A kit for detecting African swine fever virus and a fluorescent quantitative PCR detection method

Abstract

The present invention discloses a kit for detecting African swine fever virus and a fluorescence quantitative PCR detection method, which belongs to the technical field of kits and virus detection. The kit includes an African swine fever virus capture probe (VP72-CP), a [P 6,6,6,14 + ][Co(hfacac)3 ‑ ] materials, and specific probes for fluorescent quantitative PCR detection of African swine fever virus. In the present invention, the capture probe is anchored and bound to the magnetic ionic liquid material. The combined capture material can specifically recognize and adsorb and capture African swine fever virus nucleic acid, and can be used to detect the content of African swine fever virus through fluorescent quantitative PCR detection method with good sensitivity, specificity and repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of kits and virus detection, and relates to a kit for detecting African swine fever virus and a fluorescent quantitative PCR detection method. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease caused by the African swine fever virus (ASFV). It highly infects domestic pigs and Eurasian wild boars, characterized by rapid onset, a short course, and a mortality rate as high as 100%. Clinical manifestations include fever, difficulty breathing, coughing, viscous or serous discharge from the eyes and nose, bleeding, redness, and cyanosis of the skin, severe bleeding from multiple internal organs, and severe spleen enlargement in some pigs. Conventional ASF testing, which primarily involves sampling serum, plasma, anticoagulant blood, oral mucus, and lung, lymph node, kidney, bone marrow, and tonsil tissue, falls short of preventive measures. Currently, there is no effective commercial vaccine or specific treatment for ASFV globally. Residual ASFV in the environment can infect pigs through insect vectors, severely impacting pig farmers and the entire supply chain. Therefore, early prevention is crucial for controlling ASF, and environmental detection of ASFV plays a crucial role in disease prevention and control.

[0003] As a functional material, ionic liquids have been successfully applied in various fields, such as catalysis, synthesis, electrochemistry, and extraction. They have been widely used in gas capture, solvents, catalysts, and extraction, achieving excellent results. Magnetic ionic liquids are a new type of material with high thermal stability, easy modification, reusability, and extremely low solubility in water. These properties make magnetic ionic liquids suitable as extraction reagents for a wide range of applications, including the extraction of DNA from the environment. Magnetic ionic liquids combined with probes have the characteristics of low background DNA co-extraction, high extraction efficiency, and strong specificity. Compared with the extraction methods of commercial environmental DNA extraction kits, the magnetic ionic liquid DNA extraction method is simple to operate, does not require complex reagents and expensive instrumentation, and greatly shortens the extraction time. This shows that the use of magnetic ionic liquids as environmental DNA extraction reagents has many advantages and great development prospects.

[0004] Xitian Peng et al. (Coupling oligonucleotides possessing a poly-cytosine tag with magnetic ionic liquids for sequence-specific DNA analysis; Xitian Peng et al., 2018. Chem Commun. 54, 73. DOI: 10.1039 / c8cc05954c) prepared a magnetic ionic liquid [P 6,6,6,14 + ][Co(hfacac)3 - ] material, and reported that the material can enrich the target DNA in animals, but did not disclose that it can be used to detect pathogenic nucleic acids. Summary of the Invention

[0005] In view of the technical deficiencies, the first purpose of the present invention is to provide a kit for extracting and detecting African swine fever virus, and the second purpose is to provide a fluorescent quantitative PCR detection method for African swine fever virus.

[0006] To achieve the first technical objective above, the inventors, based on years of research experience in the field of virus detection, conducted extensive experimental research and unremitting exploration, ultimately obtained the following technical solution:

[0007] A kit for detecting African swine fever virus, comprising an African swine fever virus capture probe (VP72-CP), and a [P 6,6,6,14 + ][Co(hfacac)3 - ] materials, and specific primers (VP72-F / R) and probe (VP72-P) for fluorescent quantitative PCR detection of non-swine fever virus;

[0008] The present invention [P 6,6,6,14 + ][Co(hfacac)3 - The material is a magnetic ionic liquid with the following chemical structure:

[0009]

[0010] The nucleotide sequence of the African swine fever virus capture probe is shown in SEQ ID NO.1 in the sequence table, and the specific sequence is: 5'-C (20) -CACAAGCCGCACCAAAGCAAACCT-3';

[0011] The nucleotide sequences of the specific primers for fluorescent quantitative PCR detection of non-swine fever virus are shown in SEQ ID NO.2 and SEQ ID NO.3 in the sequence table. The specific sequences are:

[0012] Upstream primer: 5'-GGCCCTCTCCTATGCAACATTC-3'

[0013] Downstream primer: 5'-GGGTTGGTATGGCTACACGTTC-3'

[0014] The nucleotide sequence of the probe for fluorescent quantitative PCR detection of non-swine fever virus is shown in SEQ ID NO. 4 in the sequence table, and its specific sequence after modification with a fluorescent reporter group and a quencher group is:

[0015] 5'-FAM-CACAAGCCGCACCAAAGCAAACCT-BHQ1-3'.

[0016] It should be noted that the large and complex genome of ASFV can encode up to 200 proteins, of which more than 50 are structural proteins. Structural proteins play an important role in the processes of viral DNA complexation, assembly, transcription, and immune evasion. The p72 protein accounts for about 31%-33% of the total mass of the virus particles and is about 73.2kD in size. It is one of the structural proteins of African swine fever virus. The p72 protein has good immunogenicity, and the B646L gene sequence encoding the p72 protein is highly conserved. Therefore, the present invention uses the B646L gene sequence as the target sequence to design a capture probe (VP72-CP), fluorescent quantitative PCR specific primers (VP72-F / R) and a probe (VP72-P). At the same time, there is a continuous sequence of 20 cytosine nucleotides at the 5' end of the CP to facilitate the interaction with the capture probe and [P 6,6,6,14 + ][Co(hfacac)3 - ] materials, the materials combine to form CP-[P 6,6,6,14 + ][Co(hfacac)3 - ] Composite capture material. P72 protein is the main structural protein of ASFV, encoded by the B646L gene. This gene is highly conserved and has strong antigenicity. The amino acid sequence of different virus strains is not much different. Compared with other structural proteins of ASFV, it has better stability and is the most commonly used target gene in virus detection. In the present invention, the screening effect of the capture probe VP72-CP on African swine fever virus enables the constructed African swine fever virus nucleic acid extraction method to have the ability to specifically identify African swine fever virus. The minimum detection limit of the prepared African swine cancer virus detection is 1.05×10 0 copies / μL.

[0017] Further preferably, the African swine fever virus detection kit as described above further comprises a DNA desorption solution, wherein the DNA desorption solution is preferably RNase-free water.

[0018] Further preferably, the African swine fever virus detection kit as described above also includes a fluorescent quantitative PCR reaction system.

