An african swine fever virus pathogen detection kit and detection method

Abstract

This invention relates to the field of detection kit technology, specifically to an African swine fever virus pathogen detection kit and detection method. This invention provides an African swine fever virus detection kit and detection method integrating specific primers and probes, a multiplex quality control system, and standardized operating procedures. By optimizing the nucleic acid extraction process, establishing a dual-channel fluorescent PCR detection system, and defining clear judgment criteria, it achieves high sensitivity and high specificity detection of African swine fever virus nucleic acid and full-process quality monitoring. It can not only accurately identify virus-positive samples but also effectively exclude false negative results through an internal standard system. This solves the key problems of insufficient specificity and low reliability of traditional detection methods, providing a stable and reliable complete technical solution for the clinical diagnosis and prevention and control of African swine fever.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to a detection kit and detection method for African swine fever virus pathogen. Background Technology

[0002] In contemporary animal disease control, African swine fever virus (ASFV) has become a major threat to the global pig industry due to its high infectivity, high mortality rate, and primary cause of fever and hemorrhagic lesions. Since there is currently no effective vaccine, rapid and sensitive diagnostic techniques are crucial for controlling the disease. Existing pathogen detection mainly relies on nucleic acid detection and immunological techniques. However, these methods all have significant limitations. Immunological detection depends on antibody production, which suffers from latency and a high false-positive rate. While nucleic acid detection can identify the virus in the early stages of infection, conventional PCR and real-time quantitative PCR are time-consuming, cumbersome, and require high-quality experimental conditions and equipment. Furthermore, the presence of trace amounts of viral nucleic acid or sample inhibitors can lead to false negatives, or cross-reactions with pathogens can cause false positives. More importantly, current technologies lack full-process monitoring of nucleic acid extraction and amplification, making the reliability of test results highly dependent on operational experience and the experimental environment, hindering the implementation of standardized, verifiable, and accurate diagnosis at the grassroots level. Studies have shown that combining highly specific primer-probe systems with endogenous quality control systems can effectively improve the accuracy and stability of molecular detection. However, existing detection schemes mostly involve the simple stacking of functional modules or step-by-step preparation, failing to achieve deep integration at the system level in terms of detection sensitivity, process controllability, and objectivity of result interpretation. Specifically, there is currently a lack of a complete technical system that integrates target-specific amplification, full-process quality monitoring, and standardized operating procedures into a unified design and procedural system. This leads to problems such as disconnected quality control, complex operation, and subjective result interpretation in the detection process, making it impossible to ensure stable and reliable detection results under different conditions. This severely restricts the practical effectiveness of early diagnosis, epidemic monitoring, and precise prevention and control of African swine fever.

[0003] To address the above problems, the present invention provides a solution. Summary of the Invention

[0004] The purpose of this invention is to provide a detection kit and method for African swine fever virus pathogens, which can quickly, conveniently and stably detect African swine fever virus pathogens.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: an African swine fever virus pathogen detection kit includes a PCR amplification primer pair for African swine fever virus pathogen nucleic acid, a detection probe for African swine fever virus pathogen nucleic acid, a positive control, a negative control, an internal standard, an internal standard PCR amplification primer pair, an internal standard detection probe, a nucleic acid extraction reagent, a nucleic acid amplification system, and a PCR reaction solution; Furthermore, the nucleotide sequences of the PCR amplification primer pairs for the African swine fever virus pathogen nucleic acid are shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer shown in SEQ ID NO: 1 is a forward PCR amplification primer for the African swine fever virus pathogen nucleic acid, and the primer shown in SEQ ID NO: 2 is a reverse PCR amplification primer for the African swine fever virus pathogen nucleic acid.

[0006] Furthermore, the nucleotide sequence of the detection probe for African swine fever virus pathogen nucleic acid is shown in SEQ ID NO:3. The 5' end of the detection probe for African swine fever virus pathogen nucleic acid is labeled with a fluorescent reporter group FAM, and the 3' end of the detection probe for African swine fever virus pathogen nucleic acid is labeled with a fluorescent quencher group TAMRA.

[0007] Furthermore, the nucleotide sequence of the positive control is shown in SEQ ID NO:4, the negative control is prepared with 10mM Tris-HCl and 1mM EDTA using ddH2O, with a pH of 8.0, and the nucleotide sequence of the internal standard is shown in SEQ ID NO:5.

