A method for the multiplex detection of ASFV, PCV2 and PCV3 suitable for rapid real-time fluorescent PCR

Abstract

The application relates to the technical field of animal virus molecular detection, and particularly discloses a multiple detection method for ASFV, PCV2 and PCV3 suitable for rapid real-time fluorescent PCR. Specific primers and TaqMan probes are designed according to the B646L gene of ASFV and the ORF1 gene conservative regions of PCV2 and PCV3, and the sequences are shown as SEQ ID NO:1-9. The primers and probes are optimized: avoiding strong GC anchoring at the 3' end of the primers, continuous AT series and G base at the 3' end of the probes, and shortening the length of the probes to adapt to rapid real-time fluorescent PCR. The minimum detection limit of the three viruses is not higher than 5 copies per muL, the detection is completed within 30 minutes, there is no cross reaction with 11 common pig pathogenic agents, the coefficient of variation is less than 2%, the sensitivity is high, the specificity is strong, the method is rapid and convenient, and is suitable for rapid diagnosis of mixed infection of ASFV, PCV2 and PCV3.

Description

Technical Field

[0001] This invention relates to the field of animal virus molecular detection technology, specifically to a multiplex detection method for ASFV, PCV2, and PCV3 suitable for rapid real-time fluorescent PCR. Background Technology

[0002] African swine fever (ASF) is a highly contagious and severe hemorrhagic disease caused by the African swine fever virus (ASFV). According to the World Organisation for Animal Health (OIE), from 2005 to January 2025, ASF has been reported in 83 countries worldwide. ASFV is classified as a Class A animal disease in my country, posing a serious threat to the pig farming industry.

[0003] Porcine circovirus type 2 (PCV2) is the main pathogen causing porcine circovirus-associated diseases (PCVAD), including multisystemic wasting syndrome in weaned piglets (PMWS), proliferative necrotizing pneumonia (PNP), porcine dermatitis and nephropathy syndrome (PDNS), and reproductive disorders. In 2016, Palinski et al. discovered porcine circovirus type 3 (PCV3) in PDNS cases. Subsequently, PCV3 was reported in China, South Korea, Thailand, the United Kingdom, Italy, Spain, Denmark, Poland, Brazil, Sweden, and other countries, and the clinical symptoms it causes are extremely similar to those of PCV2.

[0004] ASFV infection primarily targets monocytes / macrophages, affecting the function of various immune cells. Both PCV2 and PCV3 prioritize the immune system, leading to impaired both cellular and humoral immunity after infection. Co-infection with ASFV alongside PCV2 and PCV3 further amplifies the immunosuppressive effect on the host, resulting in complete paralysis of the swine immune system. Clinical studies have shown that mixed infections of ASFV, PCV2, and PCV3 occur frequently, exacerbating the disease and posing greater challenges to disease control.

[0005] Currently, although there are reports of triple PCR detection methods for ASFV, PCV2, and PCV3 both domestically and internationally, these methods are conventional PCR methods with a detection limit of approximately 1×10⁻⁶. 4 The detection limits are limited, including low sensitivity, inability to perform real-time quantification, and a detection time typically requiring 2-3 hours. Dual-quantitative real-time PCR methods for PCV2 and PCV3 have been reported, with a detection limit of 10 copies / μL, but the detection time is long, approximately 1.5 hours or more, and simultaneous detection of ASFV is not possible. Furthermore, while triple-quantitative real-time PCR methods for PCV2, PCV3, and PCV4 have been reported, their detection limit for PCV3 is only 1.38 × 10² copies / μL, indicating low sensitivity.

[0006] Currently, there are no reported methods for achieving high-sensitivity triple detection of ASFV, PCV2, and PCV3 on a rapid real-time fluorescence PCR platform. Therefore, developing a rapid multiplex detection method capable of simultaneously, quickly, with high sensitivity, and accurately detecting these three viruses is of great significance for early diagnosis, epidemiological investigation, and comprehensive prevention and control of diseases in swine farms. Summary of the Invention

[0007] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies in the detection of mixed infections of ASFV, PCV2, and PCV3, and to provide a multiplex detection method suitable for rapid real-time fluorescent PCR platforms, capable of simultaneously detecting the above three viruses, and a specific primer-probe combination to achieve high sensitivity, high specificity, rapid and convenient detection. The rapid real-time fluorescent PCR method of this invention can simultaneously detect African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3) within 30 minutes, with a detection limit as low as 5 copies / μL.

