A rapid extraction kit for virus nucleic acid in complex biological samples and application thereof

Abstract

The application relates to a rapid virus nucleic acid extraction kit for complex biological samples and application thereof, and belongs to the technical field of molecular biology. The kit comprises a lysis solution, a silica gel membrane nucleic acid adsorption column, a first washing solution, a second washing solution and an eluent; the lysis solution comprises isomeric tridecanol polyoxyethylene ether, polyvinylpyrrolidone K30, trehalose, MgCl2, glycerol, tris (hydroxymethyl) aminomethane hydrochloride and tris (2-carboxyethyl) phosphine hydrochloride. The kit can effectively overcome the problems of the existing nucleic acid extraction kit in animal virus nucleic acid detection, such as a complicated operation process, a long extraction time, insufficient adaptability to complex samples and limited removal effect of amplification inhibitors, and has the advantages of fast extraction speed, simple process, low cost and wide applicability.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a rapid extraction kit for viral nucleic acids from complex biological samples and its application. Background Technology

[0002] Nucleic acid extraction is a crucial pretreatment step in molecular biology detection. Its extraction efficiency and purity directly affect the sensitivity and accuracy of downstream detection results, such as PCR, RT-PCR, real-time quantitative reverse transcription PCR (RT-qPCR), digital PCR, isothermal amplification, and high-throughput sequencing. Therefore, establishing a rapid, efficient, stable, and applicable nucleic acid extraction method suitable for complex biological samples is of great significance for animal disease diagnosis, pathogen monitoring, and rapid on-site detection.

[0003] Currently, nucleic acid extraction mainly includes phenol / chloroform extraction, magnetic bead extraction, and silica membrane centrifuge column extraction. While phenol / chloroform extraction offers high nucleic acid recovery rates, it requires toxic organic reagents such as phenol and chloroform, is cumbersome, has a long experimental cycle, and places high demands on operators and the experimental environment, making it unsuitable for rapid on-site testing. Magnetic bead extraction boasts high automation and high throughput, but requires specialized equipment and has high reagent costs, making it difficult to meet the needs of grassroots laboratories and on-site testing. Silica membrane centrifuge column extraction is widely used in clinical and research fields due to its simple operation and good purification effect; however, traditional commercial kits typically require long lysis times and multiple centrifugations and washes, with the entire extraction process generally taking 30-60 minutes, which is insufficient for rapid detection needs.

[0004] With the development of rapid nucleic acid detection technologies such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and CRISPR and PfAgo, amplification or detection time has been significantly shortened. However, nucleic acid extraction remains one of the most time-consuming steps in the entire detection process, becoming a significant factor limiting the efficiency of rapid on-site detection.

[0005] In animal disease testing, the samples to be tested are mostly complex biological samples such as intestinal tissue, lung tissue, lymph nodes, blood, feces, and various swabs. Among them, fecal samples are particularly rich in bile salts, polysaccharides, lipids, proteins, and other PCR inhibitors. If the lysis buffer cannot fully lyse the virus particles, or if the inhibitors cannot be effectively removed during purification, it will seriously affect amplification reactions such as PCR, RT-qPCR, and RPA, and may even lead to false negative results.

[0006] Currently, some commercially available rapid lysis buffers can achieve rapid nucleic acid release, but most products are only suitable for cell culture or virus culture media, and their effectiveness in extracting complex samples such as tissue homogenates and feces is limited. Although some lysis buffers can be used directly as amplification templates, the lysis products may still contain residual proteins and amplification inhibitors, making them incompatible with real-time quantitative PCR and high-sensitivity detection platforms. In addition, some lysis systems require high-temperature heating, proteinase K digestion, or vector RNA-assisted recovery, which is not conducive to rapid on-site detection.

[0007] Therefore, developing a nucleic acid extraction technology that can rapidly lyse samples, effectively remove amplification inhibitors, and is compatible with multiple molecular detection platforms is of great significance for improving on-site detection efficiency. Summary of the Invention

[0008] This invention provides a rapid viral nucleic acid extraction kit for complex biological samples and its application, overcoming the problems of existing nucleic acid extraction kits in animal viral nucleic acid detection, such as cumbersome operation procedures, long extraction time, insufficient adaptability to complex samples, and limited removal effect of amplification inhibitors.