[0008] Furthermore, the nucleotide sequences of the internal standard PCR amplification primer pair are shown in SEQ ID NO:6 and SEQ ID NO:7, wherein the primer shown in SEQ ID NO:6 is the forward PCR amplification primer for the internal standard, and the primer shown in SEQ ID NO:7 is the reverse PCR amplification primer for the internal standard.

[0009] Furthermore, the nucleotide sequence of the internal standard detection probe is shown in SEQ ID NO:8, the 5' end of the internal standard detection probe is labeled with a fluorescent reporter group CY5, and the 3' end of the internal standard detection probe is labeled with a fluorescent quencher group BHQ2.

[0010] A method for detecting African swine fever virus pathogens includes the following steps: S1: Sample processing and nucleic acid extraction: Extracting total nucleic acid from the sample to be tested; S2: Preparation of reaction system: Mix the extracted nucleic acid with the PCR amplification primer pair of African swine fever virus pathogen nucleic acid, the detection probe of African swine fever virus pathogen nucleic acid, internal standard, internal standard PCR amplification primer pair and internal standard detection probe in the kit to prepare the PCR reaction system; S3: Real-time PCR amplification: The PCR reaction system is amplified by real-time PCR amplification program, which includes pre-denaturation and repeated denaturation, annealing and extension cycles. S4: Result determination: By analyzing the fluorescence signal during the quantitative PCR process, it is determined whether the sample contains the nucleic acid of the African swine fever virus pathogen.

[0011] Furthermore, the sample to be tested in step S1 is serum, and the nucleic acid extraction includes lysing the sample using proteinase K and lysis buffer, purifying the nucleic acid through an adsorption column bound to a silica membrane, and eluting with elution buffer to obtain purified nucleic acid.

[0012] Furthermore, the specific procedure for quantitative PCR amplification in step S3 is as follows: pre-denaturation: 95℃ for 30 seconds; cycling reaction: 44 cycles, each cycle including denaturation at 95℃ for 25 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 15 seconds; complete extension: 72℃ for 10 minutes.

[0013] Furthermore, the result judgment criteria in step S4 are as follows: Experimental failure: Negative control: FAM channel, Ct value is 0, and there is no obvious S-shaped amplification curve; CY5 channel, Ct value is present and Ct≤30, and there is an obvious S-shaped amplification curve; Positive control: FAM channel, Ct value is present and Ct≤40, and there is an obvious S-shaped amplification curve; CY5 channel, Ct value is present and Ct≤30, and there is an obvious S-shaped amplification curve. Positive: The FAM channel shows an amplification curve, and the Ct values ​​are all ≤40; Negative: The FAM channel showed no amplification curve and no Ct value was detected; the CY5 channel showed an amplification curve and the Ct values ​​were all ≤30. When the FAM channel shows an amplification curve and the Ct value is 40 < Ct value ≤ 44, and the CY5 channel shows an amplification curve and the Ct value is ≤ 30, it is recommended to repeat the experiment once. If the result is the same as above, it is determined to be positive for African swine fever virus pathogen. If there is no amplification, it is determined to be negative for African swine fever virus pathogen. Experiment invalid: No amplification curves were observed in either the FAM or CY5 channels, and no Ct value was detected.

[0014] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: 1. This invention provides a complete detection system for African swine fever virus pathogens. Through a specially designed high-efficiency primer-probe combination, a rigorous internal standard quality control system, and a standardized nucleic acid extraction and amplification process, it systematically solves the key technical problems of insufficient specificity, susceptibility to interference from inhibitors, strong subjectivity in result interpretation, and high risk of false negatives in existing detection methods. 2: This invention integrates a specific fluorescent probe detection system targeting the target gene of African swine fever virus pathogen with an independent endogenous internal standard monitoring system. The internal standard monitors the efficiency of nucleic acid extraction and amplification throughout the process, effectively distinguishing between true negative results and false negatives caused by extraction failure or the presence of PCR inhibitors, significantly improving the accuracy and reliability of the detection results. 3. This invention establishes a standardized detection method with high sensitivity, strong specificity, and good repeatability by using serum pretreatment, nucleic acid extraction, and a carefully designed qPCR reaction procedure, combined with clear objective judgment criteria based on Ct value and amplification curve. The method is convenient to operate and produces stable results, providing an efficient and reliable technical tool for the early diagnosis, epidemic monitoring, and precise prevention and control of African swine fever. Attached Figure Description

[0015] Figure 1 This is the amplification curve from the specificity detection experiment in Example 2.

