Kit for detecting African swine fever virus and fluorescent quantitative PCR (Polymerase Chain Reaction) detection method

Abstract

The invention discloses a kit for detecting African swine fever virus and a fluorescent quantitative PCR (polymerase chain reaction) detection method, and belongs to the technical field of kits and virus detection. The kit comprises an African swine fever virus capture probe (VP72-CP), a [P6, 6, 6, 14 +] [Co (hfacac) 3-] material chemically combined with the VP72-CP, and a specificity probe and a probe for non-swine fever virus fluorescent quantitative PCR (Polymerase Chain Reaction) detection. The capture probe and the magnetic ionic liquid material are anchored and combined, the combined capture material can specifically recognize, adsorb and capture African swine fever virus nucleic acid, the fluorescent quantitative PCR detection method is used for detecting the content of the African swine fever virus, and good sensitivity, specificity and repeatability are achieved.

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 is an acute, febrile, highly contagious disease caused by African swine fever virus (ASFV). It is very easy to infect domestic pigs and Eurasian wild boars. It is characterized by rapid onset, short course, and a mortality rate of up to 100%. Clinical manifestations include fever, difficulty breathing, coughing, viscous or serous fluid secretions from the eyes and nose, bleeding, redness, and cyanosis of the skin, severe bleeding in multiple internal organs, and severe enlargement of the spleen in some pigs. Conventional swine fever detection is mainly based on sampling of pig serum, plasma, anticoagulant blood, oral mucus, and lung, lymph node, kidney, bone marrow, and tonsil tissues, which cannot achieve the purpose of prevention. At present, there is no effective commercial African swine fever virus vaccine or specific medicine in the world, and the African swine fever virus remaining in the environment can infect pigs through insect vectors, bringing a heavy blow to farmers and the entire industry chain. Therefore, early prevention has become an important means to control African swine fever disease, especially the detection of African swine fever virus in the environment plays a very important role in prevention and control.

[0003] As a functional material, ionic liquids have been successfully applied in various fields, such as catalysts, synthesis, electrochemistry and extraction. They have been widely used in gas capture, solvents, catalysts and extraction, and have achieved good results. Magnetic ionic liquids are a new type of material with high thermal stability, easy modification, reusability, and extremely poor solubility in water. These characteristics enable magnetic ionic liquids to be used as widely used extraction reagents, including for the extraction of DNA in the environment. Magnetic ionic liquids combined with probes have the characteristics of co-extraction under low background DNA, high extraction efficiency and strong specificity. Compared with the extraction method of environmental DNA extraction kits on the market, the magnetic ionic liquid DNA extraction method is simple to operate, does not require complex reagents and expensive instrument processing, 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 - ] materials, and reported that the materials can enrich the target DNA in animals, but did not disclose that they 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] In order to achieve the above-mentioned first technical purpose, the inventors combined their research experience in the field of virus detection over the years, and through a large number of experimental studies and unremitting exploration, finally 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 probes (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, and 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 quenching group is:

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

[0016] It should be noted that the huge 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, with a size of about 73.2kD. 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 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. The 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, and 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 constructs a DNA purification and extraction method based on oligonucleotide probe-modified magnetic ionic liquid and applies it to the extraction of African swine fever virus nucleic acid. The method can quickly and efficiently extract and separate the ASFV target DNA sequence in environmental samples, is easy 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 designs fluorescent quantitative PCR specific primers and probes based on the conservative region of the VP72 gene of ASFV, constructs a highly sensitive and specific ASFV fluorescent quantitative PCR detection method, and serves as a monitoring, early warning and purification control means 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, unbound substances are removed, and an incubation product is obtained;

[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 African swine fever virus are added to the fluorescent quantitative PCR reaction system, and then the desorption product is added to amplify the target sequence, and the Ct value of the desorption product is determined, 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, and washing the lower layer.

[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 solution obtained after desorption to obtain a supernatant and a lower layer, 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: pH of the system is adjusted to 6-10 after thermal denaturation, and capture is performed at 56-60°C for 5min-10min (the most preferred parameters are pH of the system is adjusted to 7 after thermal denaturation, and capture is performed 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 anchor 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 could 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 an oligonucleotide probe modified with a magnetic ionic liquid 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 a commercial extraction kit, and the overall extraction efficiency is higher than that of a 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 of actual environmental samples. Moreover, the specific primers and probes of fluorescent quantitative PCR 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. Among them, 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 Are specific test results; among them: 1 is ASFV; 2-6 are PoRV, PRRSV, PEDV, and PRV, respectively; 7 is a negative control.