[0019] In addition, the present invention has constructed a DNA purification and extraction method based on a magnetic ionic liquid modified with an oligonucleotide probe and applied it to the extraction of African swine fever virus nucleic acid. This method can quickly and efficiently extract and separate ASFV target DNA sequences from environmental samples. It is simple to operate, has high extraction efficiency, and the magnetic ionic liquid material can be recycled and reused. It is superior to commercial column extraction kits in terms of minimum extraction limit and low-concentration DNA extraction efficiency. On this basis, the present invention designed fluorescent quantitative PCR-specific primers and probes based on the conserved region of the VP72 gene of ASFV, constructed a highly sensitive and specific ASFV fluorescent quantitative PCR detection method, and used it as a monitoring, early warning, and purification control method for ASFV.

[0020] Specifically, the second object of the present invention is achieved as follows: a non-diagnostic African swine fever virus fluorescence quantitative PCR detection method, the method comprising the following steps:

[0021] (1) The above-mentioned African swine fever virus capture probe and [P 6,6,6,14 + ][Co(hfacac)3 - ] materials are mixed, incubated, and unbound substances are removed to obtain an incubation product;

[0022] (2) adding a sample to be tested to the incubation product, performing DNA capture, removing unbound substances, and obtaining a captured product;

[0023] (3) adding a desorption solution to the captured product to desorb the DNA and remove the captured product to obtain a desorption product;

[0024] (4) The specific primers and probes for the fluorescent quantitative PCR detection of the above-mentioned African swine fever virus are added to the fluorescent quantitative PCR reaction system, and then the desorbed product is added to amplify the target sequence, and the Ct value of the desorbed product is measured, and the content of African swine fever virus in the sample is converted using the standard equation.

[0025] Further preferably, in the non-diagnostic African swine fever virus detection method as described above, in step (1) or step (2), the method of removing unbound matter includes: performing magnetic adsorption separation on the mixed solution obtained after incubation or capture to obtain a supernatant and a lower layer liquid, and washing the lower layer liquid.

[0026] Further preferably, in the fluorescent quantitative PCR detection method for African swine fever virus for non-diagnostic purposes as described above, the method of removing the incubation product in step 3) includes: performing magnetic adsorption separation on the mixed liquid obtained after desorption to obtain a supernatant and a lower layer liquid, and retaining the supernatant as the extraction product.

[0027] Further preferably, in the fluorescent quantitative PCR detection method for African swine fever virus for non-diagnostic purposes as described above, the parameters of the incubation step in step 1) are: incubation at 30-80°C for 5min-10min (the most preferred parameter is incubation at 50°C for 7min).

[0028] Further preferably, in the fluorescent quantitative PCR detection method for African swine fever virus for non-diagnostic purposes as described above, the parameters of the capture step in step 2) are: adjusting the pH of the system after thermal denaturation to 6-10, and capturing at 56-60°C for 5min-10min (the most preferred parameters are adjusting the pH of the system after thermal denaturation to 7, and capturing at 58°C for 5min).

[0029] Further preferably, in the fluorescent quantitative PCR detection method for African swine fever virus for non-diagnostic purposes as described above, the parameters of the desorption step in step 3) are: heating at 70-90°C for 15min-25min (the most preferred parameter is desorption at 70°C for 15min).

[0030] Compared with the prior art, the present invention provides a kit for extracting and detecting African swine fever virus nucleic acid, comprising [P 6,6,6,14 + ][Co(hfacac)3 - ] materials, as well as specific primers and probes for fluorescent quantitative PCR detection of non-swine fever virus. The kit and the corresponding detection method have the following advantages and significant improvements:

[0031] (1) In the present invention, [P 6,6,6,14 + ][Co(hfacac)3 - ] is a magnetic ionic liquid material that has a strong affinity for continuous cytosine nucleotide sequences and can be used as the sequence of the anchoring material in the capture probe. The combined capture material can specifically identify and adsorb and capture African swine fever virus nucleic acid, thereby greatly improving the accuracy of detection.

[0032] (2) In the present invention, after the material is captured, a DNA desorption solution can be obtained through a desorption step, and a fluorescent quantitative PCR amplification reaction is carried out with specific primers and probes for fluorescent quantitative PCR detection of African swine fever virus to measure its Ct value, thereby realizing quantitative detection of African swine fever virus.

[0033] (3) Specific primers and probes were designed based on the conserved region of the ASFV VP72 gene, and an ASFV fluorescence quantitative PCR detection method was constructed. The results showed that the detection method had good sensitivity, specificity, and repeatability. Its clinical application was the same as that of the commercial ASFV detection kit, and it can be preliminarily used in clinical testing.

[0034] (4) Oligonucleotide probes were designed based on the conserved region of the ASFV VP72 gene, and an ASFV nucleic acid purification and extraction method using oligonucleotide probes modified with magnetic ionic liquids was constructed. The results showed that this extraction method can selectively extract ASFV DNA with an extraction efficiency of up to 90.51%. It also has good repeatability and reusability. The minimum extraction limit is 10 times that of the commercial extraction kit, and the overall extraction efficiency is higher than that of the commercial extraction kit.

[0035] (5) The minimum extraction limit of African swine fever virus nucleic acid by the African swine fever nucleic acid extraction kit of the present invention can reach 1.05×10 0 The detection limit is low, and it is expected to be used in the detection of samples with low virus concentration. At the same time, the kit of the present invention has good extraction efficiency and selectivity, and can be used for DNA extraction from actual environmental samples. In addition, the fluorescent quantitative PCR specific primers and probes can quickly and accurately detect African swine fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The magnetic ionic liquid material of the present invention [P 6,6,6,14 + ][Co(hfacac)3 - ]'s molecular structure diagram.

[0037] Figure 2 The polyacrylamide gel electrophoresis results are shown in Figure 2. M is the DNA standard DL5000; 1-6 are positive controls; 7-12 are negative controls.

[0038] Figure 3 is the result of sensitivity test; among them: 1-7 are 1.05×10 6 ~10 0 copies / μL; 8 for negative control.

[0039] Figure 4 is the standard curve of CT value and DNA concentration.

[0040] Figure 5 Specificity test results; 1 is ASFV; 2-6 are PoRV, PRRSV, PEDV, and PRV, respectively; 7 is a negative control.

[0041] Figure 6The results of condition optimization for the binding of oligonucleotide probes to magnetic ionic liquids; including: (a) temperature; (b) volume ratio; (c) binding time.

[0042] Figure 7 Optimization results of conditions for capturing DNA using oligonucleotide probe-modified magnetic ionic liquids; where: (a) temperature; (b) pH; (c) ionic strength.

[0043] Figure 8 Optimization results of conditions for desorption of DNA by oligonucleotide probe-modified magnetic ionic liquids; where: (a) temperature; (b) time.

[0044] Figure 9 The results of the reusability test.

[0045] Figure 10 The results of the comparative test are shown in Figure 1. Among them, samples 1-8 are 1.05×10 6 ~10 -1 copies / μL. DETAILED DESCRIPTION

[0046] The technical scheme of the present invention is described clearly and completely below in conjunction with the examples of implementation. The following examples are only used to illustrate the present invention and should not be regarded as the scope of protection of the present invention. The technical operation steps or conditions not specified in the examples are all in accordance with the techniques or conditions described in the literature of the field or in accordance with the instructions of the parent. The reagents or instruments used are the manufacturers indicated and are all conventional products that can be purchased commercially.

