and RNA viruses according to the type of genome. The key to the co-extraction of DNA and RNA lies in the lysis buffer. The methods for extracting DNA include the SDS method and the guanidine salt method. Since the extraction of RNA requires the elimination of the influence of RNase (RNase is widely present and difficult to eliminate), and high concentrations of guanidine salt can inactivate RNase, the guanidine salt method is very suitable for experiments that extract both DNA and RNA.

[0003] The application of the magnetic bead method to extract nucleic acids has been very widespread. When magnetic beads adsorb nucleic acids, they will give priority to adsorbing large fragments of nucleic acids because large fragments of nucleic acids carry more negative charges. Therefore, the fragments of viral nucleic acids are different in size, and the difficulty of being adsorbed by magnetic beads varies. The nucleic acid fragments of small viruses are relatively small, so they are more difficult to adsorb, such as porcine circovirus and HBV virus. Therefore, the extraction of nucleic acids of small viruses requires higher reagents. The kits currently on the market are difficult to take into account the extraction of all types of viruses, especially small viruses. The performance of extracting nucleic acids of small viruses is relatively poor, and there is a phenomenon of missed detection.

[0004] Moreover, the existing kits for simultaneously extracting viral DNA and RNA often require setting different elution temperatures or elution solutions, resulting in cumbersome extraction steps. Therefore, there is a need for a method that can simultaneously extract viral DNA and RNA with simple steps, has a wide range of applications, and can take into account the extraction of small viral nucleic acids. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a DNA and RNA co-extraction kit with simple operation and wide application range, comprising the following components: a lysis solution, a binding solution, a rinsing solution 1, a rinsing solution 2, an eluting solution, magnetic beads and an RNA precipitation aid; wherein the lysis solution comprises 1M-6M guanidine isothiocyanate, 2-7M guanidine hydrochloride, 10-500 mM Tris-HCL ((pH is 7.5-8.5)), 100-1000 mM sodium chloride, 5-100 mM EDTA-2Na (pH is 7.5-8.5), 1%-30% Tween 20 (v / v), and 0.5-2% (m / v) of sodium lauryl sarcosine; and the rinsing solution 1 comprises 1-3 M guanidine isothiocyanate or guanidine hydrochloride, 5-500 mM Tris-HCl (pH is 7.5-8.5), 5%-10% Tween 20 (v / v), and 100-600 mM NaCl. Preferably, the pH value of the 10-500 mM Tris-HCL is 8.0, and the pH value of the 5-100 mM EDTA-2Na is 8.0.

[0006] In an embodiment of the present invention, the rinse solution 2 includes 75-80% (v / v) ethanol. The binding solution is at least one of alcohols, preferably, the binding solution is selected from at least one of methanol, ethanol, propanol, and isopropanol, and more preferably, the binding solution is isopropanol.

[0007] In an embodiment of the present invention, the magnetic beads are silanol magnetic beads, the dosage of the magnetic beads is 1-3 mg, and the particle size of the magnetic beads is 300 nm. Preferably, the concentration of the magnetic beads is 50 mg / ml, and the dosage is 20-60 μL.

[0008] In an embodiment of the present invention, the eluent is: DEPC water.

[0009] In an embodiment of the present invention, the RNA sedimentation aid is carrier RNA, and the dosage of the sedimentation aid is 3-12 μg.

[0010] In a preferred embodiment of the present invention, a viral DNA / RNA co-extraction kit comprises the following components:

[0011] Lysis solution, the lysate comprising 2M guanidine thiocyanate, 4M guanidine hydrochloride, 50 mM Tris-HCL (pH=8), 150 mM sodium chloride, 50 mM EDTA-2Na (pH=8), 5% Tween 20 (v / v), 0.5% SLS sodium lauryl sarcosine (m / v), and 100 mM NaCl;

[0012] Binding solution: isopropanol;

[0013] Magnetic beads: Silane magnetic beads, concentration is 50 mg / mL, dosage is 30 μL, particle size of magnetic beads is 300 nm;

[0014] Carrier RNA: For each reaction, add 8 μg of carrier RNA to the lysate, shake and mix well as the lysate working solution; the carrier RNA here is a product of "Shanghai Yisheng Biotechnology Co., Ltd.", product number: 19704ES01.

