Method and product for detecting African swine fever virus nucleic acid based on liquid chip

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method and a product for detecting African swine fever virus nucleic acid based on a liquid chip. According to the specific technical scheme, the number of the primer pairs for detecting the African swine fever is eight, and the primer pairs are designed on the basis of ASFV 360-12L, ASFV 505-1R, ASFV 360-13L, ASFV 360-14L, ASFV 505-2R, ASFV 505-3R, ASFV I177L and ASFV B646L genes in the African swine fever viruses respectively.

Description

Technical Field

[0001] The present invention belongs to the field of virus detection, and specifically relates to a method and product for detecting African swine fever virus nucleic acid based on a liquid phase chip. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by the African swine fever virus (ASFV). Domestic pigs and wild boars are generally susceptible. ASF has a high incidence rate, a short course of disease once it breaks out, and a high mortality rate. The clinical symptoms are complex and difficult to distinguish from other diseases, and are mostly manifested as high fever, skin congestion, abortion, edema, and organ bleeding.

[0003] African swine fever is a double-stranded DNA virus and the only member of the Asfrican swine fever virus family. Currently, ASFV has been divided into 24 genotypes or 8 serogroups. Its genome is between 170-193kbp, encoding 68 structural proteins and more than 100 non-structural proteins. It consists of a four-layer protein shell and an inner genome. The structure is significantly more complex than many other viruses, and its multi-layer structure plays different roles in the replication and survival of ASFV.

[0004] Currently, the main methods for clinical detection of African swine fever virus are: conventional PCR method, fluorescent PCR method and immunological method.

[0005] The PCR method is a technology that uses DNA polymerase to amplify the African swine fever virus DNA in the sample and then captures the amplified product signal. The ordinary PCR method and the fluorescent PCR method use DNA dyes and fluorescent groups to measure the PCR amplification products, respectively, and have the characteristics of high sensitivity and good accuracy. However, in ordinary PCR detection, if the lengths of the amplified product fragments are close, it is impossible to distinguish between specific products and non-specific products, which can easily lead to false positive results. When fluorescent PCR is used to detect multiple target genes at the same time, each target gene uses a different fluorescent group, and the emission wavelengths of the groups cannot be too close, otherwise the number of targets for multiple detection will be limited; even after conditions are optimized, it is generally difficult to exceed triple detection.

[0006] Immunological methods are technologies that use specific antibodies that can bind to African swine fever virus antigens to detect viral antigens. The more common ones include sandwich ELISA, indirect ELISA, blocking ELISA, and indirect immunofluorescence. When using these technologies, since the detection signal is not amplified by amplification like PCR, the detection sensitivity is low and cannot be used for early detection of viral infection. At the same time, the immune response of antigens and antibodies is easily interfered by impurities in the sample, and the false positive rate is high.

[0007] Therefore, establishing a highly sensitive detection method that can simultaneously detect more than three deletion variant sites is crucial for early detection and prevention of the disease, in-depth research on the pathogenic mechanism of ASFV, and future differential diagnosis of vaccine strains, variants and wild virus infections. Summary of the invention

[0008] The purpose of the present invention is to provide a method and product for detecting African swine fever virus nucleic acid based on a liquid phase chip.

[0009] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a primer pair for detecting African swine fever, the primer pair comprising 8 pairs, which are designed based on ASFV 360-12L, ASFV 505-1R, ASFV 360-13L, ASFV 360-14L, ASFV 505-2R, ASFV 505-3R, ASFV I177L and ASFV B646L genes in African swine fever virus, respectively.