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

[0042] Figure 7 The optimization results of conditions for capturing DNA with oligonucleotide probe-modified magnetic ionic liquid; wherein: (a) temperature; (b) pH; (c) ionic strength.

[0043] Figure 8 The results of condition optimization for desorption of DNA by magnetic ionic liquid modified with oligonucleotide probes; wherein: (a) temperature; (b) time.

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

[0045] Fig.10 The results of the comparative test are shown in Table 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 implementation cases. The following implementation cases are only used to illustrate the present invention and should not be regarded as the protection scope of the present invention. The technical operation steps or conditions not specified in the embodiments are all carried out according to the techniques or conditions described in the literature of the field or according to the product instructions. The reagents or instruments used are the manufacturers indicated, and are all conventional products that can be obtained commercially.

[0047] In this embodiment, 6 pairs of fluorescent quantitative PCR primers and TaqMan probes were designed based on the ASFV VP72 gene. The prepared ASFV standard positive plasmid was used as a conventional PCR amplification template to perform a primer screening test. The results showed that the amplification band of the VP72-F1 / R1 primer was single, brighter and clearer than other primers, and no primer dimers appeared. Therefore, VP72-F1 / R1 and VP72-P1 were selected to construct the ASFV fluorescent quantitative PCR detection method. After the fluorescent quantitative PCR system was optimized, the specificity test results proved that the method had good specificity. At the same time, the sensitivity test was carried out using the gradient diluted standard positive plasmid as the amplification template. The test results showed that the minimum nucleic acid detection concentration of the method was 10 copies / μL, and the sensitivity of the method was better than the qPCR detection method established by Yu Bin et al. And through the intra-batch and inter-batch repeatability tests, the verification method had good repeatability. Finally, 184 environmental samples from different pig farms in Hubei Province were tested and compared with three different commercial kits. The test results showed that the detection rate of this method and the remaining commercial kits reached 100%.

[0048] 1 Materials and methods

[0049] 1.1 Test materials

[0050] 1.1.1 Strain

[0051] The complete ASFVVP72 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 preserved 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 Xitian 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, and make up to 1,000 mL with ddH2O. 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 at 121°C for 20 min. After the temperature of the medium drops to about 50°C, add the corresponding antibiotics, spread the plate, and after the medium solidifies, place it upside down at 4°C for storage.

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

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

[0063] (5) Solutions with different pH values: Britton-Robinson (BR) buffer is a mixture of phosphoric acid, boric acid, and acetic acid, with the concentration of each of the three being 0.04 mol / L. Add an unused amount of 0.2 mol / L NaOH solution to the BR buffer and adjust the pH value to the desired value on 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 designed and constructed according to the experimental requirements, all of which were synthesized by Wuhan Novozymes Biotechnology Co., Ltd.

[0069] (1) ASFVVP72 fluorescent quantitative PCR primers: The African swine fever virus (ASFVVP72) gene sequence was searched on NCBI, and 6 pairs of fluorescent quantitative PCR primers and probes were designed using Oligo7.0 primer and probe design software. The 6 pairs of specific primers for ASFVVP72 gene fluorescent quantitative PCR were named VP72-F1 / 2 / 3 / 4 / 5 / 6 and VP72-R1 / 2 / 3 / 4 / 5 / 6; the TaqMan probe was named VP72-P1 / 2 / 3 / 4 / 5 / 6.

[0070] (2) Specific primers for constructing the ASFV VP72 standard positive plasmid 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] The synthesized complete fragment of ASFVVP72 gene was used as a template, and the reaction solution containing Phanta high-fidelity DNA polymerase and primers VP72-QF / VP72-QR were used for amplification by Wuhan Novozyme Biotechnology Co., Ltd. The reaction system (50.0 μL) was as follows: 2×PhantaMaxMasterMix (DyePlus) 25.0 μL, 2.0 μL of upstream and downstream primers, 19.0 μL of sterile water, and 2.0 μL of DNA. PCR reaction program: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 57°C annealing for 15 s, 72°C extension for 2 min, 35 cycles; 72°C thorough extension for 5 min, and cooling and storage at 4°C.