[0008] Furthermore, conventional qPCR conditions cannot be directly transferred to the rapid platform because short annealing times amplify multiple competition, increase background noise, and cause low-concentration false negatives. This invention solves this problem through the synergistic design of primer and probe structures.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: On the one hand, this invention provides a method for multiplex detection of ASFV, PCV2, and PCV3 suitable for rapid real-time fluorescence PCR, comprising the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Using the nucleic acid as a template, multiplex real-time PCR amplification was performed using primer and probe sets; in: The upstream primer sequence for detecting African swine fever virus is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:2, and the probe sequence is shown in SEQ ID NO:3. The upstream primer sequence for detecting porcine circovirus type 2 is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:5, and the probe sequence is shown in SEQ ID NO:6. The upstream primer sequence for detecting porcine circovirus type 3 is shown in SEQ ID NO:7, the downstream primer sequence is shown in SEQ ID NO:8, and the probe sequence is shown in SEQ ID NO:9. The primers and probes meet the following conditions: the 3′ end of the primer does not contain a continuous GC anchoring structure, the probe length is controlled to be 24-28 nucleotides, and the 3′ end of the probe is not a G base; S3. The amplification reaction was carried out using the following amplification program: 95℃ pre-denaturation for 1-3 min; 95℃ denaturation for 5-15 s; 57℃ annealing extension for 5-20 s; for a total of 40-50 cycles. S4. Collect fluorescence signals and determine the presence of African swine fever virus, porcine circovirus type 2, and porcine circovirus type 3 based on the fluorescence signals.

[0010] Preferably, the amplification program is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 8 s; 57℃ annealing extension for 10 s; for a total of 45 cycles.

[0011] Preferably, the probe for detecting African swine fever virus is labeled with CY5 fluorescent group and BHQ2 quenching group; the probe for detecting porcine circovirus type 2 is labeled with FAM fluorescent group and BHQ1 quenching group; and the probe for detecting porcine circovirus type 3 is labeled with VIC fluorescent group and BHQ1 quenching group.

[0012] Preferably, the total volume of the multiplex quantitative PCR reaction system is 20–30 μL, comprising: 2× rapid quantitative PCR amplification enzyme premix, the volume of which is half of the total reaction system volume; 0.4–1.0 μL each of African swine fever virus upstream and downstream primers; 0.4–1.0 μL each of porcine circovirus type 2 upstream and downstream primers; 0.4–1.0 μL each of porcine circovirus type 3 upstream and downstream primers; 0.4–1.0 μL of African swine fever virus probe; 0.4–1.0 μL of porcine circovirus type 2 probe; 0.4–1.0 μL of porcine circovirus type 3 probe; 1–5 μL of template; and the remainder being ddH2O.

[0013] Preferably, the total volume of the reaction system is 25 μL, comprising: 0.6 μL each of the upstream and downstream primers for African swine fever virus (ASFV) with a final concentration of 0.24 μmol / L; 0.8 μL each of the upstream and downstream primers for porcine circovirus type 2 (PCV2) with a final concentration of 0.32 μmol / L; 0.8 μL each of the upstream and downstream primers for PCV3 (PCV3) with a final concentration of 0.32 μmol / L; 0.6 μL of the ASFV probe; 0.6 μL of the PCV2 probe; 0.5 μL of the PCV3 probe; and 3 μL of template.

[0014] Preferably, the method has a minimum detection limit of no more than 5 copies / μL for African swine fever virus, porcine circovirus type 2, and porcine circovirus type 3.

[0015] Preferably, the method can complete the simultaneous detection of African swine fever virus, porcine circovirus type 2, and porcine circovirus type 3 within 30 minutes.

[0016] Preferably, the method does not show specific amplification signals for porcine deltacoronavirus, foot-and-mouth disease virus, getta virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus.

[0017] Preferably, the amplified fragment of the African swine fever virus is 134 bp in length, the amplified fragment of porcine circovirus type 2 is 154 bp in length, and the amplified fragment of porcine circovirus type 3 is 130 bp in length.