[0009] One objective of this invention is to provide a rapid viral nucleic acid extraction kit for complex biological samples. The kit includes a lysis buffer, a silica membrane nucleic acid adsorption column, a first washing buffer, a second washing buffer, and an elution buffer. The lysis buffer includes isotridecyl alcohol polyoxyethylene ether, polyvinylpyrrolidone K30, trehalose, MgCl2, glycerol, tris(hydroxymethyl)aminomethane hydrochloride, and tris(2-carboxyethyl)phosphine hydrochloride.

[0010] Furthermore, the final concentrations of each component in the lysis solution are as follows: isotridecyl alcohol polyoxyethylene ether 0.5%-3.0% (v / v), polyvinylpyrrolidone K30 0.5%-1.0% (w / v), trehalose 50mM-100mM, MgCl2 2mM-5mM, glycerol 5%-15% (v / v), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) 20mM-50mM, and tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl) 0.1mM-2.0mM.

[0011] Furthermore, the final concentrations of each component in the lysis solution are as follows: isotridecyl alcohol polyoxyethylene ether 3.0% (v / v), polyvinylpyrrolidone K30 0.8% (w / v), trehalose 60mM, MgCl2 3mM, glycerol 10% (v / v), tris(hydroxymethyl)aminomethane hydrochloride 25mM, and tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl) 1.0mM.

[0012] Furthermore, the first washing solution comprises 2M-6M guanidine isothiocyanate, 10mM-50mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 0.1% Triton X-100, and nuclease-free water, and the pH of the first washing solution is 6.4.

[0013] Further, the second washing solution comprises tris-(hydroxymethyl)aminomethane hydrochloride and ethanol, wherein the final concentration of tris-(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) is 2mM-20mM and the pH is 7.0-8.5; the final volume fraction of ethanol is 60%-90% (v / v); preferably, the final concentration of Tris-HCl in the second washing solution is 2mM and the final volume fraction of ethanol is 80% (v / v).

[0014] Furthermore, the silica membrane nucleic acid adsorption column includes a column shell, a support layer, and a silica membrane; Furthermore, the elution buffer is nuclease-free water.

[0015] A second objective of this invention is to provide an application of the above-mentioned rapid viral nucleic acid extraction kit in the extraction of viral nucleic acids from complex biological samples for non-diagnostic purposes. The complex biological samples include one or more of viral culture media, tissue homogenates, fecal suspensions, whole blood or serum samples; the viral nucleic acids include DNA viral nucleic acids and / or RNA viral nucleic acids.

[0016] Further, in use, the sample to be extracted is mixed with lysis buffer and lysed at room temperature; anhydrous ethanol is added to the lysed mixture and mixed well, then transferred to a silica membrane nucleic acid adsorption column to adsorb DNA or RNA onto the silica membrane; the silica membrane nucleic acid adsorption column is washed sequentially with the first and second washing buffers; after removing the residual washing buffer, elution is performed with elution buffer to obtain DNA or RNA.

[0017] Furthermore, the volume ratio of the sample to be tested to the lysis buffer is 1:0.5-3, preferably 1:1; the lysis time is 3 min-15 min, preferably 5 min-10 min.

[0018] Furthermore, the volume of anhydrous ethanol added is 0.5 times the total volume of the lysed sample and the lysis buffer mixture; after adding anhydrous ethanol and mixing, the resulting mixture is transferred to a silica membrane nucleic acid adsorption column for centrifugation, so that DNA or RNA is adsorbed onto the silica membrane.

[0019] Further, the amount of the first washing solution added is 1.5 to 3.5 times the volume of the extracted sample; the amount of the second washing solution added is 2.5 to 4 times the volume of the extracted sample, and the sample is washed twice; the amount of the elution solution added is 30 μL to 50 μL.

[0020] Furthermore, the DNA or RNA extracted using the kit of this invention can be directly used for molecular detection methods such as PCR, RT-PCR, RT-qPCR, RPA, and PfAgo.