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

[0077] a. After electrophoresis is completed and observed by the gel system, use a surgical blade to cut out the single target DNA band from the agarose gel, put it into a clean centrifuge tube and weigh it;

[0078] b. Add an equal volume of sol solution to the centrifuge tube (if the gel weight is 0.1g, its volume can be regarded as 100uL, then add 100μL of sol solution), place it in a 50℃-55℃ water bath for 10min, and gently turn the centrifuge tube upside down to ensure that the gel block is fully dissolved;

[0079] c. Add the solution obtained in the previous step to an adsorption column (the adsorption column is placed in a collection tube), centrifuge at 12,000 r / min for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0080] d. Add 600 μL of rinse solution to the adsorption column, centrifuge at 12,000 r / min 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 r / min for 2 minutes, remove the washing solution as much as possible, and place the adsorption column open at room temperature or in a 50°C incubator for several minutes to remove the residual washing solution in the adsorption column and prevent the ethanol in the washing solution from affecting subsequent experiments;

[0083] g. Place the adsorption column in a clean centrifuge tube, drop 50 μL of the eluent preheated in a 65°C water bath into the center of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge it at 12,000 r / min for 1 minute;

[0084] h. DNA product was stored at -20℃

[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 Novogene Biotechnology Co., Ltd. The reaction system consisted of 1.0μL 5×TA / Blunt-Zero Cloning Mix, 2.0μL PCR product, and 2.0μL sterile water. The reaction procedure was: 25°C (temperature controlled by PCR instrument), 5min.

[0087] (4) Transformation of ligation products

[0088] a. Cells were transformed with DH5α chemically competent cells from Nanjing Novozyme Biotechnology Co., Ltd. The competent cells were taken out from ultra-low temperature (-80°C) and quickly thawed on ice.

[0089] b. Add the plasmid to be transformed into 100 μL competent cells, flick the tube wall to mix (avoid using a pipette), and let stand on ice for 30 min.

[0090] c. After the ice bath, place the competent cells in a 42°C water bath, heat shock for 45 seconds, and then quickly place them on ice for 2 minutes. Add 900 μL LB liquid culture medium (without antibiotics) to the centrifuge tube, mix well, and place in a shaking incubator at 37°C, 200 r / min for 1 hour to recover.

[0091] d. After recovery, place the competent cells in a centrifuge at 5,000 r / min and centrifuge for 3 min. Discard 900 μL of supernatant, resuspend the cells with the remaining culture medium, and evenly spread them 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 culture overnight.

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

[0094] The single colonies grown on the LB solid medium were inoculated into 2 mL of liquid medium LB (containing antibiotics) and placed in a constant temperature incubator at 37°C and 180 r / min for 8h-12h. Then the cultured bacterial solution was used as a PCR template and amplified with VP72-QF / QR specific primers to identify the recombinant plasmid by PCR. The reaction components are as follows: PCR reaction system (25 μL): 1.0 μL of upstream and downstream primers, 12.5 μL of 2×Phanta MaxMasterMix (DyePlus), 1.0 μL of template, and 9.5 μL of sterile water. PCR reaction program: 95°C pre-denaturation for 3min, 95°C / 15s, 57°C / 15s, 72°C / 2min, a total of 35 cycles; 72°C extension for 2min, 10°C cooling for 2min. After amplification, the PCR product containing a single target DNA band was sequenced according to the results of agarose gel electrophoresis.

[0095] (6) Plasmid extraction

[0096] a. Pick up a single colony cultured on the LB plate with a sterile pipette tip, inoculate it into 5mL of liquid culture medium LB containing ampicillin resistance, and culture it in a constant temperature shaker at 37℃ and 180r / min for 8h-12h. Centrifuge the cultured bacterial solution at 12,000r / min for 1min and discard the supernatant. Invert the centrifuge tube on sterile absorbent paper and let it stand for 30s to drain the residual liquid.

[0097] b. Take 600 μL of reagent A (precooled to 0℃-4℃), add it to the centrifuge tube with 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 place on ice for 2 min (the solution in the tube will gradually become clear and transparent).

