阅读说明：本技术 非洲猪瘟病毒的锁核酸探针荧光定量pcr检测组合物、检测方法及检测试剂盒 (Locked nucleic acid probe fluorescent quantitative PCR detection composition, detection method and detection kit for African swine fever virus ) 是由 李艳 李春玲 郭怡德 勾红潮 蔡汝健 蒋智勇 宋帅 楚品品 卞志标 徐民生 于 2019-12-17 设计创作，主要内容包括：本发明公开一种非洲猪瘟病毒的锁核酸探针荧光定量PCR检测组合物、检测方法及检测试剂盒,属于分子生物学领域。本发明的检测引物、探针和检测试剂盒,利用锁核酸的特点,设计含有锁核酸的特异性探针,该探针对DNA有强大的识别能力和强大的亲和力,从而能够很好的应用于非洲猪瘟病毒精准检测,提高了非洲猪瘟病毒的检测效率,漏检的概率降低,为非洲猪瘟病毒检测提供了一种新的检测方法和检测试剂。本发明的探针、引物和试剂盒,与现有的常规检测方法相比,具有特异性强、灵敏度高等优点,特别适用于科研及临床应用,具有很好的商业应用价值。(The invention discloses a locked nucleic acid probe fluorescent quantitative PCR detection composition, a detection method and a detection kit of African swine fever virus, belonging to the field of molecular biology. According to the detection primer, the probe and the detection kit, the characteristic of locked nucleic acid is utilized to design the specific probe containing the locked nucleic acid, and the probe has strong recognition capability and strong affinity to DNA, so that the detection primer, the probe and the detection kit can be well applied to the accurate detection of the African swine fever virus, the detection efficiency of the African swine fever virus is improved, the probability of missed detection is reduced, and a novel detection method and a novel detection reagent are provided for the detection of the African swine fever virus. Compared with the conventional detection method, the probe, the primer and the kit have the advantages of strong specificity, high sensitivity and the like, are particularly suitable for scientific research and clinical application, and have good commercial application value.)

1. A locked nucleic acid probe fluorescent quantitative PCR detection composition of African swine fever virus is characterized in that: comprises 1 group of specific primer pairs for amplifying African swine fever virus and 1 LNA-Taqman probe:

the sequence of the specific primer pair is shown as Seq ID No. 1-2;

the sequence of the LNA-Taqman probe is shown as Seq ID No. 3.

2. The African swine fever virus locked nucleic acid probe fluorescent quantitative PCR detection composition according to claim 1, which is characterized in that: in the probe with the sequence shown by the LNA-Taqman probe, the base 'G' at the 7 th position, the base 'A' at the 11 th position, the base 'G' at the 13 th position and the base 'A' at the 17 th position are all locked nucleic acids.

3. The African swine fever virus locked nucleic acid probe fluorescent quantitative PCR detection composition according to claim 1, which is characterized in that:

the LNA-Taqman probe is marked with a fluorescent substance at the 5 'end and marked with a quenching substance at the 3' end.

4. The African swine fever virus locked nucleic acid probe fluorescent quantitative PCR detection composition according to claim 3, wherein:

the 5' end labeling fluorescent substance is labeled FAM;

the 3' end is marked with a quenching substance and is marked with BHQ 1.

5. Use of the locked nucleic acid probe fluorogenic quantitative PCR detection composition for African swine fever virus according to any one of claims 1-4 in the preparation of an African swine fever detection kit.

6. A locked nucleic acid probe fluorescent quantitative PCR detection kit of African swine fever virus is characterized in that: a fluorogenic quantitative PCR detection composition comprising the locked nucleic acid probe of African swine fever virus according to any one of claims 1-4.

7. The fluorescence quantitative PCR detection kit for locked nucleic acid probe of African swine fever virus according to claim 6, which is characterized in that:

the detection kit also comprises a positive control;

the positive control is a recombinant plasmid containing the main structural protein P72 gene B646L of the African swine fever virus.

8. The use of the locked nucleic acid probe fluorogenic quantitative PCR detection kit for African swine fever virus according to claim 6 or 7 for identifying and/or detecting African swine fever virus.