[0021] Advantages compared to existing technologies: This invention employs a lysis buffer combined with a silica membrane nucleic acid adsorption column and a two-step washing system to achieve sample lysis and nucleic acid purification at room temperature, with a simple and rapid process. This invention performs sample lysis at room temperature, eliminating the need for 56°C heating, proteinase K digestion, or the addition of carrier RNA, thus simplifying the sample lysis process. This invention combines room temperature lysis with purification using a silica membrane nucleic acid adsorption column. After sample lysis, anhydrous ethanol is added for the first column loading, without the need for additional binding buffer or pretreatment of the adsorption column. Subsequently, the sample is washed sequentially with a first washing buffer and a second washing buffer, and the nucleic acid is obtained by drying the column and eluting. The obtained nucleic acid can be used for corresponding amplification and detection.

[0022] This invention is applicable not only to RNA virus nucleic acid extraction but also to DNA virus nucleic acid extraction, with a wide range of sample types and pathogen types. Experimental results show that the nucleic acid extracted by the kit of this invention can obtain stable amplification results by RT-qPCR or PCR detection, and has good consistency with the detection results of commercial nucleic acid extraction kits. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the composition of the rapid nucleic acid extraction kit of the present invention; Figure 2 This is a process flow diagram of the rapid nucleic acid extraction method of the present invention; Figure 3 This is a schematic diagram illustrating the composition of the lysis solution and the function of each component in this invention; Figure 4 This invention illustrates the effect of different lysis times on nucleic acid extraction efficiency. Figure 5 This is the result of verifying the applicability of the present invention to nucleic acid extraction from different biological samples; Figure 6 This is a linear regression analysis graph showing the Ct values ​​of samples obtained from the rapid nucleic acid extraction kit of this invention and commercial nucleic acid extraction kits; Figure 7 Agarose gel electrophoresis images of RNA extracted using different methods for RT-PCR amplification are shown. Lane 1 is the negative control; lanes 2 and 3 are samples without column purification; lanes 4 and 5 are RNA extracted using commercial kits; lanes 6 and 7 are RNA extracted using the kit of this invention; and lane 8 is the positive control. Detailed Implementation

[0024] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. However, the present invention is not limited to these embodiments, nor are these embodiments limited in any way.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional techniques in molecular biology, microbiology, recombinant DNA, and immunology. Unless otherwise specified, the materials and preparations involved in the following examples are all commercially available products that can be purchased from the market.

[0026] Unless otherwise specified, all reagents used in this invention are commercially available analytical grade reagents, and all experimental water is nuclease-free water.

[0027] This invention provides a rapid viral nucleic acid extraction kit for complex biological samples, the composition of which is shown in the schematic diagram below. Figure 1 As shown, the kit includes lysis buffer, a silica membrane nucleic acid adsorption column (DNA / RNA column), a first washing buffer, a second washing buffer, an elution buffer, and centrifuge collection tubes. 1. Composition and preparation method of the lysis buffer. (1) Taking the preparation of 100mL lysis buffer as an example, its composition is shown in Table 1.

[0028] Table 1. Components and final concentration range of the lysis buffer

[0029] The lysis solution of this invention (see schematic diagram of the function of each component as shown) Figure 3 As shown, isotridecyl alcohol polyoxyethylene ether, as a nonionic surfactant, can disrupt the viral envelope and host cell membrane, promoting nucleic acid release; tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), as a reducing agent, helps to break some disulfide bonds in proteins, reducing the impact of protein impurities on nucleic acid release and stability; polyvinylpyrrolidone K30 and trehalose help maintain nucleic acid stability; glycerol helps maintain the stability of the lysis system; tris(hydroxymethyl)aminomethane hydrochloride is used to maintain a suitable pH environment for the lysis system; MgCl2 is used to adjust the ionic environment of the lysis system and, together with other components, maintains the stability of the system. The above components work together to promote the lysis of viral particles and host cells and the release of nucleic acids at room temperature, providing suitable conditions for subsequent silica membrane nucleic acid adsorption and purification. Using the lysis buffer prepared in this invention, rapid lysis of viral particles and host cells can be achieved at room temperature, without the need for 56°C heating, proteinase K digestion, or the addition of carrier RNA to release DNA or RNA, providing excellent reaction conditions for subsequent silica membrane nucleic acid adsorption column purification.