[0047] In this example, six pairs of fluorescent quantitative PCR primers and TaqMan probes were designed based on the ASFV VP72 gene. A primer screening test was performed using a prepared ASFV standard positive plasmid as a conventional PCR amplification template. The results showed that the VP72-F1 / R1 primers produced a single, brighter, and clearer amplification band compared to the other primers, with no primer dimers present. Therefore, VP72-F1 / R1 and VP72-P1 were selected to construct an ASFV fluorescent quantitative PCR detection method. After optimizing the fluorescent quantitative PCR system, specificity tests demonstrated the method's high specificity. A sensitivity test was also conducted using a serial dilution of the standard positive plasmid as the amplification template. The results showed that the method's minimum nucleic acid detection concentration was 10 copies / μL, surpassing the sensitivity of the qPCR detection method established by Yu Bin et al. Intra- and inter-batch reproducibility tests verified the method's high reproducibility. Finally, 184 environmental samples from different pig farms in Hubei Province were tested and compared with three different commercial kits. The results showed that the detection rate of this method and the other commercial kits all reached 100%.

[0048] 1 Materials and Methods

[0049] 1.1 Test materials

[0050] 1.1.1 Strain

[0051] The complete ASFV VP72 gene fragment was synthesized by Wuhan Bioengineering Biological Co., Ltd. Porcine group A rotavirus (PoRV, HuB2020 strain), porcine reproductive and respiratory syndrome virus (PPRSV, H-1 strain), porcine epidemic diarrhea virus (PEDV, DY2 strain), and pseudorabies virus (PRV, HuB2020 strain) were all stored by the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences.

[0052] The 184 environmental samples used in this study were collected from four breeding pig farms in Hubei Province.

[0053] The magnetic ionic liquid material used in this study was preserved by Hubei Academy of Agricultural Sciences and has the chemical formula [P 6,6,6,14 + ][Co(hfacac)3 - ], molecular structure such as Figure 1 As shown, the preparation method refers to Xi Tian Peng et al., 2018. Chem Commun. 54, 73. DOI: 10.1039 / c8cc05954c.

[0054] 1.1.2 Main reagents

[0055] The reagents and consumables used in this study are shown in Table 1.

[0056] Table 1 Reagents and consumables used in this study

[0057]

[0058] 1.1.3 Preparation of main solution

[0059] (1) LB liquid medium: Dissolve 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl in an appropriate amount of sterile deionized water. After complete dissolution, adjust the pH to 7.0-7.2 with 5 mol / L NaOH. Add ddH2O to 1,000 mL. Sterilize with high-pressure steam at 121°C for 20 min and store at room temperature until use.

[0060] (2) LB solid medium: Add 1.5% agar powder to LB liquid medium, dissolve in an appropriate amount of sterile deionized water, and sterilize by high-pressure steam sterilization at 121°C for 20 min. After the medium temperature drops to about 50°C, add the appropriate antibiotics and spread the medium onto plates. After the medium solidifies, place it upside down and store at 4°C until ready for use.

[0061] (3) Ampicillin (Amp): Prepare the stock concentration to 200 mg / mL with sterile deionized water, sterilize by filtration through a 0.22 μm pore size filter, and aliquot at -20°C for use.

[0062] (4) 80% glycerol: Pour 80 mL of glycerol into a 100 mL volumetric tube, adjust the volume to 100 mL, sterilize with high pressure steam for 20 min, and store for later use.

[0063] (5) Solutions of Different pH Values: Britton-Robison (BR) buffer is a mixture of phosphoric acid, boric acid, and acetic acid, all at a concentration of 0.04 mol / L. Add the desired amount of 0.2 mol / L NaOH solution to the BR buffer and adjust the pH to the desired value using a pH meter. The configuration is shown in Table 2.

[0064] Table 2 Preparation of different pH solutions

[0065]

[0066] 1.2 Experimental methods

[0067] 1.2.1 ASFV fluorescent quantitative PCR primer and probe design

[0068] In this study, 7 pairs of specific experimental primers and 6 TaqMan probes were constructed and designed according to the experimental requirements, all of which were synthesized by Wuhan Novozymes Biotechnology Co., Ltd.

[0069] (1) ASFV VP72 fluorescent quantitative PCR primers: The African swine fever virus (ASFV VP72) gene sequence was searched on NCBI, and 6 pairs of fluorescent quantitative PCR primers and probes were designed using Oligo7.0 primer probe design software. The 6 pairs of specific primers for ASFV VP72 gene fluorescent quantitative PCR were named VP72-F1 / 2 / 3 / 4 / 5 / 6 and VP72-R1 / 2 / 3 / 4 / 5 / 6; the TaqMan probes were named VP72-P1 / 2 / 3 / 4 / 5 / 6.

[0070] (2) Specific primers for constructing ASFV VP72 standard positive plasmids were named VP72-QF and VP72-QR. See Table 3 for details.

[0071] Table 3 Primer sequences used in the experiment

[0072]

[0073] 1.2.2 Preparation of ASFV VP72 plasmid

[0074] (1) Construction of positive plasmid

[0075] A synthetic intact fragment of the ASFV VP72 gene was used as a template for amplification using a Phanta high-fidelity DNA polymerase reaction solution and primers VP72-QF / VP72-QR (produced by Wuhan Novozymes Biotechnology Co., Ltd.). The reaction system (50.0 μL) consisted of 25.0 μL of 2× PhantaMax MasterMix (DyePlus), 2.0 μL of each upstream and downstream primer, 19.0 μL of sterile water, and 2.0 μL of DNA. The PCR reaction procedure was as follows: 95°C pre-denaturation for 3 min, 35 cycles of 95°C denaturation for 15 s, 57°C annealing for 15 s, and 72°C extension for 2 min; a complete extension at 72°C for 5 min, and storage at 4°C.

[0076] (2) Purification and recovery of PCR amplification products

[0077] a. After electrophoresis is complete and the gel is observed, use a scalpel blade to cut the single target DNA band from the agarose gel, place it in a clean centrifuge tube, and weigh it;

[0078] b. Add an equal volume of sol solution to the centrifuge tube (if the gel weighs 0.1g and its volume can be considered as 100uL, then add 100μL of sol solution). Place the tube in a 50℃-55℃ water bath for 10 minutes, gently turning the tube upside down to ensure that the gel is fully dissolved.

[0079] c. Add the solution obtained in the previous step to an adsorption column (place the adsorption column in a collection tube), centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube;

[0080] d. Add 600 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube;

[0081] e. Repeat the steps

[0082] f. Centrifuge at 12,000 rpm for 2 minutes to remove as much wash solution as possible. Place the column open at room temperature or in a 50°C incubator for several minutes to remove any remaining wash solution and prevent ethanol in the wash solution from affecting subsequent experiments.