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

[0100] e. Centrifuge at 12,000r / min for 10 minutes at 4℃, transfer the supernatant to a new centrifuge tube, taking care not to suck up the precipitate. Add 2 times the volume of pre-cooled anhydrous ethanol, mix well, and incubate at -20℃ for 20 minutes.

[0101] f. Centrifuge at 12,000 r / min 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 r / min for 5 min at 4°C and discard the supernatant. Invert the centrifuge tube on sterile absorbent paper and let it stand for 30 seconds to drain the remaining liquid and dry it 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 Screening of primers for ASFV fluorescence quantitative PCR

[0105] The standard positive plasmid constructed by method 1.2.2 was used as a template, and the 6 pairs of fluorescent quantitative PCR primers designed by method 1.2.1 were used for conventional PCR amplification. Positive and negative controls were set for each pair of fluorescent quantitative PCR primers. The amplification products of each pair of primers were detected by polyacrylamide gel electrophoresis, and a pair of primers with better amplification effect was selected. The reaction system and reaction procedure of amplification are shown in Table 4 and Table 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] The standard positive plasmid constructed by the method of 1.2.2 was used as a template. The concentrations of primers and probes were screened according to the reaction system in Table 6. The final concentrations of primers and probes were set to 4 gradients (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L), 3 replicates for each gradient, and the matrix method was used to determine the best amplification system. After the system was determined, 5 annealing temperature gradients (54°C, 56°C, 58°C, 60°C, 62°C) were set according to the reaction program in Table 7, 3 replicates for each gradient, and the best annealing temperature was determined.

[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 phase 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 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 and detected using the established method. Each gradient was repeated 3 times, and a negative control was set up to construct a standard curve 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 ASFVVP72 positive plasmids were used as reaction templates, with 3 replicates in each group, and ddH2O was used as a negative control. ASFVVP72 fluorescence quantitative PCR method was constructed for amplification. 1.2.7 Repeatability test of ASFVVP72 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. Each sample was repeated three times, and the coefficient of variation within and between batches was calculated to test the stability of the established method.

[0121] 1.2.8 Detection of clinical samples of ASFV VP72 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 compliance rate of the new method with that of the commercial kits.

[0123] 1.2.9 Design of oligonucleotide probes

[0124] According to the strong affinity of magnetic ionic liquid materials to 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 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 5 μL of MIL was vortexed to mix. The oligonucleotide probe-modified magnetic ionic liquid (MIL-Probe) was dispersed 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 surface of MIL. The MIL-Probe in the solution was adsorbed by a magnetic rod and washed twice with ddH2O to prepare the 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 by NanoPhotometerN50 spectrophotometer, and the binding amount of the probe (Qng / μL) was calculated. The calculation formula is as shown in 3-1. The probe binding amounts under different conditions were 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] In the formula, 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 the 200 μL standard plasmid constructed in method 2.3.1 and vortex to mix, place in a PCR instrument for temperature control and heating, set the PCR program: 95℃ denaturation for 3min, 60℃ binding for 5min. After the capture is completed, the MIL-Probe in the solution is adsorbed by a magnetic rod and washed twice with ddH2O.

[0132] According to the above steps, the capture temperature, pH value and ionic strength conditions in the capture experiment 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 DNA concentration of the solution before capture, and C5 (ng / μL) is the DNA concentration of the solution after capture. 1.2.12 Desorption of DNA by oligonucleotide probe-modified magnetic ionic liquid

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

[0136] According to the above steps, the desorption temperature and time conditions in the desorption test were changed, and the DNA desorption efficiency of each group was compared to explore the influence of various factors on the capture of DNA by MIL-Probe. The Ct value of the desorption solution and the positive plasmid was determined by the constructed fluorescent quantitative PCR method, and then the copy number concentration of the positive plasmid before and after capture was calculated according to the constructed standard curve equation (Formula 3-3), and the DNA capture efficiency (W2%) was calculated. The calculation formulas are shown in 3-4 and 3-5. The DNA capture efficiency under different conditions was compared to explore the experimental conditions for the maximum 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 repeated 10 times under the same conditions, 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 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 test material, which was repeated four times, 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 diluted 10 times to a concentration of 1.05×10 0 The positive plasmids of each gradient were extracted by the optimized extraction method and the instructions of the commercial extraction kit. Three replicates were set for each gradient. The Ct values ​​of the DNA solutions after extraction by the two methods were determined according to the constructed ASFV fluorescent quantitative PCR method.