[0030] (2) Preparation method First, add approximately 70 mL of nuclease-free water, then add tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.8) and MgCl2 sequentially, and mix thoroughly. Next, add glycerol, trehalose, polyvinylpyrrolidone K30, and isotridecyl alcohol polyoxyethylene ether, and continue to stir slowly until the system is homogeneous. Then, add tris(2-carboxyethyl)phosphine hydrochloride. Finally, add nuclease-free water to bring the volume to 100 mL, mix thoroughly, filter sterilize using a 0.22 μm microporous membrane, aliquot, and store at 4 °C for later use.

[0031] 2. Composition and preparation method of the first washing solution (1) The first washing buffer is used to remove proteins, lipids, polysaccharides and other impurities, while maintaining the stable adsorption of DNA or RNA on the silica membrane, thereby improving the subsequent nucleic acid purification effect. Taking the preparation of 100mL of the first washing buffer as an example, its composition is shown in Table 2.

[0032] Table 2 Composition of the first washing solution

[0033] Note: Adjust the pH of the first washing solution to 6.4.

[0034] In the first washing solution of this invention, guanidine isothiocyanate, as a high-concentration denaturing salt, can further denature proteins and inhibit nuclease activity, while maintaining the adsorption between nucleic acids and the silica membrane; a tris(hydroxymethyl)aminomethane hydrochloride buffer system is used to maintain a suitable washing environment; Triton X-100, as a nonionic surfactant, can help remove lipids, membrane components, and some protein impurities from the sample. The synergistic effect of these components effectively removes proteins, cell debris, and some amplification inhibitors from the lysis buffer, while maintaining the stable adsorption of DNA or RNA on the silica membrane, providing a basis for the subsequent second washing solution to further remove residual salt ions and other inhibitors.

[0035] (2) Preparation method First, add approximately 60 mL of nuclease-free water to a clean container, then add guanidine isothiocyanate to bring the final volume to 100 mL, achieving a concentration of 4 M. Stir thoroughly until completely dissolved. Next, add tris(hydroxymethyl)aminomethane hydrochloride to a final concentration of 20 mM, and then add Triton X-100 to a final volume fraction of 0.1% (v / v). Mix gently. Adjust the pH of the solution to 6.4 using hydrochloric acid. Finally, add nuclease-free water to a final volume of 100 mL and mix thoroughly before use. If necessary, filter the solution through a 0.22 μm microporous membrane.

[0036] 3. Composition and preparation method of the second washing solution (1) The second washing solution is used to further remove salt ions, protein residues, lipids and other nucleic acid amplification inhibitors, while maintaining the stable adsorption of DNA or RNA on the silica membrane. Taking the preparation of 100 mL of the second washing solution as an example, its composition is shown in Table 3.

[0037] Table 3 Composition of the second washing solution

[0038] In the second washing solution of this invention, ethanol removes residual salt ions, small molecule impurities, and some amplification inhibitors from the silica membrane, and promotes the dehydration of the silica membrane, allowing DNA or RNA to continue to be stably adsorbed onto the silica membrane. The tris(hydroxymethyl)aminomethane hydrochloride buffer system maintains a suitable pH environment, reduces nucleic acid loss, and improves the final elution efficiency. Washing twice with the second washing solution prepared in this invention further removes residual proteins, salt ions, and other amplification inhibitors from complex biological samples, improving the purity and stability of DNA or RNA, enabling the obtained nucleic acids to be directly used for molecular detection such as PCR, RT-PCR, RT-qPCR, RPA, and PfAgo. Using the first and second washing solutions together balances nucleic acid recovery rate and purification effect, improving the extraction efficiency of viral nucleic acids from complex biological samples. The second washing solution provided in this invention, used in conjunction with the lysis buffer and the first washing solution, can further improve the extraction efficiency and purification effect of viral nucleic acids from complex biological samples.

[0039] (2) Preparation method First, prepare 20 mL of 10 mM tris-HCl buffer and adjust its pH to 7.5. Then, add 80 mL of anhydrous ethanol and mix thoroughly to obtain the second washing buffer. The final concentration of Tris-HCl in the second washing buffer is 2 mM, and the final volume fraction of anhydrous ethanol is 80% (v / v). After preparation, store in a tightly sealed container for later use to prevent ethanol evaporation from affecting the washing effect.