[0016] Figure 2 The amplification curve is shown for the product concentration of 1000 copies / ml in the lowest detection limit in Example 3.

[0017] Figure 3 The amplification curve is shown for the product concentration of 100 copies / ml in the lowest detection limit in Example 3.

[0018] Figure 4 The amplification curve is shown for the product concentration of 10 copies / ml in the lowest detection limit in Example 3.

[0019] Figure 5 The amplification curve is shown for the product concentration of 1 copy / ml in the lowest detection limit in Example 3.

[0020] Figure 6 This is the amplification curve of the negative control in the minimum detection in Example 3. Detailed Implementation

[0021] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Example 1 This embodiment tests African swine fever pathogens in domestic pigs to evaluate the detection effectiveness of the African swine fever virus pathogen detection kit.

[0024] One hundred domestic pigs with no history of genetic diseases were selected, including 48 normal pigs, 10 pigs infected with African swine fever, and 42 pigs suspected of being infected with African swine fever for sample collection. Draw 5 ml of venous blood into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000 rpm for 5-10 minutes. Take the supernatant into an enzyme-free EP tube and centrifuge again at 1000 rpm for 2-4 minutes. Take the supernatant into a new enzyme-free EP tube to obtain a serum sample, which should be stored at -80℃ for later use. 1. Sample processing and nucleic acid extraction: 1.1: Take 1 ml of serum sample into an EP tube, add 100 μl of Proteinase K, and mix by repeatedly pipetting. Then add 1 ml of Lysis Buffer and immediately vortex vigorously for 15 seconds to ensure thorough mixing. After mixing, incubate at room temperature for 10 minutes. 1.2: After incubation, add 1 ml of anhydrous ethanol and vortex again for 15 seconds to mix. Then transfer the entire mixture to the adsorption column, place the adsorption column in a centrifuge tube, and centrifuge at 12000 rpm for 60 seconds. The centrifugal force causes the liquid to pass through the silica membrane, and the nucleic acid is specifically captured on the membrane. Impurities and waste liquid are centrifuged to the bottom of the collection tube. 1.3: Carefully remove the adsorption column, discard the waste liquid in the collection tube, put the adsorption column back into the same collection tube, add 500ul Wash Buffer 1 to the center of the adsorption column membrane, centrifuge at 12000rpm for 60s, discard the waste liquid, then add 500ul Wash Buffer 2 to the center of the adsorption column membrane, centrifuge at 12000rpm for 60s, discard the waste liquid; 1.4: Place the empty adsorption column back into the collection tube, open the cap, and centrifuge at 12000 rpm for 2 min. After centrifugation, remove the adsorption column and place it in a new sterile 1.5 mL centrifuge tube. Carefully add 50 μL of preheated Elution Buffer (65 °C) to the center of the adsorption column membrane. Let it stand at room temperature for 5 min to allow the liquid to fully wet the membrane and dissolve the DNA. Then centrifuge at 12000 rpm for 60 s. Collect the elution buffer containing purified DNA at the bottom of the centrifuge tube and label it as the sample. 2: Preparation of the reaction system: Sample / Negative Control / Positive Control 5.0 μL 1.5 μL primer-probe premix 2X PCR MIX 10ul Internal standard 1.0ul ddH2O2.5ul 3: Real-time PCR amplification: It consists of 44 cycles of denaturation, annealing, and extension; Preheat to 50℃ for 60 seconds Pre-denaturation 95℃ for 30s Denaturation at 95℃ for 25 seconds Annealing at 55℃ for 30 seconds Extended to 72℃ for 15 seconds Extend completely at 72℃ for 10 minutes Cool to 4°C for 15 seconds 4. Result determination: The criteria for judgment are as follows: Experimental failure: Negative control: FAM channel, Ct value is 0, and there is no obvious S-shaped amplification curve; CY5 channel, Ct value is present and Ct≤30, and there is an obvious S-shaped amplification curve; Positive control: FAM channel, Ct value is present and Ct≤40, and there is an obvious S-shaped amplification curve; CY5 channel, Ct value is present and Ct≤30, and there is an obvious S-shaped amplification curve. Positive: The FAM channel shows an amplification curve, and the Ct values ​​are all ≤40; Negative: The FAM channel showed no amplification curve and no Ct value was detected; the CY5 channel showed an amplification curve and the Ct values ​​were all ≤30. When the FAM channel shows an amplification curve and the Ct value is 40 < Ct value ≤ 44, and the CY5 channel shows an amplification curve and the Ct value is ≤ 30, it is recommended to repeat the experiment once. If the result is the same as above, it is determined to be positive for African swine fever virus pathogen. If there is no amplification, it is determined to be negative for African swine fever virus pathogen. Experiment invalid: No amplification curves were observed in either the FAM or CY5 channels, and no Ct value was detected.