[0083] g. Place the adsorption column in a clean centrifuge tube and dropwise add 50 μL of eluent preheated in a 65°C water bath to the center of the adsorption membrane. Let stand at room temperature for 2 minutes and centrifuge at 12,000 rpm for 1 minute.

[0084] h. DNA products were stored at -20°C

[0085] (3) Connecting carrier

[0086] The purified and recovered PCR product was connected to the pCE2TA / Blunt-Zero vector using the 5minTA / Blunt-Zero Cloning Kit of Nanjing Novozymes Biotechnology Co., Ltd. The reaction system was 5×TA / Blunt-Zero Cloning Mix 1.0μL, PCR product 2.0μL, and sterile water 2.0μL. The reaction procedure was: 25℃ (PCR instrument temperature control) for 5min.

[0087] (4) Transformation of ligation products

[0088] a. Cells were transformed with DH5α chemically competent cells from Nanjing Novozymes Biotechnology Co., Ltd. The competent cells were frozen at -80°C and quickly thawed on ice.

[0089] b. Add the plasmid to be transformed into 100 μL of competent cells, gently tap the tube to mix (avoid pipetting), and let it stand on ice for 30 minutes.

[0090] c. After the ice bath, place the competent cells in a 42°C water bath. Heat shock them for 45 seconds, then quickly place them on ice for 2 minutes. Add 900 μL of LB liquid medium (without antibiotics) to the centrifuge tube, mix thoroughly, and then shake at 37°C, 200 rpm for 1 hour to recover.

[0091] d. After recovery, centrifuge the competent cells at 5,000 rpm for 3 minutes. Discard 900 μL of the supernatant, resuspend the cells in the remaining culture medium, and evenly spread the cells on an LB solid culture medium plate containing ampicillin resistance.

[0092] e. Place the plate upright in a 37°C incubator for 10 minutes. After the bacterial solution is completely absorbed, invert the plate and incubate overnight.

[0093] (5) Identification and sequencing of recombinant plasmids

[0094] Individual colonies grown on LB solid medium were inoculated into 2 mL of LB liquid medium (containing antibiotics) and incubated at 37°C in an incubator at 180 rpm for 8–12 hours. The culture was then used as a PCR template for amplification using VP72-QF / QR-specific primers. The recombinant plasmid was identified by PCR. The reaction components were as follows: PCR reaction system (25 μL): 1.0 μL each of the upstream and downstream primers, 12.5 μL of 2× Phanta MaxMasterMix (DyePlus), 1.0 μL of template, and 9.5 μL of sterile water. The PCR protocol was as follows: 95°C initial denaturation for 3 minutes, followed by 35 cycles of 95°C / 15 seconds, 57°C / 15 seconds, and 72°C / 2 minutes; extension at 72°C for 2 minutes, and cooling at 10°C for 2 minutes. After amplification, PCR products containing a single target DNA band were sequenced based on agarose gel electrophoresis.

[0095] (6) Plasmid extraction

[0096] a. Use a sterile pipette to pick a single colony from the LB plate and inoculate it into 5 mL of liquid LB medium containing ampicillin. Incubate the culture in a shaker at 37°C at 180 rpm for 8-12 hours. Centrifuge the culture at 12,000 rpm for 1 minute and discard the supernatant. Invert the centrifuge tube onto sterile absorbent paper and let it sit for 30 seconds to allow any remaining liquid to drain.

[0097] b. Take 600 μL of Reagent A (pre-cooled to 0℃-4℃), add it to the centrifuge tube containing the bacterial pellet, and vortex to mix thoroughly.

[0098] c. Add 200 μL of freshly prepared Reagent B, gently invert the tube to mix thoroughly, and let it stand on ice for 2 minutes (the solution in the tube will gradually become clear and translucent).

[0099] d. Add 150 μL of Reagent C and gently invert to mix. A white flocculent precipitate will be formed.

[0100] e. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant to a fresh centrifuge tube, taking care not to aspirate the precipitate. Add 2 volumes of pre-chilled anhydrous ethanol, mix thoroughly, and incubate at -20°C for 20 minutes.

[0101] f. Centrifuge at 12,000 rpm for 10 min at 4°C and discard the supernatant. Add 100 μL of 70% ethanol to the centrifuge tube to wash the precipitate and place on ice for 2 min.

[0102] g. Centrifuge at 12,000 rpm for 5 minutes at 4°C and discard the supernatant. Invert the tube onto sterile absorbent paper and let it sit for 30 seconds to drain any remaining liquid. Dry at room temperature.

[0103] h. Add 50 μL TE buffer (containing 0.5 μL RNase A), dissolve the precipitate, and store at -20°C.

[0104] 1.2.3 ASFV fluorescent quantitative PCR primer screening

[0105] Using the standard positive plasmid constructed in 1.2.2 as a template, perform conventional PCR amplification with the six pairs of fluorescent quantitative PCR primers designed in 1.2.1. Positive and negative controls were set for each pair of fluorescent quantitative PCR primers. The amplification products of each primer pair were examined by polyacrylamide gel electrophoresis, and the primer pair with the best amplification effect was selected. The amplification reaction system and reaction procedures are shown in Tables 4 and 5.

[0106] Table 4 Reaction system

[0107]

[0108] Table 5 Reaction procedure

[0109]

[0110] 1.2.4 Optimization of ASFV VP72 Fluorescence Quantitative PCR Reaction Conditions

[0111] Using the standard positive plasmid constructed according to 1.2.2 as a template, the primer and probe concentrations were screened according to the reaction system in Table 6. Four gradients of primer and probe final concentrations were set (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, and 0.4 μmol / L), with three replicates for each gradient. The optimal amplification system was determined using the matrix method. Once the system was determined, five annealing temperature gradients (54°C, 56°C, 58°C, 60°C, and 62°C) were set according to the reaction program in Table 7, with three replicates for each gradient, to determine the optimal annealing temperature.

[0112] Table 6 Reaction system

[0113] Components Volume (μL) 2×PerfectStartⅡProbeqPCRSuperMix 10.0 VP72-F 0.4 VP72-R 0.4 VP72-P 0.4 template 1.0 <![CDATA[ddH2O]]> 7.8 Total volume 20.0

[0114] Table 7 Reaction procedure

[0115] Reaction stage temperature time Number of cycles Pre-denaturation 94℃ 30s 1 transsexual 94℃ 5s 40 annealing 54 / 56 / 58 / 60 / 62℃ 30s 40

[0116] 1.2.5 Sensitivity test of ASFV VP72 by fluorescence quantitative PCR

[0117] The concentration of the positive plasmid stock solution was measured by NanoPhotometerN50 spectrophotometer, and the positive plasmid stock solution was diluted 10 times in a gradient manner. The established method was used for detection, with 3 replicates for each gradient. A negative control was set up, and a standard curve was constructed to determine the minimum detection limit of the established method.