[0015] Rinsing solution 1, comprising 1 M guanidine isothiocyanate or guanidine hydrochloride, 50 mM Tris-HCl (pH=8), and 5% Tween 20 (v / v) (the function of Tween 20 is to reduce the aggregation of magnetic beads and the phenomenon of sticking to the wall of a 1.5 mL centrifuge tube during the rinsing process);

[0016] Rinsing solution 2, wherein the rinse solution 2 is 80% ethanol (v / v);

[0017] The eluent is DEPC water.

[0018] Another aspect of the present invention provides a manual extraction method for extracting DNA and RNA simultaneously, characterized in that it comprises the following steps:

[0019] 1) Prepare the total system of lysis working solution: according to the extraction of 350μL sample volume, 396μL of lysis solution and 132μL of binding solution, as well as RNA precipitation agent, are added and mixed to obtain the total system of lysis working solution. Preferably, the RNA precipitation agent is carrier RNA, and the amount is 3-12μg. In a preferred embodiment of the present invention, the amount is 8μg. In a specific embodiment of the present invention, the configuration of the total system of lysis working solution also requires redundancy calculation: for example, if a total of 5 samples are extracted, 396×5.03, i.e., 1992μL of lysis solution, 132×5.03μL, i.e., 665μL of isopropanol, and 40μL of carrier RNA (1μg / μL, carrier RNA does not need to be calculated redundantly) need to be added;

[0020] 2) Add 10 μL proteinase K (40 mg / mL), 350 μL sample, and 525 μL lysis working solution prepared in step 1) to the centrifuge tube in sequence, shake and mix, perform short centrifugation, and lyse on a metal bath at 65-75°C and 800 rpm for 10-15 min. Short centrifugation means using a handheld centrifuge for centrifugation for less than 5 seconds to gently centrifuge the liquid on the tube wall and tube cap;

[0021] 3) Let stand for 5 minutes, wait until the temperature drops to room temperature, add 30μL magnetic beads, shake and mix evenly, leave at room temperature for 10 minutes, invert and mix from time to time to keep the magnetic beads suspended. Remove from the tube, place on a magnetic stand and let stand, wait until the magnetic beads are completely adsorbed, and discard the supernatant;

[0022] 4) First rinse: Continue to add 500 μL of rinse solution 1, shake and mix evenly for 30 seconds, centrifuge briefly, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0023] 5) Second rinse: Continue to add 500 μL of rinse solution 2, shake and mix evenly for 30 seconds, centrifuge briefly, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0024] 6) Third rinse: Repeat step 5) once;

[0025] 7) Drying: Remove the centrifuge tube from the previous step, let it stand, discard the remaining liquid, open the lid, and air dry for 7-10 minutes;

[0026] 8) Elution: Add 50-100 μL of elution solution, mix with a pipette, and incubate on a metal bath at 1600 rpm and 50-60°C for 10 min. Centrifuge briefly to centrifuge the evaporated liquid on the tube cap. Finally, place it on a magnetic rack, let it stand and then aspirate the supernatant to obtain a supernatant containing viral DNA and RNA. Preferably, the elution temperature is 56°C. In an embodiment of the present invention, the sample includes a cell-free body fluid, and preferably, the sample is selected from at least one of the centrifuged supernatants of serum, plasma, and tissue grinding fluid.

[0027] The beneficial effects of this application include but are not limited to:

[0028] 1. The sensitivity is improved by using carrier RNA. By comparing the extraction effects of different amounts of carrier RNA, this kit found that when 8μg was added to each sample, the CT value of virus detection was the smallest;

[0029] 2. By optimizing the combination of components in the lysis buffer and the optimal concentration of each component, the viral nucleic acid can be released to the maximum extent;

[0030] 3. By optimizing the ratio of lysate and isopropanol and the amount of magnetic beads, the binding of magnetic beads and viral nucleic acid is maximized;

[0031] 4. Since magnetic beads preferentially adsorb large nucleic acid fragments, smaller viruses have shorter nucleic acid lengths and are not easily adsorbed by magnetic beads. To address this problem, we selected porcine circovirus (a virus with the smallest virus particles) as the research object. The results showed that our kit has good sensitivity in detecting small viruses and performs better than competing products.