[0040] The present invention will be further described in detail below with reference to specific embodiments: Example 1: Extraction of viral nucleic acid The following solutions were used: lysis buffer (3.0% (v / v) isotridecyl polyoxyethylene ether, 0.8% (w / v) polyvinylpyrrolidone K30, 60 mM trehalose, 3 mM MgCl2, 10% (v / v) glycerol, 1.0 mM tris(2-carboxyethyl)phosphonic acid hydrochloride, 25 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.8), nuclease-free water to a final volume of 100 mL), and first wash buffer (20 mM tris(hydroxymethyl)aminomethane hydrochloride, 4 M guanidine isothiocyanate, Triton X-100). Viral nucleic acids were extracted from complex biological samples using a combination of 0.1% (v / v) nuclease-free water to a final volume of 100 mL, pH adjusted to 6.4, a second wash buffer (Tris-HCl 2 mM, 80% (v / v) ethanol, and nuclease-free water to a final volume of 100 mL, with the Tris-HCl buffer prepared at pH 7.5), and a silica gel membrane nucleic acid adsorption column. The samples included viral culture media, tissue homogenates, fecal suspensions, whole blood, serum, and cell samples.

[0041] (1) Sample lysis Take 200 μL of the sample to be tested into a 1.5 mL nuclease-free centrifuge tube, add 200 μL of the lysis buffer prepared in Example 1, mix thoroughly by pipetting, and lyse at room temperature for 10 min to allow the virus particles and host cells to be fully lysed and release DNA or RNA.

[0042] (2) Nucleic acid binding and adsorption Add 0.5 times the total volume of anhydrous ethanol to the lysed mixture and mix thoroughly. In this example, after mixing 200 μL of sample with 200 μL of lysis buffer and completing lysis, add 200 μL of anhydrous ethanol. No other binding buffer is added before the first column loading, and the silica membrane nucleic acid adsorption column is not pretreated. Transfer the entire mixture to the silica membrane nucleic acid adsorption column, centrifuge at 12000 rpm for 1 min, discard the filtrate, and place the adsorption column back into the collection tube, followed by the first washing step.

[0043] (3) First step washing Add 500 μL of the first washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the filtrate, and put the adsorption column back into the collection tube.

[0044] (4) Second step washing Add 600 μL of the second washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate; add another 600 μL of the second washing solution and repeat the above centrifugation steps.

[0045] (5) Dry column Centrifuge at 12000 rpm for 2 min. Remove residual washing solution, then incubate the adsorption column at room temperature for 2-5 min to further reduce ethanol residue and minimize its impact on subsequent PCR or RT-qPCR amplification.

[0046] (6) Nucleic acid elution Place the adsorption column in a new 1.5 mL nuclease-free centrifuge tube, add 30 μL-50 μL of nuclease-free water to the center of the silica membrane, let it stand at room temperature for 3-5 minutes, then centrifuge at 12000 rpm for 2 minutes and collect the eluent to obtain purified DNA or RNA.

[0047] The process flow diagram of the rapid nucleic acid extraction method established by this invention is shown below. Figure 2 As shown, viral nucleic acid was obtained through room temperature lysis, silica membrane adsorption, washing, and elution. No 56°C heating, proteinase K digestion, or carrier RNA addition was required during extraction. The following examples validated the suitability of the obtained nucleic acid for RT-qPCR, RT-PCR, and real-time fluorescent PCR detection.

[0048] Example 2: Effect of different lysis times on nucleic acid extraction efficiency To optimize the lysis conditions of the rapid nucleic acid extraction kit of this invention and investigate the effect of different lysis times on nucleic acid extraction efficiency, lysis times of 3 min, 5 min, 10 min, and 15 min were set while keeping other experimental conditions consistent with those in Example 1. 200 μL of PDCoV virus culture medium was added to 200 μL of the lysis buffer of this invention, and lysis was performed at room temperature for 3 min, 5 min, 10 min, and 15 min, respectively. Nucleic acid extraction was then carried out according to the nucleic acid extraction method established in this invention. The extracted nucleic acid was detected by RT-qPCR, and the Ct value was used as an indicator to evaluate the nucleic acid extraction efficiency at different lysis times. Three replicates were set for each group.