[0018] On the other hand, the present invention provides a kit for rapid real-time fluorescent PCR detection of ASFV, PCV2 and PCV3, comprising the above-mentioned primer set and probe set, as well as 2× rapid fluorescent quantitative PCR amplification enzyme premix, positive control and negative control; wherein the positive control is a recombinant plasmid containing target amplification fragments of African swine fever virus, porcine circovirus type 2 and porcine circovirus type 3; and the negative control is ddH2O.

[0019] The beneficial effects achieved by this invention are as follows: 1. This invention is the first to achieve triple detection of ASFV, PCV2, and PCV3 on a rapid real-time fluorescence PCR platform, solving the technical challenge of compatibility between rapid platforms and multiple detection. 2. The annealing time of this invention is shortened from the conventional 30-60 s to 10 s, a reduction of 83%, while still achieving a high sensitivity of 5 copies / μL, which is comparable to the optimal sensitivity of conventional qPCR; 3. The detection limit for all three viruses in this invention is 5 copies / μL. Compared to the previously reported PCV2 / PCV3 dual real-time PCR method, where the PCV3 detection limit is 10 copies / μL, the detection limit for PCV3 in this application is reduced to 5 copies / μL, resulting in a 1-fold increase in sensitivity; compared to the previously reported PCV2 / PCV3 / PCV4 triple real-time PCR method, where the PCV3 detection limit is 138 copies / μL, the sensitivity of this application is increased by 27.6 times. 4. This invention has good repeatability (CV<2%) and high specificity, with no cross-reactivity with 11 common porcine pathogens. The total detection time is about 28 minutes, which is significantly better than the 1.5-2 hours of conventional fluorescent PCR methods and also better than the 1-2 hours of detection time of PCV2 / PCV3 dual fluorescent PCR kits. It achieves a breakthrough in the time of triple detection on a rapid platform and plays an important role in rapid clinical diagnosis. Attached Figure Description

[0020] Figure 1 The figure shows the standard curve results of this invention; where A: ASFV; B: PCV2; C: PCV3.

[0021] Figure 2 The following is a diagram showing the results of the specificity test of this invention: 1-7 are PDCoV, FMDV, GETV, CSFV, PRRSV, JEV, and TGEV, respectively; 8 is the positive control pUC57-ASFV-PCV2-PCV3; 9-12 are PEDV, negative porcine spleen, negative porcine lymph node, and ddH2O, respectively; 13 is PCV2; 14 is ASFV; and 15 is PCV3. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] In this invention, rapid real-time fluorescence PCR refers to real-time fluorescence PCR technology in which the annealing extension time for each cycle does not exceed 20 seconds.

[0025] Example 1: Primer and probe design and synthesis 1.1 Instruments and Equipment The Pangu Super8 / Pro portable rapid real-time fluorescence quantitative PCR system was purchased from Aipu Biotechnology (Suzhou) Co., Ltd., the NanoDrop micro spectrophotometer was purchased from Thermo Fisher Scientific (China) Co., Ltd., and the rapid fluorescence quantitative PCR amplification enzyme premix (UNG) and 2x were purchased from Aipu Biotechnology (Suzhou) Co., Ltd.

[0026] 1.2 Viral nucleic acid and samples Positive nucleic acid samples for specificity validation of African swine fever virus (ASFV), porcine deltacoronavirus (PDCoV), foot-and-mouth disease virus (FMDV), getta virus (GETV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3), as well as the tandem plasmid standard pUC57-ASFV-PCV2-PCV3 containing the target amplification fragments of ASFV, PCV2, and PCV3, and 36 clinical samples, were all known laboratory materials.

[0027] 1.3 Primer and probe design and synthesis Based on the ASFV B646L gene, PCV2 Rep gene, and PCV3 Rep gene sequences in the GenBank database, their conserved regions were analyzed using SnapGene software. Candidate primers and probes were designed for the conserved regions of the three viruses using Oligo 7 software. Specifically, four pairs of candidate primers and probes were designed for ASFV, two pairs for PCV2, and three pairs for PCV3.