[0118] 1.2.6 Specificity test of ASFV VP72 fluorescence quantitative PCR

[0119] PoRV, PRRSV, PEDV, PRV and ASFV VP72 positive plasmids were used as reaction templates, with 3 replicates per group, and ddH2O was used as a negative control. ASFV VP72 fluorescence quantitative PCR method was used for amplification. 1.2.7 Repeatability test of ASFV VP72 fluorescence quantitative PCR

[0120] The established method was used to detect the same sample in the same batch and the same samples in different batches using a 10-fold gradient dilution of the reference plasmid as a template. Three replicates were set for each sample, and the intra- and inter-batch coefficients of variation were calculated to test the stability of the established method.

[0121] 1.2.8 Detection of ASFV VP72 in Clinical Samples by Fluorescence Quantitative PCR

[0122] A total of 184 environmental samples from four different breeding pig farms in Hubei Province were collected and tested using the optimized ASFVVP72 fluorescent quantitative PCR method. The results were compared with those of three other commercial kits to determine the consistency of the new method with the commercial kit sample detection.

[0123] 1.2.9 Design of oligonucleotide probes

[0124] According to the strong affinity of magnetic ionic liquid materials for 20 cytosine base sequences, 20 cytosine bases were added to the 5' end of the VP72-P sequence as oligonucleotide probes and magnetic ionic liquid anchoring parts, and it was named VP72-CP:C 20 -CACAAGCCGCACCAAAGCAAACCT.

[0125] 1.2.10 Synthesis of Oligonucleotide Probe-Modified Magnetic Ionic Liquid Composites

[0126] The synthesized oligonucleotide probe VP72-CP dry powder was dissolved in ddH2O and vortexed to mix. The final concentration of the probe solution was 10 mmol / L. 10 μL of the probe was taken and vortexed with 5 μL of MIL to disperse the oligonucleotide probe-modified magnetic ionic liquid (MIL-Probe) in the solution. The solution was placed in a PCR instrument and incubated at 35°C for 5 minutes to allow the probe to bind to the MIL surface. The MIL-Probe in the solution was adsorbed by a magnetic rod and washed twice with ddH2O to prepare an oligonucleotide probe-modified magnetic ionic liquid (MIL-Probe).

[0127] According to the above steps, the temperature, volume ratio of probe to MIL, and binding time conditions in the experiment were changed. The concentration of the probe before and after binding was measured using a NanoPhotometer N50 spectrophotometer. The binding amount of the probe (Qng / μL) was calculated using formula 3-1. The binding amount of the probe under different conditions was compared to explore the effect of single-factor changes on probe binding and the experimental conditions for the maximum binding amount of the probe.

[0128] Q=(C1-C2)×V1(3-1)

[0129] Where C1 (ng / μL) is the probe concentration before binding, C2 (ng / μL) is the probe concentration after binding, and V1 (μL) is the volume of the probe solution.

[0130] 1.2.11 DNA capture by oligonucleotide probe-modified magnetic ionic liquids

[0131] Add the washed MIL-Probe to 200 μL of the standard plasmid prepared in Method 2.3.1 and vortex to mix thoroughly. Place the probe in a PCR instrument and heat it with a controlled temperature setting: denaturation at 95°C for 3 minutes, binding at 60°C for 5 minutes. After capture, attach the MIL-Probe to the solution using a magnetic rod and wash twice with ddH2O.

[0132] According to the above steps, the capture temperature, pH value and ionic strength conditions in the capture test were changed, the DNA capture efficiency was compared, and the influence of various factors on the capture of DNA by MIL-Probe was explored. The concentration of positive plasmid before and after binding was measured by NanoPhotometerN50 spectrophotometer, and the DNA capture efficiency (W1%) was calculated. The calculation formula is shown in 3-2. The DNA capture efficiency under different conditions was compared to explore the experimental conditions for the maximum DNA capture efficiency.

[0133] W1%=(C4-C5) / C4×100%(3-2)

[0134] In the formula, C4 (ng / μL) is the concentration of DNA in the solution before capture, and C5 (ng / μL) is the concentration of DNA in the solution after capture. 1.2.12 Desorption of DNA by Magnetic Ionic Liquid Modified with Oligonucleotide Probes

[0135] Prepare 20 μL of ddH2O as the desorption solution, add the washed MIL-Probe, place it in a PCR instrument and heat it at 70°C for 10 minutes. After heating, cool it down quickly in an ice bath, and separate the MIL-Probe and the desorption solution by magnetic adsorption.

[0136] Following the above steps, the desorption temperature and time conditions were varied during the desorption experiment, and the DNA desorption efficiency of each group was compared to explore the impact of various factors on DNA capture by the MIL-Probe. The Ct values ​​of the desorption solution and the positive plasmid were measured using a constructed fluorescence quantitative PCR method. The copy number concentration of the positive plasmid before and after capture was calculated using the constructed standard curve equation (Formula 3-3). The DNA capture efficiency (W2%) was calculated using the formulas shown in 3-4 and 3-5. The DNA capture efficiency under different conditions was compared to explore the experimental conditions that maximized DNA desorption efficiency.

[0137] X=(38.459-Y) / 3.33(3-3)

[0138] C = 1.05 × 10 X (3-4)

[0139] W2%=(C6×20) / [(C4-C5)×200]×100(3-5)

[0140] In the formula, Y represents the Ct value of fluorescence quantitative PCR, and C6 (copies / μL) represents the DNA copy number concentration after desorption.

[0141] 1.2.13 Repeatability test

[0142] (1) Intragroup repeat test: DNA extraction of the same batch of the same positive plasmid sample was performed under the same conditions. Ten samples were extracted repeatedly and the extraction efficiency of each sample and the relative standard deviation (RSD) within the batch were calculated.

[0143] (2) Repeated test between groups: DNA extraction was performed on different batches of the same sample. The extraction interval for each positive plasmid sample was 12 h. Ten samples were extracted for 5 consecutive days. The extraction efficiency of each sample and the RSD value between batches were calculated.

[0144] 1.2.14 Reusability test

[0145] The reusability of the MIL was evaluated by performing four consecutive probe binding, DNA capture, and desorption experiments. After desorption, the regenerated MIL was used again as binding-capture-desorption material for four repeats, and the DNA extraction efficiency was calculated each time.

[0146] 1.2.15 Comparative test

[0147] The concentration was 1.05×10 6 The positive standard with 10 copies / μL was serially diluted 10-fold to a concentration of 1.05×10 0 The positive plasmids of each gradient were extracted using the optimized extraction method and the instructions of the commercial extraction kit. Three replicates were set for each gradient, and the Ct values ​​of the DNA solutions after extraction by the two methods were determined according to the constructed ASFV fluorescence quantitative PCR method.

[0148] 2 Results

[0149] 2.1 ASFV fluorescence quantitative PCR detection method

[0150] 2.1.1 Identification of ASFV VP72 Plasmid

[0151] The synthesis size of ASFVVP72 plasmid is the full length of VP72 gene. The plasmid was sent to Wuhan Qingke Biotechnology Co., Ltd. for genome sequencing. The obtained gene sequencing results were sequenced in NCBIBLAST. The results showed that the homology with the nucleic acid sequence of the target gene was 100%, confirming that the ASFVVP72 standard positive plasmid was successfully constructed.