[0032] 5. When detecting larger viruses, such as porcine blue ear virus, the sensitivity is also very good, and compared with excellent products on the market, the sensitivity is better;

[0033] 6. The method of the present invention can directly extract DNA and RNA when extracting nucleic acids, without the need to distinguish the types of nucleic acids, and taking different experimental steps according to the types of nucleic acids, which makes the operation simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The appendix described herein is used to provide a further understanding of the present application and constitutes a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the accompanying drawings:

[0035] Figure 1 Schematic diagram of the amplification curve of PCV2 in the comparative experiment of guanidine salt combination in the lysate of the embodiment;

[0036] Figure 2 Schematic diagram of the amplification curve of PRRSV in the comparative experiment of guanidine salt combination in the lysate of the embodiment;

[0037] Figure 3 This is a schematic diagram of the amplification curve of PCV2 in a further comparative experiment of the guanidine salt combination in the embodiment;

[0038] Figure 4 Schematic diagram of the amplification curve of PRRSV in a further comparative experiment of the guanidine salt combination in the embodiment;

[0039] Figure 5 Schematic diagram of the amplification curve of PCV2 in the composition optimization experiment of washing solution 1 in Example;

[0040] Figure 6 Schematic diagram of the amplification curve of PRRSV in the composition optimization experiment of washing solution 1 in Example;

[0041] Figure 7 A schematic diagram of the amplification curve of PCV2 in an experiment to determine whether adding carrier RNA is effective;

[0042] Figure 8 A schematic diagram of the amplification curve of PRRSV in an experiment to determine whether adding carrier RNA is effective;

[0043] Fig. 9 This is a schematic diagram of the amplification curve of PCV2 in the experiment comparing the amount of carrier RNA added in the example;

[0044] Fig.10 This is a schematic diagram of the amplification curve of PRRSV in the experiment comparing the amount of carrier RNA added in the example;

[0045] Fig.11 This is a schematic diagram of the amplification curve of PCV2 in the experiment comparing the effects of 8ug / 12ug carrier RNA in Example;

[0046] Fig.12 This is a schematic diagram of the amplification curve of PRRSV in the experiment comparing the effects of 8ug / 12ug carrier RNA in Example;

[0047] Fig.13 Schematic diagram of the amplification curve of PCV2 in the competitive product comparison experiment of the embodiment;

[0048] Fig.14 Schematic diagram of the amplification curve of PRRSV in the competitive product comparison experiment of the embodiment. DETAILED DESCRIPTION

[0049] The present invention will be described in further detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] Porcine circovirus type 2 (PCV2) virus culture: Binzhou Animal Husbandry and Veterinary Research Institute;

[0052] Porcine reproductive and respiratory syndrome virus (PRRSV) virus culture: Binzhou Animal Husbandry and Veterinary Research Institute;

[0053] Competitor's kit: Magnetic Bead Viral DNA / RNA Extraction Kit (DP438) of Tiangen Biochemical Technology (Beijing) Co., Ltd. It should be noted that the short centrifugation in the embodiments of the present invention refers to the use of a handheld centrifuge for centrifugation within 5 seconds, the purpose of which is to gently centrifuge the liquid on the tube wall and the tube cover.

[0054] Example 1. DNA / RNA co-extraction experiment

[0055] Manual extraction experimental steps:

[0056] 1) Prepare the total system of lysis working solution: according to the extraction of 350μL sample volume, you need to add 396μL lysis solution and 132μL isopropanol (here is the isopropanol binding solution), and 8μL carrier RNA (1μg / μL). Calculate redundancy: for example, if a total of 5 samples are extracted, you need to add 396×5.03, that is, 1992μL lysis solution, 132×5.03μL, that is, 665μL isopropanol, and 40μL carrier RNA (1μg / μL, carrier RNA does not need to be calculated redundantly);

[0057] 2) Add 10 μL proteinase K (40 mg / mL), 350 μL sample, and 525 μL lysis working solution to the centrifuge tube in sequence, shake and mix, perform short centrifugation, and lyse on a metal bath at 65°C and 800 rpm for 10 min. Short centrifugation means using a handheld centrifuge for centrifugation within 5 seconds to gently centrifuge the liquid on the tube wall and tube cap;