[0010] Preferably, the ASFV 360-12L upstream primer sequence is shown in SEQ NO ID: 1, and the ASFV 360-12L downstream primer sequence is shown in SEQ NO ID: 2; the ASFV 505-1R upstream primer sequence is shown in SEQ NO ID: 3, and the ASFV 505-1R downstream primer sequence is shown in SEQ NO ID: 4; the ASFV 360-13L upstream primer sequence is shown in SEQ NO ID: 5, and the ASFV360-13L downstream primer sequence is shown in SEQ NO ID: 6; the ASFV 360-14L upstream primer sequence is shown in SEQ NO ID: 7, and the ASFV 360-14L downstream primer sequence is shown in SEQ NO ID: 8; the ASFV 505-2R upstream primer sequence is shown in SEQ NO ID: 9, and the ASFV 505-2R downstream primer sequence is shown in SEQ NO ID: 10; the ASFV 505-3R upstream primer sequence is shown in SEQ NO ID: ID: 11, the ASFV 505-3R downstream primer sequence is shown in SEQ NO ID: 12; the ASFV I177L upstream primer sequence is shown in SEQ NO ID: 13, and the ASFV I177L downstream primer sequence is shown in SEQ NO ID: 14; the ASFV B646L upstream primer sequence is shown in SEQ NO ID: 15, and the ASFV B646L downstream primer sequence is shown in SEQ NO ID: 16.

[0011] Correspondingly, the probe used to detect African swine fever in combination with the primer pair is a double-binding probe, one end of which is the reverse complementary sequence of the microsphere TAG, and the other end is the reverse complementary sequence of the target PCR product.

[0012] Preferably, the probe sequence corresponding to the ASFV 360-12L gene is shown in SEQ NO ID: 25; and / or; the probe sequence corresponding to the ASFV505-1R gene is shown in SEQ NO ID: 26; and / or; the probe sequence corresponding to the ASFV 360-13L gene is shown in SEQ NO ID: 27; and / or; the probe sequence corresponding to the ASFV 360-14L gene is shown in SEQ NO ID: 28; and / or; the probe sequence corresponding to the ASFV 505-2R gene is shown in SEQ NO ID: 29; and / or; the probe sequence corresponding to the ASFV 505-3R gene is shown in SEQ NO ID: 30; and / or; the probe sequence corresponding to the ASFV I177L gene is shown in SEQ NO ID: 31; and / or; the probe sequence corresponding to the ASFV B646L gene is shown in SEQ NO ID: 32.

[0013] Accordingly, a method for detecting African swine fever using primer pairs and probes comprises the following steps:

[0014] (1) preparing fluorescent microspheres coupled with TAG probes;

[0015] (2) performing PCR amplification on the sample to be tested using the primer pair described in claim 1 or 2 to obtain a PCR amplification product;

[0016] (3) incubating the PCR amplification product, fluorescent microspheres and the probe according to claim 3 or 4, and after the incubation, adding streptavidin-phycoerythrin to obtain a "microsphere-PCR amplification product-SAPE" complex;

[0017] (4) Detecting the fluorescence signal value of the complex, and determining whether the sample to be tested is infected with African swine fever based on the fluorescence signal value.

[0018] Preferably, in step (1), a TAG probe is introduced into the magnetic fluorescent microspheres to prepare fluorescent microspheres coupled with the TAG probe.

[0019] Preferably, in step (2), in the PCR amplification system, 8 pairs of primers are mixed in equal proportions, and the total volume of the downstream primers is 3 times that of the upstream primers.

[0020] Preferably, in step (3), the incubation conditions are: incubate at 95°C for 5 min, then incubate at 60°C for 15 min, then incubate at 45°C for 15 min.

[0021] Correspondingly, a method for determining whether a pig is infected with African swine fever using the method is provided.

[0022] If the sample fluorescence signal value is ≤4000, it is judged as negative and not infected with African swine fever;

[0023] If the sample fluorescence signal value is greater than 12000, it is judged as positive and infected with African swine fever;

[0024] If the sample fluorescence signal value is 4000<12000, it is judged as a suspicious sample and needs to be repeated or other detection methods are used for further verification.

[0025] Correspondingly, a kit comprising the primer pair and the probe.

[0026] The present invention has the following beneficial effects:

[0027] MASA liquid chip (Multi-Analyte Suspension Array, multifunctional suspension array) technology combines flow detection technology with molecular hybridization technology, and has the characteristics of high sensitivity, high specificity, high throughput and simple operation. The liquid chip system is composed of round microspheres of uniform size. Different probe molecules are fixed on each microsphere and labeled with different coded fluorescent dyes. Molecular hybridization is carried out in a suspension solution. During the detection process, the target molecule can specifically bind to the probe coupled to the microsphere, so that the microsphere of the cross-linked probe carries the reporter molecule (phycoerythrin). When the microsphere is detected by the flow array instrument (NovaHT), the red and green lasers on the instrument detect the coded fluorescence and reporter molecules on a single microsphere respectively, and the detection results are directly interpreted by the fluorescence value.