[0148] 2 Results

[0149] 2.1 ASFV fluorescence quantitative PCR detection method

[0150] 2.1.1 ASFV VP72 plasmid identification

[0151] The synthetic size of ASFVVP72 plasmid was 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 positive control wells 1-6, except for the VP72-F2 / R2 primer, all other primers were effectively amplified, but non-specific bands appeared in the VP72-F3 / R3 primer, and primer dimers appeared in the corresponding negative control wells. The amplification band of the VP72-F1 / R1 primer was brighter than that of other primers, and the band was single, and no primer dimers appeared in the negative control wells. Therefore, VP72-F1 / R1 and VP72-P1 were selected as specific primers and probes for constructing the ASFV fluorescent quantitative PCR method.

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

[0155] Referring to the amplification system and reaction program in the PerfectStartⅡProbeqPCRSuperMix manual, the final concentrations of primers and probes with the smallest Ct value and the strongest fluorescence intensity were screened by the matrix method, and the optimal final concentrations of primers and probes were both 0.1μmol / L, as shown in Table 8. The temperature gradient amplification results screened the optimal annealing temperature to be 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 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. The results are 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 ASFVVP72 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 Figure 5 As shown, 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 standard positive plasmid with 1000 copies / μL was used for intra-batch and inter-batch detection. Three replicates were set for each group, and the coefficient of variation was calculated to determine the stability of the established method. The coefficient of variation results are shown in Tables 10 and 11. As shown in the results, the intra-batch coefficients of variation of 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-batch and inter-batch coefficients of variation were less than 0.5%, indicating that the method has good repeatability.

[0166] Table 10 Intra-batch repeatability results

[0167]

[0168] Table 11 Inter-batch repeatability results

[0169]

[0170]

[0171] 2.1.7 Results of ASFVVP72 fluorescence quantitative PCR clinical sample testing

[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 test 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 process of oligonucleotide probe binding. At too high or too low temperatures, the binding performance of MIL will be affected. Therefore, except for temperature, other conditions are kept unchanged (pH = 7, binding time is 5 minutes), and different binding temperatures of MIL and probe are set (30℃-90℃). Each group has 3 replicates. According to the method of 3.3.2, the probe binding test is carried out, 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 when the binding temperature is 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 greatly affected by temperature. The binding amount between 30℃ and 80℃ is higher than 4,000ng, so the binding temperature of 50℃ is 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 to form small droplets in a certain volume of solution, increasing its contact area with the probe in the solution, allowing MIL to 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), while other conditions remained unchanged. The temperature was controlled at 50°C, and the binding time was incubated on a shaker for 5 minutes. Three replicates were set for each group. After the magnetic rod was used to adsorb and separate 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 (b), it can be seen from the figure that when the volume ratio of the two increases from 1:2 to 1:10, the probe binding amount gradually increases. When the volume ratio is 1:10, the probe binding amount reaches a maximum of 44,621ng, and then the binding amount gradually decreases. It is observed that floccules appear on the surface of MIL after binding, which is difficult to remove by washing and will reduce the subsequent DNA extraction efficiency. Therefore, it is speculated that a certain volume of MIL has a maximum limit for the binding amount of the probe. Beyond this maximum limit, the capture probe will aggregate on the surface of MIL, thereby affecting the binding of MIL to the probe in the solution. Therefore, a volume ratio of MIL to probe of 1:10 was selected for the conditions of 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, the binding time of MIL and the probe was set to 1min-10min in this experiment. Other conditions were the same (temperature 50°C, volume ratio of the two was 1:10). According to the established separation method, 10 parallel experiments were carried out to determine and calculate the average probe concentration before and after binding, and calculate the binding amount of the probe. The results are shown in Figure 6(c) shows that the amount of probe binding gradually increases with time. After 3-6 minutes, the binding amount increases at a faster rate. At 7 minutes, the maximum binding amount (52,660 ng) is reached, and then the binding amount is stable. Therefore, the binding time of 7 minutes is selected for the 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 the hybridization efficiency. Therefore, in order to explore the effect of temperature on DNA capture efficiency, in this work, the temperature gradient was set around the Tm (58°C) of the probe-DNA binding fragment. By setting the temperature to 54°C-62°C, other conditions remained unchanged, and 3 replicates were set for each group, parallel experiments were performed according to method 1.2.3, the solution DNA concentration before and after capture was measured, and the solution DNA capture efficiency (W1%) was calculated. The results are shown in Figure 1. Figure 7 As shown in (a), the capture efficiency gradually increased within the temperature range of 54°C-58°C, reaching the maximum capture efficiency (83.10%) at 58°C, and then the capture efficiency 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 min), and each group was set up with 3 replicates. Parallel experiments were performed according to method 1.2.3 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 detachment of the probe; at the same time, the extreme acid-base environment will affect the binding of the probe to the single-stranded DNA, resulting in a decrease in the 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 interactions play a major role in the extraction process. In this experiment, a DNA sodium salt solution with a NaCl concentration of 0.1 mol / L-3.0 mol / L was prepared, and other conditions remained unchanged (temperature was 58°C, pH value was 7). Three replicate groups were set up for each test, and the test was carried out according to method 3.2.3. 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 MIL-Probe capture of DNA. The results are as follows: Figure 7 As shown in (c), when the NaCl concentration is 0.1 mol / L, the capture efficiency is as high as 87.20%. As the ionic strength gradually increases, the DNA capture efficiency decreases significantly. When the NaCl concentration is 3.0 mol / L, MIL-Probe only captures 51.30%.