[0025] Table 1. Detection results of swine serum samples in Example 1

[0026] As can be seen from the analysis of Table 1, the kit and detection method used in Example 1 can accurately detect healthy pigs and pigs infected with African swine fever during the detection process. When faced with pigs suspected of being infected with African swine fever, it can normally detect African swine fever positive and African swine fever negative, and its application performance is good.

[0027] Example 2 This embodiment mainly tests the interference of different DNAs on the detection of African swine fever virus. Three DNA types were set up: CHO, HEK293, and E. coli genomic DNA. Specificity detection was performed simultaneously with African swine fever virus DNA samples and negative controls. The reaction system used in this embodiment is as follows: Sample / Negative Control / Positive Control 5.0 μL 1.5 μL primer-probe premix 2X PCR MIX 10ul Internal standard 1.0ul ddH2O2.5ul The PCR amplification procedure used in this embodiment is as follows: It consists of 44 cycles of denaturation, annealing, and extension; Preheat to 50℃ for 60 seconds Pre-denaturation 95℃ for 30s Denaturation at 95℃ for 25 seconds Annealing at 55℃ for 30 seconds Extended to 72℃ for 15 seconds Extend completely at 72℃ for 10 minutes Cool to 4°C for 15 seconds Table 2, Specificity Detection Results of Example 2

[0028] By analyzing Table 2 and Figure 1 The detection value of African swine fever virus DNA in the FAM channel was 33.35. CHO, HEK293, and E. coli genomic DNA were not detected in the FAM channel. There was no cross-reactivity during African swine fever virus amplification detection. The results indicate that the kit and detection method of the present invention have good specificity.

[0029] Example 3 This embodiment mainly tests the lowest detection limit of the present invention by diluting the known concentration of the extract to 1000 copies / mL, 100 copies / mL, 10 copies / mL, and 1 copy / mL, and setting up a negative control; The reaction system used in this embodiment is as follows: Sample / Negative Control / Positive Control 5.0 μL 1.5 μL primer-probe premix 2X PCR MIX 10ul Internal standard 1.0ul ddH2O2.5ul The PCR amplification procedure used in this embodiment is as follows: It consists of 44 cycles of denaturation, annealing, and extension; Preheat to 50℃ for 60 seconds Pre-denaturation 95℃ for 30s Denaturation at 95℃ for 25 seconds Annealing at 55℃ for 30 seconds Extended to 72℃ for 15 seconds Extend completely at 72℃ for 10 minutes Cool to 4°C for 15 seconds Table 3. Results of detection at the limit of detection

[0030] By analyzing Table 3 and Figure 2-6 It is evident that the detection method of the present invention has high sensitivity, reaching 100 copies / ml.

[0031] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0032] SEQ ID NO:1

[0033] SEQ ID NO:2

[0034] SEQ ID NO:3

[0035] SEQ ID NO:4

[0036] SEQ ID NO:5

[0037] SEQ ID NO:6

[0038] SEQ ID NO:7

[0039] SEQ ID NO:8

Claims

1. A detection kit for African swine fever virus pathogen, characterized in that, The detection kit includes PCR amplification primer pairs for African swine fever virus pathogen nucleic acid, detection probes for African swine fever virus pathogen nucleic acid, positive control, negative control, internal standard, internal standard PCR amplification primer pairs, internal standard detection probes, nucleic acid extraction reagents, nucleic acid amplification system, and PCR reaction solution.

2. The African swine fever virus pathogen detection kit according to claim 1, characterized in that, The nucleotide sequences of the PCR amplification primer pairs for the African swine fever virus pathogen nucleic acid are shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer shown in SEQ ID NO: 1 is the forward PCR amplification primer for the African swine fever virus pathogen nucleic acid, and the primer shown in SEQ ID NO: 2 is the reverse PCR amplification primer for the African swine fever virus pathogen nucleic acid.