[0058] 3) Magnetic bead binding: let stand for 5 minutes, wait until the temperature drops to room temperature, add 30μL magnetic beads, shake and mix evenly, let stand at room temperature for 10 minutes, invert and mix from time to time to keep the magnetic beads suspended. Shortly separate, place on a magnetic stand and let stand, wait for the magnetic beads to be completely adsorbed, and then discard the supernatant. Magnetic beads: Silane hydroxy magnetic beads, concentration is 50mg / mL, dosage is 20-60μL;

[0059] 4) First rinse: Continue to add 500 μL of rinse solution 1, shake and mix evenly for 30 seconds, centrifuge briefly, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0060] 5) Second rinse: Continue to add 500 μL of rinse solution 2, shake and mix evenly for 30 seconds, spin, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant. Rinse solution 2 is 75% to 80% ethanol, preferably 80% ethanol;

[0061] 6) Third rinse: Continue to add rinse solution 2, shake and mix evenly for 30 seconds, centrifuge briefly, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant;

[0062] 7) Drying: Remove the centrifuge tube from the previous step and place it on a magnetic rack. Let it stand, remove the remaining liquid, and open the lid to air dry for 5-10 minutes.

[0063] 8) Elution: Add 50-100 μL of elution buffer, mix well by pipetting, incubate on a metal bath at 1600 rpm and 56°C for 10 min, centrifuge briefly, centrifuge the evaporated liquid on the tube cap, and finally, place on a magnetic rack, let stand and aspirate the supernatant to obtain a liquid containing viral DNA / RNA. The elution buffer is DEPC water.

[0064] Automated extraction steps:

[0065] 1) Set up the program: Set up the program on the automated extractor. The specific contents are shown in Table 1.

[0066] Table 1

[0067]

[0068] 2) Add reagents and samples to the 96-well plate. Add reagents first and samples last, as shown in Table 2.

[0069] Table 2

[0070]

[0071] 3) After the program is completed, aspirate the extracted nucleic acid from the 5th well and transfer it to a 1.5 ml enzyme-free centrifuge tube and store it in a -20℃ refrigerator.

[0072] Example 2 Optimization of lysate composition

[0073] Comparison of guanidine salt combinations in lysate The components of each lysate are shown in Table 3.

[0074] Table 3

[0075]

[0076] The experimental reagents are as follows: lysis buffer is lysis buffer 1 and lysis buffer 2 in the above table, rinse buffer 1, the rinse buffer 1 includes 1M guanidine isothiocyanate or guanidine hydrochloride, 50mM Tris-HCl, and 5% Tween 20;

[0077] Rinsing solution 2, wherein the rinse solution 2 comprises 80% ethanol;

[0078] The eluent is DEPC water.

[0079] The detection reagents used primer probes synthesized according to the national standards for porcine blue ear virus (GBT35912-2018) and porcine circovirus (GBT35901-2018), and the fluorescent PCR reaction system used Yisheng's DNA amplification reagent and RNA amplification reagent. The samples were porcine blue ear virus (PRRSV) virus culture and porcine circovirus type 2 (PCV2) virus culture purchased from Binzhou Animal Husbandry and Veterinary Research Institute.

[0080] The extraction process was carried out using the automated extraction steps in Example 1.

[0081] The RT-qPCR detection process is as follows:

[0082] (1) Prepare the amplification system as shown in Table 4.

[0083] Table 4

[0084]

[0085] (2) The amplification procedure is shown in Table 5.

[0086] Table 5

[0087]

[0088] After extraction and amplification, the results are shown in Table 6.

[0089] Table 6

[0090]

[0091] The amplification curve is shown in the attached figure Figure 1 and Figure 2 The above results show that lysis buffer 1 is better for extracting PCV2, and lysis buffer 2 is better for extracting PRRSV. Since both large and small viruses need to be taken into account, the nucleic acid of small viruses is relatively more difficult to extract, and PCV2 is a very small virus, so lysis buffer 1 was selected for subsequent research.

[0092] Further comparison of guanidine salt combinations in lysate The specific lysate compositions are shown in Table 7.

[0093] Table 7

[0094]

[0095] The lysate in step 1 of Example 2 was replaced with the lysate in Table 7, and the experiment was repeated. The experimental results are shown in Table 8.