[0028] The present invention combines virological detection technology with liquid chip technology to establish a nucleic acid liquid chip detection method for detecting African swine fever virus. The MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, MGF505-3R, B646L, and I177L genes on the African swine fever virus are selected in a targeted manner, and an ASFV liquid chip detection method that can simultaneously detect 8 deletion mutation sites is established. The microspheres for detection are obtained by coupling the carboxylated microspheres used for detection with the probes; then the African swine fever virus plasmid is prepared as a PCR template, and the PCR product is hybridized with the detection microspheres and double-binding probes, and the flow array instrument is used for on-machine detection, and the fluorescence signal value (MFI) of the "microsphere-nucleic acid amplification product-SAPE" complex obtained after sample hybridization is detected and judged, and the result judgment of the detection sample can be quickly and effectively performed.

[0029] The present invention combines liquid phase chip technology to detect African swine fever virus nucleic acid, which not only effectively shortens the detection time, but also has the characteristics of rapid, sensitive and specific detection, and can detect multiple test samples at one time, or detect multiple targets in a single sample. At the same time, the present invention can also perform fluorescence detection on PCR amplification products and liquid phase chip hybridization products, verify each other, improve the detection rate, and effectively reduce the occurrence of missed detection of false negative samples. DETAILED DESCRIPTION

[0030] The present invention specifically designs amplification primers and probes for ASFV MGF 360-12L, ASFV MGF 505-1R, ASFV MGF360-13L, ASFV MGF 360-14L, ASFV MGF 505-2R, ASFV MGF 505-3R, ASFV I177L and ASFVB646L genes in African swine fever virus. The sequences of each primer pair are shown in Table 1. The length of each primer is controlled to be as close as possible, and the GC content is controlled to be within the range of 40% to 60%; and the Tm of each primer is similar, which can ensure the specificity and stability of the primer and the target DNA sequence, thereby improving the efficiency and specificity of PCR amplification; and by repeatedly screening and optimizing each group of primer pairs, the length of the target band (PCR product) amplified by the primer is controlled to be 100bp. The shorter the PCR product, the better the specificity, the higher the subsequent amplification efficiency and sample hybridization efficiency, and more target bands can be generated under the same reaction conditions. At the same time, considering that the present invention requires eight-fold PCR amplification, by controlling the length of each PCR product to be 100 bp, the detection specificity can be improved. However, the length difference between the PCR products obtained by other multiplex PCR in the prior art may reach 100 bp, resulting in poor detection specificity.

[0031] Table 1 Sequence list of primer pairs

[0032]

[0033]

[0034] The present invention also provides TAG probes for each gene, the length of the probes is controlled at about 30 bp, the GC content is controlled within the range of 40% to 60%, and the lengths of the probes are as equal or close as possible.

[0035] Table 2 TAG probe sequence list

[0036]

[0037] In order to connect the PCR product with the NovaStar-TAG fluorescent microspheres for sample hybridization, the present invention further provides a double-binding probe for each gene. When used, one end of the double-binding probe is the reverse complementary sequence of the microsphere TAG, and the other end is the reverse complementary sequence of the target PCR product. After repeated screening and optimization, the 8 sets of primer pairs are designed to have low similarity and low overlap with each other, so as to avoid mutual binding between probes as much as possible. The double-binding probe finally provided by the present invention can only bind to the corresponding coupled microspheres and PCR products, and will not bind to other non-specific microspheres or PCR products. At the same time, in order to avoid probe interference during sample hybridization, the present invention also performs the following probe optimization: (1) Control the Tm value of the double-binding probe to be close to the Tm value of the TAG probe sequence to facilitate product connection and facilitate temperature (Tm) control at 50-55°C. (2) The probe and primer are specifically analyzed by base content, spatial structure, etc., mainly analyzing their secondary structure and interaction, and finally selecting a probe sequence that will not have dimers and hairpin structures. The details are shown in Table 3.