[0189] 2.2.3 Optimization results of conditions for desorption of DNA by oligonucleotide probe-modified magnetic ionic liquid

[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 in 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 measured and the DNA desorption rate of each group was calculated. Figure 8 As shown in (a), the desorption temperature is between 30℃ and 70℃, and the desorption efficiency gradually increases with the increase of temperature, and reaches 90% at 70℃. The desorption efficiency after 70℃ is lower than 90.00%, and the binding and desorption of DNA reach equilibrium. Therefore, the desorption temperature of 70℃ is selected as the subsequent experimental condition.

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

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

[0194] 2.2.4 Repeatability test results

[0195] The copy number concentration was 1.05×10 4 The positive plasmid with 100 copies / μL was used as a template to perform intra-group and inter-group repeatability tests. The results are shown in Tables 13 and 14. The calculated intra-group and inter-group relative standard deviation (RSD) values ​​were 2.61% and 3.93%, respectively. The intra-group and inter-group RSD values ​​were both less than 4.00%, proving that the optimized extraction method has good repeatability.

[0196] Table 13 Results of intra-group repeatability test

[0197]

[0198] Table 14 Results of intergroup repeatability test

[0199]

[0200]

[0201] 2.2.5 Results of reusability test

[0202] After four consecutive binding-capture-desorption DNA experiments, the results are as follows Fig. 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: Fig.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 has a Ct value that is always lower than that of the kit extraction method, and the amount of extracted DNA is higher than that of the kit extraction method. Therefore, the MIL-Probe extraction method of the present invention is superior to the commercial kit extraction method in terms of both the amount of extracted DNA and the minimum extraction limit.

[0205] Through the above test examples, it can be seen that the present invention has established a method for specific purification and extraction of ASFV based on oligonucleotide probe-modified magnetic ionic liquid (MIL-Probe) and ASFV fluorescence quantitative PCR detection. The synthesized MIL-Probe is used as a specific extraction material, the oligonucleotide probe selectively captures viral DNA, and then the MIL-Probe is quickly separated from the extracted sample by magnetism, and finally a high-quality DNA sample is obtained through a desorption step, which is used for fluorescence quantitative PCR detection.