[0096] Table 8

[0097]

[0098] QPCR amplification diagram is shown in the attached figure Figure 3 and Figure 4 The results showed that lysis buffers 3 to 7 could successfully amplify the corresponding viruses. Among them, lysis buffer 6 for PCV2 was the best, and the results for PRRSV were also very good. Therefore, lysis buffer 6 was selected for further research.

[0099] The specific lysate information for the detection of other lysate concentrations is shown in Table 9.

[0100] Table 9

[0101]

[0102] The results are shown in Table 10.

[0103] Table 10

[0104]

[0105] As shown in Table 10, lysis buffer 8 and lysis buffer 9 can also achieve the purpose of extracting DNA and RNA simultaneously.

[0106] Example 3 Optimization of the composition of rinse solution 1

[0107] The ratio of the rinse solution 1 to anhydrous ethanol was fixed at 1:1, and the concentration of guanidine isothiocyanate in the rinse solution 1 was optimized, and the case where guanidine salt was not used at all and NaCl was added was used, as shown in Table 11.

[0108] Table 11

[0109]

[0110] The experimental steps of Example 2 were repeated by replacing the rinsing liquid 1 in Example 2 with the rinsing liquid 1 in Table 11. The experimental results are shown in Table 12.

[0111] Table 12

[0112]

[0113] The QPCR amplification curve is shown in the attached Figure 5 and 6 From the results, when the concentration of guanidine salt in the rinse solution 1 was 1-3M, the results of PCV2 and PRRSV were good. Based on the principle of saving the raw materials, the concentration of guanidine salt in the rinse solution 1 was preferably 1M.

[0114] Other concentration tests of rinse solution 1

[0115] The components and concentrations are shown in Table 13.

[0116] Table 13

[0117]

[0118] The test results are shown in Table 14.

[0119] Table 14

[0120]

[0121] The results showed that washing solutions 1-5 to 1-7 can be used to extract DNA and RNA simultaneously.

[0122] Example 4 Screening of carrier RNA and determination of the amount to be added

[0123] 1. Comparison of the effects of adding or not adding carrier RNA

[0124] The specific experimental process is as described in Example 2, 1. During the extraction process, the total system of lysis working solution is prepared: according to the extraction of 350 μL of sample volume, 396 μL of lysis solution and 132 μL of isopropanol, and 5 μL of carrier RNA (1 μg / μL) need to be added. Calculation redundancy: For example, if a total of 5 samples are extracted, 396x5.03, that is, 1992 μL of lysis solution, 665 μL (132x5.03) of isopropanol, and 25 μL of carrier RNA (1 μg / μL, carrier RNA does not need to be calculated redundantly) need to be added; 525 μL of this system is taken as lysis working solution for each reaction.

[0125] The specific extraction steps are as described in Example 2, and the experimental results are shown in Table 15:

[0126] Table 15

[0127]

[0128] The QPCR amplification curve is shown in the attached Figure 7 and 8 The results showed that the extraction effect was better when 5μg carrier RNA was added than when it was not added.

[0129] Comparison of the effect of carrier RNA addition amount The manufacturer of carrier RNA is "Yisheng Biotechnology Shanghai Co., Ltd.", catalog number: 19704ES01

[0130] The amount of carrier RNA added was compared, and the specific extraction process and detection process were the same as 1 in Example 2. The results are shown in Table 16.

[0131] Table 16

[0132]

[0133] The QPCR amplification curve is shown in the attached Fig. 9 , 10 , 11, 12. The results showed that adding 3-12μg carrier RNA can achieve the effect of virus amplification, among which 8μg carrier RNA had the best effect.

[0134] Example 5 Comparison of Competitive Products

[0135] A competitive kit: Tiangen Biochemical Technology (Beijing) Co., Ltd. magnetic bead virus DNA / RNA extraction kit (YDP438) was used to extract 200 μL of plasma simulation samples with PCV2 virus culture and PRRSV virus culture. The final kit formula obtained in Examples 1 and 2 was compared with the best lysate formula obtained in Example 2 and the best rinse solution formula obtained in Example 3.