[0152] 2.1.2 ASFV fluorescence quantitative PCR primer screening results

[0153] Primer screening was performed according to the method in 1.2.3. The results were as follows Figure 2 As shown in the figure, in the positive control wells 1-6, except for the VP72-F2 / R2 primer, all other primers were effectively amplified, but the VP72-F3 / R3 primer showed a non-specific band, and the corresponding negative control wells showed primer dimers. The VP72-F1 / R1 primer amplification band was brighter than that of other primers, and the band was single. No primer dimers appeared in the negative control wells. Therefore, VP72-F1 / R1 and VP72-P1 were selected as the specific primers and probes for constructing the ASFV fluorescent quantitative PCR method.

[0154] 2.1.3 Results of ASFV VP72 fluorescence quantitative PCR reaction condition optimization

[0155] Referring to the amplification system and reaction program in the PerfectStart II Probe qPCR SuperMix instruction manual, a matrix method was used to screen for primer and probe final concentrations with the lowest Ct value and highest fluorescence intensity. The optimal final concentration of primer and probe was 0.1 μmol / L, as shown in Table 8. Temperature gradient amplification results revealed an optimal annealing temperature of 58°C, and the optimal amplification program was determined, as shown in Table 9.

[0156] Table 8 Reaction system

[0157]

[0158] Table 9 Reaction procedure

[0159]

[0160] 2.1.4 Results of ASFV VP72 fluorescence quantitative PCR sensitivity test

[0161] The positive plasmid was diluted 10-fold in series to a final concentration of 1.05×10 6 -1.05×10 0 The test was carried out according to the established fluorescence quantitative PCR method, and the results were as follows. Figure 3 As shown, the minimum detection concentration range is 1.05×10 0 copies / μL, the sensitivity is high. A standard curve is established based on the sensitivity test results, such as Figure 4 As shown, the standard curve equation is y = -3.33x + 38.459, and its correlation coefficient (R 2 ) was 0.9987, the amplification efficiency was 99.66%, and the CT value showed an obvious linear relationship with the concentration of the diluted template.

[0162] 2.1.5 Results of ASFV VP72 fluorescence quantitative PCR specificity test

[0163] According to the method in 1.2.6, PoRV, PRRSV, PEDV, PRV and ASFV VP72 were used as reaction templates, and the ASFV fluorescence quantitative PCR method after the reaction system was optimized was used for amplification. The results are shown in the figure. Figure 5 As shown in the results, the ASFVVP72 positive plasmid was effectively amplified, while PoRV, PRRSV, PEDV, and PRV were not effectively amplified, indicating that the constructed fluorescence quantitative PCR method is specific for the detection of ASFV.

[0164] 2.1.6 Results of ASFV VP72 fluorescence quantitative PCR repeatability test

[0165] Select dilution to 1.05 × 10 5copies / μL, 1.05×10 4 copies / μL, 1.05×10 3 The stability of the established method was determined using a standard positive plasmid containing 100 copies / μL of plasmid. Within- and inter-batch testing was performed with three replicates per group, and the coefficient of variation was calculated. The results are shown in Tables 10 and 11. As shown, the intra-batch coefficients of variation for the established method were 0.095%, 0.131%, and 0.120%, respectively, and the inter-batch coefficients of variation were 0.390%, 0.401%, and 0.422%, respectively. Both the intra- and inter-batch coefficients of variation were less than 0.5%, demonstrating good reproducibility of the method.

[0166] Table 10 Intra-batch repeatability results

[0167]

[0168] Table 11 Inter-batch repeatability results

[0169]

[0170]

[0171] 2.1.7 Results of ASFV VP72 Fluorescence Quantitative PCR Detection of Clinical Samples

[0172] 184 pig farm environmental samples were tested using the optimized method. As shown in Table 12, 57 samples were ASFV positive by fluorescence quantitative detection, which was consistent with the test results of three commercial kits.

[0173] Table 12 Pig farm environmental sample test results

[0174]

[0175] 2.2 ASFV nucleic acid extraction method based on oligonucleotide probe modified magnetic ionic liquid

[0176] 2.2.1 Optimization of conditions for binding oligonucleotide probes to magnetic ionic liquids

[0177] (1) Effect of temperature on the binding of oligonucleotide probes to magnetic ionic liquids

[0178] Temperature is one of the factors that need to be considered in the oligonucleotide probe binding process. At too high or too low temperatures, the binding performance of MIL will be affected. Therefore, except for the temperature, other conditions are kept unchanged (pH = 7, binding time is 5 minutes), and different binding temperatures (30℃-90℃) are set for MIL and probe. Each group has 3 replicates. The probe binding test is carried out according to the method in 3.3.2, the probe binding amount is calculated, and the probe binding amount at different temperatures is compared. The results are as follows Figure 6(a) shows that the binding amount gradually increases between 30℃ and 50℃, and reaches the maximum binding amount (45,861ng) at 50℃. As the temperature continues to rise, the binding amount of the probe tends to decrease, but the overall binding amount is not significantly affected by temperature. The binding amount between 30℃ and 80℃ is higher than 4,000ng, so a binding temperature of 50℃ was selected as the subsequent experimental condition.

[0179] (2) Effect of volume ratio on the binding of oligonucleotide probes to magnetic ionic liquids

[0180] Due to the insolubility of MIL, MIL will disperse into small droplets in a certain volume of solution, increasing its contact area with the probe in the solution, and MIL will bind to the probe more fully. However, an excessively high volume of probe solution can easily lead to difficulties in collecting magnetic materials and waste of reagents. Therefore, in order to explore the appropriate volume ratio of MIL to probe solution, different volume ratios of MIL to probe were set (1:2, 1:5, 1:10, 1:20, 1:25). Other conditions remained unchanged, the temperature was controlled at 50°C, the shaking incubation binding time was 5 minutes, and 3 repetitions were set for each group. After the magnetic rod adsorbed and separated the MIL-Probe and the probe solution, the average probe concentration before and after binding was measured and calculated, and the binding amount of the probe was calculated. The results are shown in the figure. Figure 6 As shown in Figure (b), the amount of probe bound gradually increases as the volume ratio increases from 1:2 to 1:10. At a volume ratio of 1:10, the amount of probe bound reaches a maximum of 44,621 ng. The amount of bound probe then decreases. Floccules are observed on the MIL surface after binding, making them difficult to remove by washing and reducing the efficiency of subsequent DNA extraction. Therefore, it is hypothesized that a given volume of MIL has a maximum limit on the amount of probe that can be bound. Beyond this limit, the capture probe aggregates on the MIL surface, affecting MIL binding to the probe in solution. Therefore, a 1:10 MIL to probe volume ratio was selected for subsequent experiments.