[0049] The results are as follows Figure 4 As shown, PDCoV nucleic acid could be successfully extracted at all lysis times. When the lysis time was increased from 3 min to 10 min, the Ct value detected by RT-qPCR gradually decreased, reaching its lowest value at 10 min; when the lysis time was further extended to 15 min, the Ct value did not continue to decrease. Considering both nucleic acid extraction effect and detection efficiency, room temperature lysis for 10 min is the preferred lysis condition for the kit of this invention.

[0050] Example 3: Applicability verification of nucleic acid extraction from different biological samples To verify the applicability of the rapid nucleic acid extraction kit of this invention to various complex biological samples, different types of samples, including viral culture medium, tissue homogenate, fecal suspension, whole blood, and serum, were used to evaluate the nucleic acid extraction performance of the kit. 200 μL of each sample was added to an equal volume of the lysis buffer described in Example 1, mixed thoroughly, and lysed at room temperature for 10 min, followed by nucleic acid extraction. The obtained nucleic acids were detected using RT-qPCR, and the applicability of this method to different types of biological samples was evaluated by whether stable amplification signals were obtained for each type of sample and the corresponding Ct values.

[0051] The results are as follows Figure 5 As shown, the kit of the present invention can obtain target nucleic acids that can be used for RT-qPCR detection from virus culture medium, tissue homogenate, fecal suspension, whole blood and serum samples. Stable amplification signals and corresponding Ct values ​​were obtained for all types of samples, indicating that the kit of the present invention has good applicability to different types of complex biological samples.

[0052] Example 4: Comparison of extraction performance between rapid nucleic acid extraction kit and commercial nucleic acid extraction kit To evaluate the nucleic acid extraction performance of the rapid nucleic acid extraction kit of this invention, nine PDCoV-positive clinical samples (including pig intestinal tissue samples and pig fecal samples) were selected. 200 μL of each of the nine clinical samples was taken, and nucleic acid extraction was performed according to the method described in Example 1 and the instructions of the commercial TaKaRa viral RNA extraction kit. Both groups were eluted with 30 μL of nuclease-free water; and PDCoV was detected using the same RT-qPCR system. The Ct values ​​of each sample were recorded, and ΔCt (Ct of this invention – Ct of the commercial kit) was calculated. Subsequently, linear regression analysis was performed with the Ct value of the commercial kit on the x-axis and the Ct value of the rapid nucleic acid extraction kit of this invention on the y-axis.

[0053] The results are shown in Table 4. All nucleic acids extracted using the rapid nucleic acid extraction kit of this invention yielded stable RT-qPCR amplification results. In the nine clinical samples, the Ct values ​​of the nucleic acids obtained by the two extraction methods generally showed a consistent trend, with most samples exhibiting small differences in Ct values, while a few samples showed some variation. Linear regression analysis showed a strong linear correlation between the Ct values ​​obtained from the rapid nucleic acid extraction kit of this invention and the commercially available kit, with a coefficient of determination R0. 2 It is 0.9741 (e.g.) Figure 6 As shown in the figure, the kit of the present invention can obtain viral nucleic acid that meets the requirements of subsequent RT-qPCR detection.

[0054] Table 4 Comparison of Ct values ​​from 9 positive samples

[0055] Example 5: RT-PCR amplification and verification of RNA extracted by a rapid nucleic acid extraction kit To further verify the applicability of the RNA extracted by the rapid nucleic acid extraction kit of this invention to subsequent RT-PCR detection, RNA was extracted from PDCoV-positive pig fecal samples using the method described in Example 1. A control group without column purification was set up; that is, the samples were directly used for subsequent RT-PCR amplification after lysis without silica membrane nucleic acid adsorption and washing steps to evaluate the impact of column purification on the nucleic acid purification effect of complex samples. A control group extracted using a commercial viral RNA extraction kit was also set up.