[0038] Table 3 Sequence list of each double binding probe

[0039]

[0040] The present invention also provides a method for detecting African swine fever using the primer pair and the probe, which specifically comprises the following steps:

[0041] 1. Prepare fluorescent microspheres coupled with TAG probes. This step is carried out in a light-proof environment to avoid fluorescence quenching. Commercially available NoveStar magnetic fluorescent microspheres are vortexed to prevent microsphere adhesion. Then, according to the instructions for the microspheres, introduce the TAG probes shown in Table 2 to prepare fluorescent microspheres that are respectively coupled with different TAG probes. When processing fluorescent microspheres, the following points should be noted: (1) EDC solution should be prepared and used immediately; (2) After preparing 1.5×TMAC buffer diluent (tetramethylammonium chloride aqueous solution), it should be filtered using a syringe filter; (3) When coupling microspheres, it is necessary to avoid light to prevent the fluorescence of the fluorescent microspheres from weakening or even bleaching; (4) Use 1.5×TMAC buffer to dilute and adjust the concentration of each microsphere to 100 / μL.

[0042] 2. PCR amplification: Extract the DNA nucleic acid of the sample to be tested, and perform PCR amplification using the 8 sets of primer pairs shown in Table 1 to obtain PCR products.

[0043] 3. Sample hybridization. Incubate the PCR amplification product, fluorescent microspheres and dual-binding probes in the detection buffer (1.5×TMAC buffer diluent). After the incubation, add SAPE (streptavidin-phycoerythrin) to obtain the "microsphere-amplification product-SAPE" complex.

[0044] 4. Detect the fluorescence signal value of the complex. Determine whether the sample to be tested is infected with African swine fever based on the fluorescence signal value (MFI). The judgment reference is as follows:

[0045] Samples with MFI ≤ 4000 were judged as negative samples and considered not to be infected with African swine fever;

[0046] If the sample MFI is greater than 12,000, it is judged as a positive sample and is considered to be infected with African swine fever;

[0047] 4000<Sample MFI<12000, set as grayscale interval, judged as suspicious sample, need to repeat the experiment or take other detection methods for further verification.

[0048] The primer pairs and probes provided by the present invention can also be further prepared into kits, detection reagents, test strips, etc. in combination with existing technologies, so as to achieve convenient and rapid detection.

[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0050] Example 1: Demonstration of the effect of primer pairs and probes combined to detect African swine fever

[0051] 1. Preparation of fluorescent microspheres coupled with TAG probes

[0052] Take 100 μL (1×10 7) NoveStar magnetic fluorescent microspheres developed by Hubei Xinzongke Virus Disease Engineering Technology Co., Ltd., the commercially available test tubes containing microspheres were mixed with a vortex mixer for 20 seconds, placed on a magnetic stand for adsorption for 2 minutes, the supernatant was removed, 300 μL of 0.1M MES solution (2-(N-morpholino)ethanesulfonic acid) with a pH of 4.5 was added to the precipitate, and then 1 μL of 0.1mM of each TAG probe (as shown in Table 2) was added after mixing, and then 2.5 μL of 10 mg / mL of EDC (1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride) was added, and then the mixture was kept away from light for 30 minutes. Then 2.5 μL of 10 mg / mL of EDC was added, and then the mixture was kept away from light for 45 minutes after mixing. The microspheres were washed once with 0.2 mL of 0.02% (v / v) Tween-20 and 0.1% (w / v) SDS (sodium dodecyl sulfate) solution, respectively, and then resuspended in 10 μL of 1×TE (pH = 8.0, composition: 1M Tris-HCl, 1 mL; 0.5 mM EDTA, 0.2 mL, plus 100 mL of double distilled water) solution to obtain fluorescent microspheres coupled with different TAG probes.

[0053] Each fluorescent microsphere was shaken and mixed, the number of microspheres was counted using a hemocytometer, and the concentration of each microsphere was adjusted to 100 / μL using 1.5×TMAC buffer. 10 μL of each microsphere was taken and mixed to obtain 80 μL of mixed microsphere solution, which was stored at 4°C away from light.