[0181] (3) Effect of binding time between MIL and oligonucleotide probe on binding

[0182] The binding of MIL to the oligonucleotide probe requires a certain amount of time. In order to fully bind to the probe and shorten the binding time, this experiment sets the binding time of MIL to the probe to 1min-10min. Other conditions are the same (temperature 50℃, volume ratio of the two is 1:10). According to the established separation method, 10 parallel experiments are carried out, and the average probe concentration before and after binding is measured and calculated to calculate the binding amount of the probe. The results are as follows Figure 6(c) shows that the amount of probe bound gradually increases over time. After 3-6 minutes, the amount of bound increases rapidly, reaching a maximum amount (52,660 ng) at 7 minutes, after which the amount of bound stabilizes. Therefore, a binding time of 7 minutes was selected for subsequent experiments.

[0183] 2.2.2 Optimization results of conditions for capturing DNA using oligonucleotide probe-modified magnetic ionic liquids

[0184] (1) Effect of temperature on DNA capture efficiency

[0185] Too low a temperature can easily lead to non-specific binding of oligonucleotide probes to single-stranded DNA, while too high a temperature can reduce hybridization efficiency. Therefore, in order to explore the effect of temperature on DNA capture efficiency, in this work, a temperature gradient was set around the Tm (58°C) of the probe-DNA binding fragment. By setting the temperature to 54°C-62°C, with other conditions unchanged, 3 replicates were set for each group, and parallel experiments were performed according to the 1.2.3 method. The solution DNA concentration before and after capture was measured, and the solution DNA capture efficiency (W1%) was calculated. The results are as follows Figure 7 As shown in (a), the capture efficiency gradually increased within the temperature range of 54°C to 58°C, reaching a maximum capture efficiency (83.10%) at 58°C, and then showed a downward trend. Therefore, 58°C was selected for subsequent experiments.

[0186] (2) Effect of pH on DNA capture efficiency

[0187] Usually, the pH value of the DNA solution will be different in different environmental matrices or biological samples. The presence of interfering matrix components may affect the capture of DNA by MIL-Probe. In this study, DNA sodium salt solutions with a pH value of 2-12 were used as samples for DNA capture to study the effect of pH on DNA capture efficiency. Other conditions remained unchanged (temperature was 58°C, capture time was 5 minutes), and 3 replicates were set up for each group. Parallel experiments were performed according to the 1.2.3 method to measure the solution DNA concentration before and after capture, and calculate the solution DNA capture efficiency (W1%). The results are shown in Figure 7 In (b), when the pH value increases from 2 to 7, the DNA capture efficiency gradually increases; the highest capture efficiency (84.50%) is achieved when the pH value reaches 7, and then the DNA capture efficiency decreases with the increase of pH value. At the same time, when the pH value is 2 and 12, the color of MIL is observed to change from the original red to green, and the hydrophobicity becomes weaker. Therefore, it is speculated that too high or too low pH value will affect the ionic structure of the MIL material, resulting in the shedding of the probe; at the same time, the extreme acid-base environment will affect the binding of the probe to the DNA single strand, resulting in a decrease in DNA capture efficiency. Therefore, pH = 7 was selected as the subsequent experimental condition. (3) Effect of ionic strength on DNA capture efficiency

[0188] Ionic strength is one of the important factors affecting the extraction performance of biomacromolecules, and electrostatic interaction plays a major role in the extraction process. In this experiment, a DNA sodium salt solution with a NaCl concentration of 0.1mol / L-3.0mol / L was prepared, and other conditions remained unchanged (temperature was 58°C, pH value was 7). Three replicate groups were set up for each experiment, and the experiment was carried out according to the 3.2.3 method. The average concentration of solution DNA before and after capture was measured and calculated, and the capture efficiency of solution DNA (W1%) was calculated to explore the effect of sodium ion concentration on the capture of DNA by MIL-Probe. The results are as follows Figure 7 As shown in (c), when the NaCl concentration is 0.1 mol / L, the capture efficiency is the highest at 87.20%. As the ionic strength gradually increases, the DNA capture efficiency decreases significantly. When the NaCl concentration is 3.0 mol / L, the MIL-Probe only captures 51.30%.

[0189] 2.2.3 Optimization of DNA desorption conditions using oligonucleotide probe-modified magnetic ionic liquids

[0190] (1) Effect of temperature on desorption efficiency

[0191] Temperature is one of the important factors affecting desorption efficiency. The DNA desorption time of MIL-Probe was set to 30℃-90℃, with 3 replicates per group. Other conditions remained unchanged. Parallel experiments were performed according to the method in 1.2.4. The DNA concentration of the desorption solution was measured and the DNA desorption rate of each group was calculated. Figure 8 As shown in (a), the desorption efficiency gradually increases with increasing temperature between 30°C and 70°C, reaching 90% at 70°C. After 70°C, the desorption efficiency drops below 90.00%, indicating equilibrium between DNA binding and desorption. Therefore, a desorption temperature of 70°C was selected as the desorption condition for subsequent experiments.

[0192] (2) Effect of time on desorption efficiency

[0193] The temperature of DNA desorption by MIL-Probe was set to 1-25min, and 3 replicates were set for each group. Other conditions were kept unchanged. Parallel experiments were performed according to the method in 1.2.4. The DNA concentration of the desorption solution was determined and the desorption rate of each group was calculated. Figure 8 As shown in (b), the desorption efficiency increases exponentially when the desorption time is between 1 and 15 minutes, reaching a maximum desorption efficiency (90.51%) at 15 minutes, and then the desorption efficiency stabilizes. Therefore, a desorption time of 20 minutes was selected as the desorption test condition.

[0194] 2.2.4 Repeatability test results

[0195] At a copy number concentration of 1.05×10 4 Reproducibility tests were performed within and between groups using a positive plasmid containing 100 copies / μL of DNA as a template. The results are shown in Tables 13 and 14. The calculated relative standard deviations (RSDs) within and between groups were 2.61% and 3.93%, respectively. Both RSDs were less than 4.00%, demonstrating that the optimized extraction method has good reproducibility.

[0196] Table 13 Intra-group repeatability test results

[0197]

[0198] Table 14 Results of intergroup repeatability test

[0199]

[0200]

[0201] 2.2.5 Reusability test results

[0202] After four consecutive binding-capture-desorption DNA tests, the results were as follows Figure 9 As shown, the DNA extraction efficiency was still 90.20±2.2%, indicating that the optimized extraction method has good reusability.

[0203] 2.2.6 Comparative test results

[0204] The Ct values ​​of the DNA solutions extracted by the two methods were determined by the optimized extraction method. The results are as follows: Figure 10 As shown in the figure, the minimum extraction limits of the two extraction methods were 1.05×10 1 copies / μL and 1.05×10 0 The MIL-Probe extraction method of the present invention achieved a significantly lower Ct value than the kit extraction method, while also yielding a higher amount of extracted DNA than the kit extraction method. Therefore, the MIL-Probe extraction method of the present invention outperformed the commercial kit extraction method in both the amount of extracted DNA and the minimum extraction limit.