[0056] RNA obtained from each group was used as a template for reverse transcription to obtain cDNA. The cDNA was then used as a template for PCR amplification targeting the PDCoV N gene, with an expected amplified fragment size of 329 bp. The upstream primer PDCoV-N1F sequence used for PCR amplification was 5′-CCAAACGCAACCCCAACAATCC-3′ (SEQ ID NO.1), and the downstream primer PDCoV-N1R sequence was 5′-CTTCTCAGTGTCTGCAGAGCCG-3′ (SEQ ID NO.2). The PCR reaction system was prepared according to the PCR amplification kit instructions, including 2×PCR Master Mix, upstream primer, downstream primer, cDNA template, and nuclease-free water. The PCR amplification program followed the PDCoV N gene amplification conditions. After amplification, the PCR products were analyzed by 1.5% agarose gel electrophoresis to observe the amplification of the target band.

[0057] The results are as follows Figure 7 As shown, the unpurified group did not obtain a clear target amplification band, while RNA extracted using both the commercial viral RNA extraction kit and the rapid nucleic acid extraction kit of this invention amplified a PDCoV N gene-specific band of approximately 329 bp. No specific amplification band was observed in the negative control. The RT-PCR amplification band of RNA obtained using the rapid nucleic acid extraction kit of this invention was clear and essentially consistent with the amplification results of the commercial kit extraction group, indicating that the RNA extracted using the kit of this invention can meet the requirements for subsequent RT-PCR amplification. Furthermore, the difference in amplification results between the unpurified and column-purified groups indicates that the silica membrane nucleic acid adsorption and two-step washing purification steps help reduce the impact of residual impurities and amplification inhibitors in complex samples on subsequent amplification reactions, improving the applicability of the obtained nucleic acid for RT-PCR detection.

[0058] Example 6: DNA extraction performance verification of the rapid nucleic acid extraction kit To verify the extraction capability of the rapid nucleic acid extraction kit of this invention for DNA viral nucleic acids and its applicability to DNA viral nucleic acid extraction, PCV5 positive clinical samples were used as the detection targets. Nucleic acid extraction was performed using both the rapid nucleic acid extraction kit of this invention and a commercially available DNA extraction kit. Both groups used the same volume of sample for extraction, maintained consistent elution volumes, and used the same volume of elution buffer as a template for real-time fluorescence PCR detection. Each sample was tested in triplicate. The detection results are shown in Table 5.

[0059] Table 5 Comparison of Ct values ​​in real-time fluorescence PCR of PCV5 positive samples

[0060] The results showed that both the rapid nucleic acid extraction kit of this invention and the commercial DNA extraction kit could successfully extract PCV5 genomic DNA and obtain stable real-time fluorescence PCR amplification results. Compared with the commercial kit, the DNA extracted by the kit of this invention had a lower overall Ct value, indicating that the obtained DNA could be effectively used for subsequent real-time fluorescence PCR detection. These results further demonstrate that the kit of this invention is not only suitable for the extraction of nucleic acids from RNA viruses but also for the extraction of nucleic acids from DNA viruses, indicating that the kit of this invention has good applicability to nucleic acids from different types of viruses.

[0061] Example 7: Evaluation of the sensitivity of the rapid nucleic acid extraction kit To evaluate the performance of the rapid nucleic acid extraction kit of this invention in nucleic acid extraction and downstream detection in samples with low viral load, Getah virus (GETV) material with a pre-determined infectious titer was used. PBS was used as the dilution medium to evaluate the complete workflow of the kit combined with the GETV reverse transcription and quantitative real-time PCR methods. The initial titer of the GETV material used was 10. 8.75 TCID 50 / mL, the total dilution factor of each sample relative to the original material is 10, respectively. 1 10 2 10 3 10 4 10 5 10 6 10 7 and 10 8 The nominal titer of the sample before extraction is calculated by dividing the original titer by the total dilution factor.

[0062] RNA was extracted independently from each gradient sample using the rapid nucleic acid extraction kit of this invention. The obtained RNA was reverse transcribed to obtain cDNA, which was then detected using GETV quantitative PCR. Five technical replicates were set up for each cDNA sample. The original Ct value of each well was recorded, and the number of replicates with Ct ≤ 35 was counted; wells for which the instrument did not provide a definite Ct value were marked as ND. The detection results for each gradient are shown in Table 6. All wells in the negative control were marked as ND.

[0063] Table 6. Detection results of GETV samples with different nominal titers

[0064] Note: ND indicates that the instrument did not provide a definite Ct value. Each concentration is a nominal value calculated based on the original titer and total dilution factor, retained to approximately three significant figures. One RNA sample was extracted for each gradient; the five Ct values ​​in the table correspond to five PCR replicates of the same cDNA sample.