[0028] To address the high heating / cooling rates and extremely short annealing times of the rapid real-time fluorescence PCR platform, the following optimizations were made during primer and probe design: (1) avoiding strong GC anchoring at the 3′ end of the primer; (2) ensuring the probe sequence does not contain continuous AT tandem structures; (3) ensuring the 3′ end of the probe is not a G base; and (4) shortening the probe length to improve its binding efficiency, keeping the probe length around 26 nucleotides. The optimal primer-probe combination was ultimately determined by comparing the smoothness of the amplification curve, fluorescence intensity, Ct value, and specificity.

[0029] The final primer and probe sequences are shown in Table 1. ASFV-F is 23 nucleotides, ASFV-R is 20 nucleotides, and ASFV-P is 26 nucleotides; PCV2-F is 18 nucleotides, PCV2-R is 20 nucleotides, and PCV2-P is 26 nucleotides; PCV3-F is 18 nucleotides, PCV3-R is 19 nucleotides, and PCV3-P is 26 nucleotides. The ASFV probe is labeled with the CY5 fluorescent group and quenched with the BHQ2 group; the PCV2 probe is labeled with the FAM fluorescent group and quenched with the BHQ1 group; and the PCV3 probe is labeled with the VIC fluorescent group and quenched with the BHQ1 group. The ASFV probe uses BHQ2 alone because of its superior absorption matching in the CY5 channel (640-680 nm emission wavelength), enabling more efficient quenching of the CY5 fluorescence signal. The PCV2 and PCV3 probes use BHQ1 because it exhibits more ideal quenching effects in the FAM (518 nm emission) and VIC (555 nm emission) channels. This differentiated configuration of quenching groups effectively avoids non-specific fluorescence crosstalk between the three channels.

[0030] Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and stored at -20°C for later use.

[0031] Table 1 Primer and probe sequence information

[0032] Example 2: Determination of the reaction system Using the Pangu Super8 / Pro portable rapid real-time quantitative PCR system, with the tandem plasmid standard pUC57-ASFV-PCV2-PCV3 as a template, the primer concentration, probe concentration, annealing temperature, and annealing time were tested.

[0033] 2.1 Determination of the reaction system The effects of different final primer concentrations (0.16 μmol / L, 0.24 μmol / L, 0.32 μmol / L) and different final probe concentrations (0.12 μmol / L, 0.16 μmol / L, 0.2 μmol / L, 0.24 μmol / L, 0.28 μmol / L) on the amplification curve and Ct value were tested under the conditions of 12.5 μL of 2× rapid real-time PCR amplification enzyme premix (UNG), 3 μL of template, and a total system volume of 25 μL.

[0034] The results showed that when the final concentration of ASFV primer was 0.24 μmol / L, the final concentration of PCV2 and PCV3 primers was 0.32 μmol / L, and the final concentrations of ASFV and PCV2 probes were 0.24 μmol / L and the final concentration of PCV3 probe was 0.2 μmol / L, the amplification curves of each target showed high fluorescence intensity, low Ct value, and no non-specific amplification.

[0035] The reaction system determined accordingly was as follows: 25 μL: 12.5 μL of 2× rapid real-time PCR amplification enzyme premix; 0.6 μL each of ASFV-F / R (final concentration 0.24 μmol / L); 0.8 μL each of PCV2-F / R (final concentration 0.32 μmol / L); 0.8 μL each of PCV3-F / R (final concentration 0.32 μmol / L); 0.6 μL of ASFV-P; 0.6 μL of PCV2-P; 0.5 μL of PCV3-P; 3 μL of template; and ddH2O to a final volume of 25 μL.

[0036] Example 3: Validation of the reaction procedure and sensitivity testing The effect of annealing temperature on amplification was tested at five temperatures: 56℃, 57℃, 58℃, 59℃, and 60℃. The results are shown in Table 2. When the annealing temperature was 57℃, the fluorescence signal of the three targets was the strongest and the Ct value was the lowest.

[0037] Table 2 Effect of different annealing temperatures on amplification efficiency

[0038] The annealing extension time was further tested within the range of 5-20 s. Annealing extension times were set to 20 s, 10 s, and 5 s, respectively, with a concentration of 1×10⁻⁶. 8 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 Using plasmid standards of 5, 1, and 0.1 copies / μL as templates, eight replicates were set for each concentration, and the limit of detection (LOD) was determined at each annealing time (based on the lowest concentration that can be detected 100%). The results are shown in Table 3.