[0205] The above test examples show that the present invention has established a method for the specific purification and extraction of ASFV based on an oligonucleotide probe-modified magnetic ionic liquid (MIL-Probe) and the detection of ASFV by fluorescence quantitative PCR. By using the synthesized MIL-Probe as a specific extraction material, the oligonucleotide probe selectively captures the viral DNA, and then uses magnetic rapid separation from the extracted sample. Finally, a high-quality DNA sample is obtained through a desorption step for fluorescence quantitative PCR detection.

[0206] The present invention explored the influencing factors of probe binding, DNA capture, and DNA desorption through single-factor experiments, and optimized the experimental conditions of each step:

[0207] (1) Optimization of conditions for the binding of oligonucleotide probes to MIL: By exploring the effects of binding temperature, volume ratio, and binding time on the probe binding amount, and optimizing the binding conditions, the results showed that under the optimal conditions of a binding temperature of 50°C, a volume ratio of MIL to probe solution of 1:10, and a binding time of 7 min, the probe binding amount reached a maximum value (52,660 ng). The results of the binding temperature optimization experiment showed that when the temperature continued to rise above 50°C, the probe binding amount tended to decrease, but the overall binding amount was not significantly affected by temperature; the results of the volume ratio optimization experiment of MIL to probe solution showed that when the volume ratio of MIL to probe solution exceeded 1:10, floccules appeared on the MIL surface and affected the binding of the probe. It is speculated that when the number of probes bound to MIL exceeds a certain amount, they tend to aggregate on the MIL surface, reducing the affinity between MIL and the probe. The results of the binding time optimization experiment showed that the binding of 5 μL MIL to the probe reached saturation at 7 min, and then the probe binding amount no longer increased, reaching the maximum probe binding amount (52,660 ng).

[0208] (2) Optimization of DNA capture conditions by MIL-Probe: By exploring the effects of capture temperature, pH, and ionic strength on DNA capture and optimizing the capture conditions, the results showed that under the optimal conditions of a capture temperature of 58°C, a sample solution pH of 7, and an ionic strength of 0.1 mol / L, the DNA capture efficiency of MIL-Probe reached 87.20%. The capture temperature optimization test results showed that the capture efficiency was highest when the temperature reached 58°C, which was consistent with the Tm value of the probe. Considering the presence of a large number of non-target nucleic acids in actual clinical samples, too low a capture temperature can easily lead to an increase in nonspecific products, while too high a capture temperature can cause the probe to bind weakly to the target nucleic acid. Therefore, 58°C was selected as the capture temperature of the probe. During pH optimization experiments, it was observed that extreme pH conditions caused the MIL color to change from red to green and significantly reduced capture efficiency. It is speculated that excessively high or low pH can easily affect the ionic structure of the MIL, altering its physicochemical properties. Results from pH optimization experiments showed that DNA capture efficiency decreased significantly with increasing ionic strength. At a NaCl concentration of 3.0 mol / L, the MIL-Probe captured only 51.30%. It is speculated that increasing ionic strength increases base stacking forces, leading to an increase in the annealing temperature of the oligonucleotide probe and a weakening of its DNA binding ability, resulting in a decrease in capture efficiency. It is also possible that ionic strength affects the binding of the MIL to the probe, causing probe detachment. Therefore, the MIL-Probe's ability to capture DNA gradually weakened with increasing ionic strength.

[0209] (3) Optimization of DNA desorption conditions by MIL-Probe: By exploring the effects of desorption temperature and desorption time on DNA desorption and optimizing the conditions, the results showed that under the optimal conditions of a desorption temperature of 70°C and a desorption time of 15 min, the DNA desorption efficiency could reach 90.51%. Repeatability tests were conducted using the optimized extraction method, and the results showed that the intra-group standard deviation (RSD) was 2.61%, and the inter-group standard deviation was 3.93%. Both RSD values ​​were lower than 4%, indicating that this method had good repeatability. The results of repeated use tests showed that although the extraction efficiency decreased with the increase in the number of tests, it was speculated that the MIL material suffered a slight loss during the extraction process, but the extraction efficiency remained above 88%, proving that the MIL material used in the present invention has good reusability in DNA extraction. The comparative test results showed that the minimum extraction limit of the optimized extraction method reached 1.05×10 0 The optimized extraction method achieved 100 copies / μL, 10-fold higher than commercial extraction kits. Furthermore, at low DNA concentrations, this method achieved Ct values ​​2-3 times earlier than commercial kits, demonstrating superior enrichment capabilities. Furthermore, the optimized extraction time was significantly shorter than that of commercial kits, requiring only 15 minutes, and no toxic chemical reagents were required. Furthermore, the MIL material used in this study is relatively inexpensive to prepare. Combined with the developed ASFV fluorescence quantitative PCR detection method, it enables early nucleic acid extraction and diagnosis of ASFV, providing technical support for ASF disease monitoring and prevention.

Claims

1. A non-diagnostic African swine fever virus fluorescence quantitative PCR detection method, characterized in that: The method comprises the following steps: (1) Combine the African swine fever virus capture probe and [P 6,6,6,14 + ][Co(hfacac)3 - The materials were mixed in a volume ratio of 10:1, incubated at 30-80° C. for 5-10 minutes, and unbound substances were removed to obtain an incubation product; the nucleotide sequence of the African swine fever virus capture probe was: 5’-C (20) - CACAAGCCGCACCAAAGCAAACCT-3’; (2) adding the sample to be tested to the incubation product, adjusting the pH of the system to 6-10 after heat denaturation, the NaCl concentration to 0.1 mol / L, capturing at 56-60°C for 5-10 minutes, removing unbound substances, and obtaining a captured product; (3) adding a desorption solution to the captured product, heating at 70-90°C for 15-25 minutes to desorb the DNA, remove the captured product, and obtain a desorption product; (4) adding specific primers and probes for fluorescent quantitative PCR detection of non-swine fever virus to the fluorescent quantitative PCR reaction system, then adding the desorbed product to amplify the target sequence, and determining the Ct value of the desorbed product; The nucleotide sequence of the specific primers for fluorescent quantitative PCR detection of non-swine fever virus is: Upstream primer: 5'-GGCCCTCTCCTATGCAACATTC-3' Downstream primer: 5'-GGGTTGGTATGGCTACACGTTC-3' The nucleotide sequence of the probe for fluorescent quantitative PCR detection of non-swine fever virus is: 5'-FAM-CACAAGCCGCACCAAAGCAAACCT-BHQ1-3'.

2. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 1, characterized in that: In step (1) or step (2), the method for removing unbound substances includes: performing magnetic adsorption separation on the mixed solution obtained after incubation or capture to obtain a supernatant and a lower layer, and washing the lower layer.

3. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 1, characterized in that: The method for removing the captured product in step 3) includes: performing magnetic adsorption separation on the mixed solution obtained after desorption to obtain a supernatant and a lower layer, and retaining the supernatant as the extraction product.

4. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 1, characterized in that: The desorption solution described in step 3) is RNase-free water.

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