9. A locked nucleic acid probe fluorescence quantitative PCR method for detecting African swine fever virus is characterized by comprising the following steps:

(1) extracting genomic DNA of a sample;

(2) preparing a nucleic acid locking probe fluorescent quantitative PCR reaction system;

(3) placing the locked nucleic acid probe fluorescent quantitative PCR reaction system prepared in the step (2) into a fluorescent quantitative PCR instrument for amplification reaction;

(4) and (3) comparing the cycle threshold Ct of the genome DNA with the standard curve to obtain the copy concentration of the African swine fever virus gene fragment in the genome DNA.

10. The fluorescence quantitative PCR method for detecting locked nucleic acid probe of African swine fever virus according to claim 9, which is characterized in that:

the locked nucleic acid probe fluorescent quantitative PCR reaction system in the step (2) comprises the locked nucleic acid probe fluorescent quantitative PCR composition, and the reaction system is as follows: premix Ex Tag (2X) 10uL, upstream primer 0.6uL at a concentration of 10. mu. mol/L, downstream primer 0.6uL at a concentration of 10. mu. mol/L, probe 0.3uL at a concentration of 10. mu. mol/L, template DNA 1uL, and sterilized water to 20 uL;

the reaction conditions of the amplification reaction in the step (3) are as follows: pre-denaturation at 95 deg.C for 10 min; amplification was carried out at 95 ℃ for 15s, 59 ℃ for 30s, for 45 cycles.

Technical Field

The invention belongs to the field of molecular biology, and relates to a locked nucleic acid probe fluorescent quantitative PCR detection composition, a detection method and a detection kit for African swine fever virus.

Background

African Swine Fever (ASF) is an acute, virulent and highly contagious infectious disease of domestic and wild pigs caused by African Swine Fever Virus (ASFV), with a mortality rate of 100%. The African swine fever causes a catastrophic attack to the pig industry, belongs to the A-type epidemic disease of the International animal health organization, and is classified as an animal epidemic disease by the rural part of agriculture in China. The African swine fever virus is the only member of the African swine fever virus family, is a double-stranded DNA virus with an envelope and is also the only DNA arbovirus. The total length of the viral genome is about 170-193 kb, and the genome encodes 150-200 proteins. The P72 protein is the main structural protein of ASFV and is coded by gene B646L, which is highly conserved and is the most common target gene in virus detection.

At present, the African swine fever still has no effective prevention vaccine and specific treatment medicine, so that a rapid and accurate detection method for the African swine fever is urgently needed to be established, and the early elimination is effectively realized. The PCR technique has become a molecular biological method widely used in nucleic acid detection, but has a disadvantage of generating false positive results due to non-specific amplification. Therefore, based on PCR technology, the application of improved primer probe design strategy has become a new direction for nucleic acid detection method.

A Locked Nucleic Acid (LNA) probe selectively modifies some basic groups in the probe into LNA basic groups on the basis of a TaqMan probe, greatly improves the affinity of the probe and a target sequence, improves the Tm value of the probe, greatly shortens the length of the probe, and has higher stability, specificity and sensitivity.

Disclosure of Invention

The invention aims to make up the defects of the prior art and provide a locked nucleic acid probe fluorescent quantitative PCR detection composition for African swine fever virus.

The invention also aims to provide a locked nucleic acid probe fluorescence quantitative PCR detection kit of the African swine fever virus, which comprises the detection composition.

The invention further aims to provide a locked nucleic acid probe fluorescent quantitative PCR method for detecting African swine fever virus by using the detection kit.

The purpose of the invention is realized by the following technical scheme: a locked nucleic acid probe fluorescence quantitative PCR detection composition of African swine fever virus comprises 1 group of specific primer pairs for amplifying the African swine fever virus and 1 LNA-Taqman probe in an amplification target region of the primer pairs.

The sequence of the specific primer pair is shown as Seq ID No. 1-2.

The sequence of the LNA-Taqman probe is shown as Seq ID No. 3.

In the probe with the sequence shown by the LNA-Taqman probe, the base 'G' at the 7 th position, the base 'A' at the 11 th position, the base 'G' at the 13 th position and the base 'A' at the 17 th position are all locked nucleic acids.

The LNA-Taqman probe is marked with a fluorescent substance at the 5 'end and marked with a quenching substance at the 3' end.