3. The African swine fever virus pathogen detection kit according to claim 1, characterized in that, The nucleotide sequence of the detection probe for African swine fever virus pathogen nucleic acid is shown in SEQ ID NO:

3. The 5' end of the detection probe for African swine fever virus pathogen nucleic acid is labeled with a fluorescent reporter group FAM, and the 3' end of the detection probe for African swine fever virus pathogen nucleic acid is labeled with a fluorescent quencher group TAMRA.

4. The African swine fever virus pathogen detection kit according to claim 1, characterized in that, The nucleotide sequence of the positive control is shown in SEQ ID NO:4, the negative control is 10 mM Tris-HCl and 1 mM EDTA prepared with ddH2O, with a pH of 8.0, and the nucleotide sequence of the internal standard is shown in SEQ ID NO:

5.

5. The African swine fever virus pathogen detection kit according to claim 1, characterized in that, The nucleotide sequences of the internal standard PCR amplification primer pair are shown in SEQ ID NO:6 and SEQ ID NO:7, wherein the primer shown in SEQ ID NO:6 is the forward PCR amplification primer of the internal standard, and the primer shown in SEQ ID NO:7 is the reverse PCR amplification primer of the internal standard.

6. The African swine fever virus pathogen detection kit according to claim 1, characterized in that, The nucleotide sequence of the internal standard detection probe is shown in SEQ ID NO:

8. The 5' end of the internal standard detection probe is labeled with a fluorescent reporter group CY5, and the 3' end of the internal standard detection probe is labeled with a fluorescent quencher group BHQ2.

7. A method for detecting African swine fever virus pathogen, characterized in that, Includes the following steps: S1: Sample processing and nucleic acid extraction: Extracting total nucleic acid from the sample to be tested; S2: Preparation of reaction system: Mix the extracted nucleic acid with the PCR amplification primer pair of African swine fever virus pathogen nucleic acid, the detection probe of African swine fever virus pathogen nucleic acid, internal standard, internal standard PCR amplification primer pair and internal standard detection probe in the kit to prepare the PCR reaction system; S3: Real-time PCR amplification: The PCR reaction system is amplified by real-time PCR amplification program, which includes pre-denaturation and repeated denaturation, annealing and extension cycles. S4: Result determination: By analyzing the fluorescence signal during the quantitative PCR process, it is determined whether the sample contains the nucleic acid of the African swine fever virus pathogen.

8. The method for detecting African swine fever virus pathogen according to claim 7, characterized in that, The sample to be tested in step S1 is serum. The nucleic acid extraction includes lysing the sample using proteinase K and lysis buffer, purifying the nucleic acid through an adsorption column bound to a silica membrane, and eluting with elution buffer to obtain purified nucleic acid.

9. The method for detecting African swine fever virus pathogen according to claim 7, characterized in that, The specific procedure for quantitative PCR amplification in step S3 is as follows: Pre-denaturation: 95℃ for 30 seconds; Cyclic reaction: 44 cycles, each cycle including denaturation at 95℃ for 25 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 15 seconds; Complete extension: 72℃ for 10 minutes.

10. The method for detecting African swine fever virus pathogen according to claim 7, characterized in that, The criteria for judging the results in step S4 are as follows: Experimental failure: Negative control: FAM channel, Ct value is 0, and there is no obvious S-shaped amplification curve; CY5 channel, Ct value is present and Ct≤30, and there is an obvious S-shaped amplification curve; Positive control: FAM channel, Ct value is present and Ct≤40, and there is an obvious S-shaped amplification curve; CY5 channel, Ct value is present and Ct≤30, and there is an obvious S-shaped amplification curve. Positive: The FAM channel shows an amplification curve, and the Ct values ​​are all ≤40; Negative: The FAM channel showed no amplification curve and no Ct value was detected; the CY5 channel showed an amplification curve and the Ct values ​​were all ≤30. When the FAM channel shows an amplification curve and the Ct value is 40 < Ct value ≤ 44, and the CY5 channel shows an amplification curve and the Ct value is ≤ 30, it is recommended to repeat the experiment once. If the result is the same as above, it is determined to be positive for African swine fever virus pathogen. If there is no amplification, it is determined to be negative for African swine fever virus pathogen. Experiment invalid: No amplification curves were observed in either the FAM or CY5 channels, and no Ct value was detected.

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