[0039] Table 3 Relationship between concentration and detection rate

[0040] Table 3 shows that when the annealing time is 20 s or 10 s, the detection rate of the three targets at a concentration of 5 copies / μL is 100%. When the annealing time is shortened to 5 s, the detection rate at a concentration of 5 copies / μL drops to 87.5%, and the detection limit increases to 10 copies / μL. Therefore, the shortest annealing time of 10 s, which does not affect the detection limit, is selected as the optimal annealing extension time.

[0041] The amplification program determined accordingly was: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 8 s; 57℃ annealing and extension for 10 s; for a total of 45 cycles. The limit of detection for ASFV, PCV2, and PCV3 was 5 copies / μL. Under the above amplification conditions, the total detection time for all 45 cycles was approximately 28 min.

[0042] Example 4: Establishment of Standard Curve Using the reaction system and amplification procedure of Example 2, at a concentration of 1×10^ 8 ~1×10^ 2 Amplification was performed using plasmid standards at 1 / μL copies as templates, with three replicates for each concentration. The ASFV standard curve equation was y = -3.32521x + 40.2796, R² = 0.99886, and amplification efficiency E = 99.86%; the PCV2 standard curve equation was y = -3.15312x + 40.22407, R² = 0.99293, and amplification efficiency E = 107.56%; the PCV3 standard curve equation was y = -3.216x + 38.98462, R² = 0.99849, and amplification efficiency E = 104.62%.

[0043] The results are as follows Figure 1 As shown, the R² values ​​of the standard curves for ASFV, PCV2, and PCV3 are all greater than 0.99, and the amplification efficiencies E are all between 90% and 110%, indicating that the three viruses can amplify at 1x10⁻¹⁰ cm⁻¹. 8 ~1x10 2 At a concentration of copies / μL, there is a good linear relationship between concentration and Ct value. The method established in this invention exhibits good amplification efficiency and linearity within the linear range.

[0044] Example 5: Specificity Test Using positive nucleic acids of porcine deltacoronavirus (PDCoV), foot-and-mouth disease virus (FMDV), getta virus (GETV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), as well as nucleic acids extracted from negative porcine spleen and lymph nodes, as templates, and using the pUC57-ASFV-PCV2-PCV3 plasmid standard as a positive control and ddH2O as a negative control, this method was used for detection.

[0045] The results are as follows Figure 2 As shown: ASFV-positive nucleic acid only showed ASFV-specific amplification curves (CY5 channel); PCV2-positive nucleic acid only showed PCV2-specific amplification curves (FAM channel); PCV3-positive nucleic acid only showed PCV3-specific amplification curves (VIC channel); the positive control showed three specific amplification curves simultaneously; positive nucleic acids, negative porcine tissue nucleic acids, and ddH2O from the other 11 viruses showed no specific amplification curves. This indicates that this method has good specificity and no cross-reactivity with the above 11 common porcine pathogens and porcine genomic nucleic acids.

[0046] Example 6: Repeatability Test With a concentration of 1×10 6 1×10 4 1×10 2 Using plasmid standards of copies / μL as templates, three intra-group and three inter-group replicates were performed for each concentration. The standard deviation (SD) and coefficient of variation (CV) of the Ct values ​​were calculated. The results are shown in Table 4: the CV values ​​for all intra-group and inter-group measurements were less than 2%, indicating that the method has good repeatability and stability.

[0047] Table 4 Results of Repeatability Tests

[0048] Example 7: Clinical Sample Testing Thirty tissue samples and six laboratory-preserved ASFV positive samples (a total of 36 samples) were collected. After nucleic acid extraction, parallel detection was performed using this method, the national standard method (GB / T 18648-2020, GB / T 21674-2025), and the PCV3 detection method reported in the literature.

[0049] As shown in Table 4, this method detected 6 positive ASFV samples, 7 positive PCV2 samples, and 6 positive PCV3 samples, which were completely consistent with the detection results of the control method, with a concordance rate of 100%.

[0050] Table 4: Detection results of this method and the control method

[0051] Where: "-" indicates not detected.

[0052] Example 8 Reagent Kit A kit for rapid real-time fluorescent PCR detection of ASFV, PCV2, and PCV3 includes: primers and probes shown in SEQ ID NO:1-9 (10 μmol / L primers and 10 μmol / L probes), 2× rapid real-time PCR amplification enzyme premix (UNG), a positive control (recombinant plasmid containing the three viral target fragments), and a negative control (ddH2O). The instruction manual includes the reaction system and procedure described in Example 2.