[0054] Preparation method of 1.5×TMAC buffer (tetramethylammonium chloride aqueous solution) (500mL): 5mol / LTMAC (tetramethylammonium chloride), 450mL; 20% (w / v) sarkosyl (sodium dodecanoate), 3.76mL; 1mol / L Tris-HCl, pH=8.0, 37.5mL; 0.5mol / L EDTA, pH=8.0, 6.0mL; double distilled water, 2.74mL; store in aliquots at 4°C.

[0055] 20% (w / v) Sarkosyl: Sarkosyl 20 g, double distilled water 100 mL, incubate in a 68°C water bath until completely dissolved, do not sterilize under high pressure, store at room temperature.

[0056] 2. PCR amplification

[0057] The PCR reaction system Master Mix was configured according to Table 4. Among them, PCR buffer is a buffer that can provide a suitable chemical environment for DNA polymerase activity, and in this embodiment, it was purchased from Takaba RR902A. The primer mixture is a mixture of the 8 pairs of primers in Table 1, and the 8 pairs of primers are mixed in equal proportions. The total volume of the upstream primers is 0.5 μL, and the total volume of the downstream primers is 1.5 μL. The template is the blood of sick pigs confirmed as ASFV positive by the qPCR method in a certain area of ​​Sichuan, and the plasmid obtained by cloning using the PCR product of ASFV positive blood; PCR amplification is performed using the plasmid as a template.

[0058] It should be noted that: when equal volumes of upstream and downstream primers (both 1 μL) were used and other conditions were the same for amplification, there was no good detection value, the positivity was not established, and the positive detection standard could not be met.

[0059] Table 4 Configuration of PCR reaction system Master Mix

[0060] Reagents Volume (μL) PCR buffer 5 Enzyme mixture 0.25 Primer mix 2 Nucleasefreewater 36.75 template 2 dNTP 4 Total volume 50

[0061] After preparing the PCR reaction system Master Mix, vortex for 15 seconds and centrifuge at 12000rpm for 15 seconds. Then add 44μL of Master Mix to the PCR reaction tube pre-cooled on the cooling plate or on ice, cover the EP tube, and put it back on ice.

[0062] Take out the nucleic acid sample to be tested (Template in Table 4), thaw to room temperature, vortex and oscillate for 15 seconds, and then centrifuge. Take 2μL of the sample to be tested and 4μL of dNTP respectively and add them to the PCR reaction tube containing 44μL of Master Mix, blow and mix well, and centrifuge for 10 seconds to ensure that there are no bubbles in the PCR reaction tube and the liquid in the tube sinks to the bottom of the tube. Then place it in the PCR instrument and run the PCR program according to Table 5.

[0063] Table 5 PCR program operating conditions

[0064]

[0065] After the reaction is completed, store at 4°C and wait for sample hybridization with the fluorescent microspheres prepared in step 1.

[0066] 3. Sample hybridization

[0067] In a light-proof environment, prepare the hybridization reaction system according to Table 6.

[0068] Table 6 Hybridization reaction system

[0069]

[0070]

[0071] Vortex the TAG microspheres for 15-30 seconds, add the vortexed mixed TAG microspheres and the diluted dual-binding probe (0.05pmol / μL) to the detection buffer, and take 51μL after vortexing and add it to the eight-tube. Vortex and centrifuge the PCR products at the end of the reaction, take 5μL of the PCR reaction products and add them to the corresponding eight-tube, blow and mix. Incubate the eight-tube at 95℃ for 5min, then incubate at 60℃ for 15min, and then incubate at 45℃ for 15min.

[0072] At the end of incubation, the 1 mg / mL SAPE stock solution was diluted 80 times, and 5 μL of the SAPE dilution solution was added to each of the eight tubes, and incubated at 45°C for 15 min in the dark to obtain the "microsphere-nucleic acid amplification product-SAPE" complex.

[0073] 4. Fluorescence signal value detection

[0074] The fluorescence signal value of the complex obtained after sample hybridization was detected according to the instruction manual of the flow array instrument (Nova HT) of Wuhan Xinzongke Virus Disease Engineering Technology Co., Ltd. The results are shown in Table 7. Among them, the PCR amplification template of the negative control was nuclease-free water, and the other conditions were the same.