[0065] The results showed that the total dilution factor was 10. 7 At that time, the nominal titer of the sample before extraction was approximately 56.2 TCID. 50 / mL, Ct values ​​were obtained in 4 out of 5 technique replicates, namely 32.31, 30.78, 33.81 and 31.31, and the other well was ND; the total dilution factor was 10. 8 At that time, the nominal titer of the sample before extraction was approximately 5.62 TCID. 50 / mL, all 5 technical duplicates were ND.

[0066] Under the conditions of this embodiment, RNA was extracted from GETV samples using the rapid nucleic acid extraction kit of this invention and detected using reverse transcription and quantitative real-time PCR methods. The lowest detectable nominal titer observed in the PBS-diluted samples was 56.2 TCID. 50 / mL. At this titer, 4 out of 5 replicates of PCR showed amplification signals with Ct≤35.

[0067] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A rapid viral nucleic acid extraction kit for complex biological samples, characterized in that, The kit includes lysis buffer, silica membrane nucleic acid adsorption column, first washing buffer, second washing buffer, and elution buffer; The final concentrations of each component in the lysis solution are as follows: isotridecyl alcohol polyoxyethylene ether 0.5%-3.0%, polyvinylpyrrolidone K30 0.5%-1.0%, trehalose 50mM-100mM, MgCl2 2mM-5mM, glycerol 5%-15%, tris(hydroxymethyl)aminomethane hydrochloride 20mM-50mM, and tris(2-carboxyethyl)phosphine hydrochloride 0.1mM-2.0mM.

2. The rapid viral nucleic acid extraction kit for complex biological samples according to claim 1, characterized in that, The first washing solution comprises 2M-6M guanidine isothiocyanate, 10mM-50mM tris(hydroxymethyl)aminomethane hydrochloride, 0.1% Triton X-100 and nuclease-free water, and the pH of the first washing solution is 6.

4.

3. The rapid viral nucleic acid extraction kit for complex biological samples according to claim 1, characterized in that, The second washing solution comprises tris(hydroxymethyl)aminomethane hydrochloride and ethanol, wherein the final concentration of tris(hydroxymethyl)aminomethane hydrochloride in the second washing solution is 2mM-20mM and the final volume fraction of ethanol is 60%-90%.

4. The rapid viral nucleic acid extraction kit for complex biological samples according to claim 1, characterized in that, The silica membrane nucleic acid adsorption column includes a column shell, a support layer, and a silica membrane.

5. The rapid viral nucleic acid extraction kit for complex biological samples according to claim 1, characterized in that, The elution buffer is nuclease-free water.

6. The application of a rapid viral nucleic acid extraction kit as described in any one of claims 1-5 in the extraction of viral nucleic acid from complex biological samples for non-diagnostic purposes, characterized in that, The complex biological sample includes one or more of the following: viral culture medium, tissue homogenate, fecal suspension, whole blood or serum; the viral nucleic acid is DNA viral nucleic acid and / or RNA viral nucleic acid.

7. The application according to claim 6, characterized in that, In use, the sample to be extracted is mixed with the lysis buffer and lysed at room temperature; anhydrous ethanol is added to the lysed mixture and mixed well, then transferred to a silica membrane nucleic acid adsorption column to adsorb DNA or RNA onto the silica membrane; the silica membrane nucleic acid adsorption column is washed sequentially with the first and second washing buffers; after removing the residual washing buffer, elution is performed with the elution buffer to obtain DNA or RNA.

8. The application according to claim 7, characterized in that, The volume ratio of the sample to be extracted to the lysis buffer is 1:0.5-3, and the lysis time is 3 min-15 min.

9. The application according to claim 7, characterized in that, The volume of anhydrous ethanol added is 0.5 times the total volume of the mixture of the lysed sample and the lysate.

10. The application according to claim 7, characterized in that, The amount of the first washing solution added is 1.5 to 3.5 times the volume of the extracted sample; the amount of the second washing solution added is 2.5 to 4 times the volume of the extracted sample, and the sample is washed twice; the amount of the elution solution added is 30 μL to 50 μL.

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