[0136] The experimental process and specific extraction steps are as described in the manual extraction experimental steps in Example 1, and the experimental results are shown in Table 17.

[0137] Table 17

[0138]

[0139] The amplification diagram corresponding to the experimental results is shown in Fig.13 and Fig.14 The results showed that the kit of the present invention had better extraction effects on PCV2 and PRRSV than the competing products.

Claims

1. A kit for co-extraction of viral DNA and RNA from cell-free body fluid samples, characterized in that: The cell-free body fluid sample is selected from at least one of serum, plasma, and centrifugal supernatant of tissue grinding fluid, and the kit is composed of the following components: Lysis buffer, binding buffer, rinse buffer 1, rinse buffer 2, elution buffer, magnetic beads and RNA precipitation aid, wherein: The lysate is composed of 1M-4M guanidine thiocyanate, 2-5M guanidine hydrochloride, 10-500 mM Tris-HCL, 100-1000 mM sodium chloride, 5-100 mM EDTA-2Na, 1%-10% (v / v) Tween 20, and 0.5%-2% (m / v) sodium lauryl sarcosine, wherein the pH value of Tris-HCL is 8, and the pH value of EDTA-2Na is 8; The rinse solution 1 is composed of 1-3 M guanidine isothiocyanate or guanidine hydrochloride, 5-500 mM Tris-HCl, 5%-10% Tween 20 (v / v), and 100-600 mM NaCl, and the pH value of Tris-HCl is 8; The RNA sedimentation aid is carrier RNA, and the amount of the sedimentation aid is 3-12 μg per 350 μL sample volume.

2. The kit according to claim 1, characterized in that The rinse solution 2 includes 75-80% ethanol (v / v); the eluent includes DEPC water.

3. The kit according to claim 1, characterized in that The combining solution includes at least one of alcohols.

4. The kit according to claim 1, characterized in that The magnetic beads are silicon hydroxy magnetic beads, with a dosage of 1-3 mg and a particle size of 300 nm.

5. The extraction method of any one of the kits of claims 1 to 4, characterized in that: The extraction method comprises the following steps: 1) Prepare the total lysis working solution system: according to the extraction volume of 350 μL of sample, add 396 μL of lysis solution and 132 μL of binding solution, as well as RNA precipitation agent, shake and mix to obtain the total lysis working solution system; 2) Add 10 μL of 40 mg / mL proteinase K, 350 μL of sample, and 525 μL of the lysis working solution prepared in step 1) to the centrifuge tube in sequence, shake and mix, perform short centrifugation, and lyse on a metal bath at 65-75°C and 800 rpm for 10-15 min. Short centrifugation means using a handheld centrifuge for less than 5 seconds to gently centrifuge the liquid on the tube wall and tube cap; 3) Let stand for 5 minutes, wait until the temperature drops to room temperature, add 30 μL of magnetic beads, shake and mix evenly, leave at room temperature for 10 minutes, invert and mix from time to time to keep the magnetic beads suspended, separate briefly, place on a magnetic stand and let stand until the magnetic beads are completely adsorbed, then discard the supernatant; 4) First rinse: Continue to add 500 μL of rinse solution 1, shake and mix evenly for 30 seconds, centrifuge briefly, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant; 5) Second rinse: Continue to add 500 μL of rinse solution 2, shake and mix evenly for 30 seconds, centrifuge briefly, place on a magnetic stand and let stand. After the magnetic beads are completely adsorbed, aspirate and discard the supernatant; 6) Third rinse: Repeat step 5) once; 7) Drying: Remove the centrifuge tube from the previous step, let it stand, discard the remaining liquid, open the lid, and air dry for 7-10 minutes; 8) Elution: Add 50-100 μL of elution buffer, mix well by pipetting, incubate at 1600 rpm, 60°C for 10 min on a metal bath, centrifuge briefly, and finally, place on a magnetic rack, let stand, and aspirate the supernatant to obtain the supernatant containing viral DNA and RNA.

6. The extraction method according to claim 5, characterized in that When testing multiple samples, redundancy of the lysis solution and the binding solution needs to be calculated when preparing the total lysis working solution system in step 1).

7. The extraction method according to claim 5, characterized in that The sample is selected from at least one of serum, plasma, and centrifugal supernatant of tissue grinding fluid.

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