[0075] Table 7 Experimental results of liquid phase chip detection of African swine fever virus

[0076] Target gene MFI Negative control results ASFV360-12L 194697 2936 ASFV505-1R 99459 3522 ASFV360-13L 122473 1509 ASFV360-14L 159042 1883 ASFV505-2R 155142 2655 ASFV505-3R 70312 2301 ASFVI177L 179095 2523 ASFVB646L 220204 3098

[0077] 5. According to the method of steps 1 to 4 of this embodiment, 100 samples from a certain area in Sichuan were tested for African swine fever. The blood samples were from castrated fattening pigs in a certain area of ​​Sichuan, 4 to 6 months old, and the species was DLY. At the same time, the common PCR method (African swine fever diagnostic technology, common PCR method in GB / T18648-2020) was used for detection as a control, and the results are shown in Table 8.

[0078] Table 8 Test results comparison table

[0079] method Positive Negative total Conventional PCR 90 10 100 Method of the present invention 94 6 100

[0080] The results showed that 90 samples were ASFV positive and 10 samples were ASFV negative using the common PCR method, with a positive rate of 90% and a negative rate of 10%; 94 samples were ASFV positive and 6 samples were ASFV negative using the method of the present invention, with a positive rate of 94% and a negative rate of 6%. The ASFV detection typing result of the positive sample was type II African swine fever. This shows that the detection method provided by the present invention has high specificity and strong sensitivity.

[0081] The detection time was then compared. Among them, the single-plex fluorescent PCR was tested using the African swine fever fluorescent PCR detection kit from Luoyang Lepson Information Technology Co., Ltd. The results are shown in Table 9.

[0082] Table 9 Detection time comparison table

[0083] Detection Methods Detection time (unit: h) Method of the present invention 2 Singleplex fluorescent PCR 2 8 single-plex fluorescence PCR 16

[0084] The results showed that the detection time of ordinary single-plex fluorescence PCR was about 2 hours. If 8 single-plex fluorescence PCRs were performed using 8 different targets, the detection time was about 16 hours. However, the total detection time was about 2 hours using the eight-plex ASFV liquid phase chip prepared by the present invention, which saved about 14 hours compared with the traditional method.

[0085] 6. Set up a control group (the operation steps and unmentioned conditions are the same as steps 1-4 of this embodiment).

[0086] The incubation conditions were as follows: incubation at 95°C for 1 min; incubation at 4°C for 1 min, incubation at 59°C for 15 min, and after adding SAPE at the end of the incubation, incubation at 59°C for 5 min.

[0087] The PCR amplification system is shown in Table 10, and the cycling conditions are shown in Table 11.

[0088] Table 10 PCR amplification system of control group

[0089] Reagents Volume (μL) PCR buffer 5 Enzyme mixture 0.25 Primer mix 2 (Use the same amount of upper and lower primers) Nucleasefreewater 36.75 template 2 dNTP 4 Total volume 50

[0090] Table 11 PCR cycle conditions for the control group

[0091]

[0092]

[0093] The test results are shown in Table 12.

[0094] Table 12 Test results of control group

[0095]

[0096] The results showed that in the control group, when the primer pairs and double-binding probes were exactly the same, the PCR reaction program, incubation conditions, and primer dosage were changed, and the on-machine detection was performed, and the test results were not good (the on-machine detection values ​​were not obvious, and the positive and negative were not established).

[0097] 7. In addition, in the early stage of the experiment, the detection results were not ideal when the direct probe was used for microsphere coupling; the double-binding probe must be used to achieve the ideal detection results. This may be because: when the direct probe is coupled with the microsphere for hybridization, there is steric hindrance on the surface of the microsphere, which makes it impossible for the target sequence to approach and bind to the probe; while the double-binding probe eliminates the steric hindrance, thereby improving the efficiency of the probe (free state) binding to the target and improving the hybridization signal.

[0098] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer pair for detecting African swine fever, characterized in that: The primer pairs include 8 pairs, which are designed based on ASFV 360-12L, ASFV 505-1R, ASFV 360-13L, ASFV 360-14L, ASFV 505-2R, ASFV505-3R, ASFV I177L and ASFV B646L genes in African swine fever virus, respectively.