[0206] The present invention explores the influencing factors of probe binding, DNA capture, and DNA desorption through single factor experiments, and optimizes 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 amount of probe binding, and optimizing the binding conditions, the results showed that the probe binding amount reached the maximum value (52,660 ng) under the optimal conditions of binding temperature of 50°C, volume ratio of MIL to probe solution of 1:10, and binding time of 7 min. The results of the binding temperature optimization experiment showed that when the temperature continued to rise above 50°C, the amount of probe binding showed a decreasing trend, but the overall binding amount was not greatly 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 surface of MIL and affected the binding of the probe. It is speculated that when the number of probes bound to MIL exceeds a certain amount, it is easy to aggregate on the surface of MIL, reducing the affinity between MIL and probe. The results of the binding time optimization experiment showed that the binding of 5μL MIL to the probe reached saturation at 7 min, and then the amount of probe binding 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 ion strength on DNA capture and optimizing capture conditions, the results showed that the DNA capture efficiency of MIL-Probe reached 87.20% under the optimal conditions of capture temperature of 58°C, sample solution pH of 7, and ion strength of 0.1 mol / L. The results of the capture temperature optimization test showed that the capture efficiency was highest when the temperature reached 58°C, which was consistent with the Tm value of the probe. Considering that there are a large number of non-target nucleic acids in actual clinical samples, too low a capture temperature is likely to lead to an increase in non-specific products, and too high a capture temperature will cause the probe to bind weakly to the target nucleic acid. Therefore, 58°C was selected as the capture temperature of the probe. During the pH optimization test, it was observed that extreme pH environments would cause the color of MIL to change from red to green, and the capture efficiency would be greatly reduced. It is speculated that too high or too low pH would easily affect the ionic structure of MIL, causing its physical and chemical properties to change. The results of the pH optimization test showed that with the gradual increase of ionic strength, the DNA capture efficiency decreased significantly. When the NaCl concentration was 3.0 mol / L, MIL-Probe only captured 51.30%. It is speculated that with the increase of ionic strength, the base stacking force increased, resulting in an increase in the annealing temperature of the oligonucleotide probe, and the ability to bind DNA became weaker, resulting in a decrease in capture efficiency. It is also possible that the ionic strength affected the binding of MIL and the probe, causing the probe to fall off. Therefore, the ability of MIL-Probe to capture DNA gradually weakened with the increase of 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%. The optimized extraction method was used for repeatability tests, and the results showed that the within-group standard deviation (RSD) was 2.61% and the between-group standard deviation was 3.93%. Both RSD values ​​were less 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 a small amount of MIL material was lost 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 extraction time of the optimized MIL material used in this study is relatively low, and combined with the constructed ASFV fluorescence quantitative PCR detection method, it can complete the early nucleic acid extraction and diagnosis of ASFV, providing technical support for the monitoring and prevention of ASF diseases.

Claims

1. A kit for detecting African swine fever virus, characterized in that: It includes an African swine fever virus capture probe, and a [P 6,6,6,14 + ][Co(hfacac)3 - ] materials, and specific primers and probes for fluorescent quantitative PCR detection of non-swine fever virus; The nucleotide sequence of the African swine fever virus capture probe is: 5’-C (20) -CACAAGCCGCACCAAAGCAAACCT-3’; 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 African swine fever virus detection kit according to claim 1, characterized in that: A DNA desorption buffer is also included.

3. The African swine fever virus detection kit according to claim 2, characterized in that: The DNA desorption solution is RNase-free Water.

4. The African swine fever virus detection kit according to claim 1, characterized in that: It also includes a fluorescent quantitative PCR reaction system.

5. A non-diagnostic African swine fever virus fluorescence quantitative PCR detection method, characterized in that: The method comprises the following steps: (1) The African swine fever virus capture probe according to claim 1 and [P 6,6,6,14 + ][Co(hfacac)3 - ] Materials are mixed, incubated, unbound substances are removed, and an incubation product is obtained; (2) adding a sample to be tested to the incubation product, performing DNA capture, removing unbound substances, and obtaining a captured product; (3) adding a desorption solution to the captured product to desorb the DNA and remove the captured product to obtain a desorption product; (4) Add the specific primers and probes for fluorescent quantitative PCR detection of non-swine fever virus as described in claim 1 to a fluorescent quantitative PCR reaction system, then add the desorbed product to amplify the target sequence, and determine the Ct value of the desorbed product.

6. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 5, 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.

7. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 5, characterized in that: The method of removing the incubation 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.

8. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 5, characterized in that: The parameters of the incubation step in step 1) are: incubation at 30-80° C. for 5 min-10 min.

9. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 5, characterized in that: The parameters of the capture step in step 2) are: after heat denaturation, the pH of the system is adjusted to 6-10, and the capture is carried out at 56-60° C. for 5 min-10 min.

10. The non-diagnostic African swine fever virus fluorescence quantitative PCR detection method according to claim 5, characterized in that: The parameters of the desorption step in step 3) are: heating at 70-90°C for 15-25 minutes.

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