[0053] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A multiplex detection method for ASFV, PCV2, and PCV3 suitable for rapid real-time fluorescent PCR, characterized in that, Includes the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Using the nucleic acid as a template, multiplex real-time PCR amplification was performed using primer and probe sets; in: The upstream primer sequence for detecting African swine fever virus is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:2, and the probe sequence is shown in SEQ ID NO:3; The upstream primer sequence for detecting porcine circovirus type 2 is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:5, and the probe sequence is shown in SEQ ID NO:

6. The upstream primer sequence for detecting porcine circovirus type 3 is shown in SEQ ID NO:7, the downstream primer sequence is shown in SEQ ID NO:8, and the probe sequence is shown in SEQ ID NO:

9. The primers and probes meet the following conditions: the 3′ end of the primer does not contain a continuous GC anchoring structure, the probe length is controlled to be 24-28 nucleotides, and the 3′ end of the probe is not a G base; S3. The amplification reaction was carried out using the following amplification program: 95℃ pre-denaturation for 1-3 min; 95℃ denaturation for 5-15 s; 57℃ annealing extension for 5-20 s; for a total of 40-50 cycles; S4. Collect fluorescence signals and determine the presence of African swine fever virus, porcine circovirus type 2, and porcine circovirus type 3 based on the fluorescence signals.

2. The multiple detection method according to claim 1, characterized in that: The amplification program was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 8 s; 57℃ annealing extension for 10 s; for a total of 45 cycles.

3. The multiple detection method according to claim 1, characterized in that: The probes used to detect African swine fever virus are labeled with CY5 fluorescent group and BHQ2 quenching group; the probes used to detect porcine circovirus type 2 are labeled with FAM fluorescent group and BHQ1 quenching group; and the probes used to detect porcine circovirus type 3 are labeled with VIC fluorescent group and BHQ1 quenching group.

4. The multiple detection method according to claim 1, characterized in that: The total volume of the multiplex quantitative PCR reaction system is 20–30 μL, comprising: 2× rapid quantitative PCR amplification enzyme premix, the volume of which is half of the total reaction system volume; 0.4–1.0 μL each of African swine fever virus upstream and downstream primers; 0.4–1.0 μL each of porcine circovirus type 2 upstream and downstream primers; 0.4–1.0 μL each of porcine circovirus type 3 upstream and downstream primers; 0.4–1.0 μL of African swine fever virus probe; 0.4–1.0 μL of porcine circovirus type 2 probe; 0.4–1.0 μL of porcine circovirus type 3 probe; 1–5 μL of template; and the remainder is ddH2O.

5. The multiple detection method according to claim 4, characterized in that: The total volume of the reaction system was 25 μL, including: 0.6 μL each of the upstream and downstream primers for African swine fever virus (ASFV), with a final concentration of 0.24 μmol / L; 0.8 μL each of the upstream and downstream primers for porcine circovirus type 2 (PCV2), with a final concentration of 0.32 μmol / L; 0.8 μL each of the upstream and downstream primers for PCV3 (PCV3), with a final concentration of 0.32 μmol / L; 0.6 μL of the ASFV probe; 0.6 μL of the PCV2 probe; 0.5 μL of the PCV3 probe; and 3 μL of template.

6. The multiple detection method according to claim 1, characterized in that: The method has a detection limit of no more than 5 copies / μL for African swine fever virus, porcine circovirus type 2, and porcine circovirus type 3.

7. The multiple detection method according to claim 1, characterized in that: The method can simultaneously detect African swine fever virus, porcine circovirus type 2, and porcine circovirus type 3 within 30 minutes.

8. The multiple detection method according to claim 1, characterized in that: The method showed no specific amplification signals for porcine deltacoronavirus, foot-and-mouth disease virus, getta virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus.

9. The multiple detection method according to claim 1, characterized in that: The amplified fragment of the African swine fever virus is 134 bp in length, the amplified fragment of porcine circovirus type 2 is 154 bp in length, and the amplified fragment of porcine circovirus type 3 is 130 bp in length.

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