2. The primer pair according to claim 1, characterized in that: The upstream primer sequence of ASFV 360-12L is shown in SEQ NO ID: 1, and the downstream primer sequence of ASFV 360-12L is shown in SEQ NO ID: 2; the upstream primer sequence of ASFV 505-1R is shown in SEQ NO ID: 3, and the downstream primer sequence of ASFV 505-1R is shown in SEQ NO ID: 4; the upstream primer sequence of ASFV 360-13L is shown in SEQ NO ID: 5, and the downstream primer sequence of ASFV 360-13L is shown in SEQ NO ID: 6; the upstream primer sequence of ASFV 360-14L is shown in SEQ NO ID: 7, and the downstream primer sequence of ASFV 360-14L is shown in SEQ NO ID: 8; the upstream primer sequence of ASFV 505-2R is shown in SEQ NO ID: 9, and the downstream primer sequence of ASFV 505-2R is shown in SEQ NO ID: 10; the upstream primer sequence of ASFV 505-3R is shown in SEQ NO ID: ID: 11, the ASFV 505-3R downstream primer sequence is shown in SEQ NOID: 12; the ASFV I177L upstream primer sequence is shown in SEQ NO ID: 13, and the ASFV I177L downstream primer sequence is shown in SEQ NO ID: 14; the ASFV B646L upstream primer sequence is shown in SEQ NOID: 15, and the ASFV B646L downstream primer sequence is shown in SEQ NO ID:

16.

3. A probe for detecting African swine fever in combination with the primer pair according to claim 1 or 2, characterized in that: The probe is a double-binding probe, one end of which is the reverse complementary sequence of the microsphere TAG, and the other end of which is the reverse complementary sequence of the target PCR product.

4. The probe according to claim 3, characterized in that: The probe sequence corresponding to the ASFV 360-12L gene is shown in SEQ NOID: 25; and / or; the probe sequence corresponding to the ASFV 505-1R gene is shown in SEQ NO ID: 26; and / or; the probe sequence corresponding to the ASFV360-13L gene is shown in SEQ NO ID: 27; and / or; the probe sequence corresponding to the ASFV 360-14L gene is shown in SEQ NO ID: 28; and / or; the probe sequence corresponding to the ASFV 505-2R gene is shown in SEQ NOID: 29; and / or; the probe sequence corresponding to the ASFV 505-3R gene is shown in SEQ NO ID: 30; and / or; the probe sequence corresponding to the ASFV I177L gene is shown in SEQ NO ID: 31; and / or; the probe sequence corresponding to the ASFV B646L gene is shown in SEQ NO ID:

32.

5. A method for detecting African swine fever using primer pairs and probes, characterized in that: The steps include: (1) preparing fluorescent microspheres coupled with TAG probes; (2) performing PCR amplification on the sample to be tested using the primer pair described in claim 1 or 2 to obtain a PCR amplification product; (3) incubating the PCR amplification product, fluorescent microspheres and the probe according to claim 3 or 4, and after the incubation, adding streptavidin-phycoerythrin to obtain a "microsphere-PCR amplification product-SAPE" complex; (4) Detecting the fluorescence signal value of the complex, and determining whether the sample to be tested is infected with African swine fever based on the fluorescence signal value.

6. The method according to claim 5, characterized in that: In step (1), a TAG probe is introduced into the magnetic fluorescent microsphere to prepare fluorescent microspheres coupled with the TAG probe.

7. The method according to claim 5, characterized in that: In step (2), in the PCR amplification system, 8 pairs of primers are mixed in equal proportions, and the total volume of the downstream primers is 3 times that of the upstream primers.

8. The method according to claim 5, characterized in that: In step (3), the incubation conditions are: incubate at 95°C for 5 min, then incubate at 60°C for 15 min, and then incubate at 45°C for 15 min.

9. A method for determining whether a pig is infected with African swine fever using the method according to any one of claims 5 to 8, characterized in that: If the sample fluorescence signal value is ≤4000, it is determined that it is not infected with African swine fever; If the sample fluorescence signal value is greater than 12000, it is determined to be infected with African swine fever; If the sample fluorescence signal value is 4000<12000, it is judged as a suspicious sample and needs to be repeated or other detection methods are used for further verification.

10. A kit comprising the primer pair according to claim 1 or 2 and the probe according to claim 3 or 4.

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