The fluorescent substance labeled at the 5' end is labeled FAM.

The 3' end is marked with a quenching substance and is marked with BHQ 1.

The locked nucleic acid probe fluorescent quantitative PCR detection composition of the African swine fever virus is applied to preparation of an African swine fever detection kit.

A locked nucleic acid probe fluorescence quantitative PCR detection kit of African swine fever virus comprises the locked nucleic acid probe fluorescence quantitative PCR detection composition.

The detection kit also comprises a positive control.

The positive control is preferably a recombinant plasmid containing the gene B646L encoding the major structural protein P72 of the African swine fever virus.

The locked nucleic acid probe fluorescent quantitative PCR detection kit for the African swine fever virus is applied to identification and/or detection of the African swine fever virus.

A locked nucleic acid probe fluorescence quantitative PCR method for detecting African swine fever virus comprises the following steps:

(1) extracting genomic DNA of a sample;

(2) preparing a nucleic acid locking probe fluorescent quantitative PCR reaction system;

(3) placing the locked nucleic acid probe fluorescent quantitative PCR reaction system prepared in the step (2) into a fluorescent quantitative PCR instrument for amplification reaction;

(4) and (3) comparing the cycle threshold Ct of the genome DNA with the standard curve to obtain the copy concentration of the African swine fever virus gene fragment in the genome DNA.

The locked nucleic acid probe fluorescent quantitative PCR reaction system in the step (2) comprises a locked nucleic acid probe fluorescent quantitative PCR composition, and the reaction system is as follows: premix Ex Tag (2X) 10uL, upstream primer 0.6uL at a concentration of 10. mu. mol/L (0.3. mu. mol/L in the system), downstream primer 0.6uL at a concentration of 10. mu. mol/L (0.3. mu. mol/L in the system), probe 0.3uL at a concentration of 10. mu. mol/L (0.15. mu. mol/L in the system), template DNA 1uL, and sterile water to 20 uL.

The reaction conditions of the amplification reaction in the step (3) are as follows: pre-denaturation at 95 deg.C for 10 min; amplification was carried out at 95 ℃ for 15s, 59 ℃ for 30s, for 45 cycles.

Compared with the prior art, the invention has the following advantages and effects:

(1) the detection composition and the detection kit for the African swine fever virus design a specific probe containing locked nucleic acid by utilizing the characteristic of the locked nucleic acid, and the probe has strong identification capability and strong affinity to DNA, so that the detection efficiency of the African swine fever virus can be effectively improved, the probability of missed detection is reduced, and a new detection method and a new detection reagent are provided for the detection of the African swine fever virus.

(2) Compared with the conventional detection method, the primer, the probe and the kit have the advantages of strong specificity, high sensitivity and the like, are particularly suitable for scientific research and clinical application, and have good commercial application value.

Drawings

FIG. 1 is a fluorescent quantitative PCR amplification curve diagram of ASFV B646L gene LNATaqMan probe obtained from different Tm values in the example.

FIG. 2 is a graph showing the fluorescent quantitative PCR amplification curve of the LNATaqMan probe of ASFV B646L gene obtained by different primer concentrations in the examples.

FIG. 3 is a graph of the fluorescent quantitative PCR amplification curve of the LNATaqMan probe of ASFV B646L gene obtained by different probe concentrations in the examples.

FIG. 4 is a graph showing the results of sensitivity detection of fluorescent quantitative PCR using LNA TaqMan probe for the ASFV B646L gene in the example; wherein 1 is 3.9 × 10 ⁹A copied positive recombinant plasmid; 2 is 3.9X 10 ⁸A copied positive recombinant plasmid; 3 is 3.9 multiplied by 10 ⁷A copied positive recombinant plasmid; 4 is 3.9 multiplied by 10 ⁶A copied positive recombinant plasmid; 5 is 3.9 multiplied by 10 ⁵A copied positive recombinant plasmid; 6 is 3.9X 10 ⁴A copied positive recombinant plasmid; 7 is 3.9X 10 ³A copied positive recombinant plasmid; 8 is 3.9X 10 ²A copied positive recombinant plasmid; 9 is 3.9 multiplied by 10 ¹A copied positive recombinant plasmid; 10 is 3.9 multiplied by 10 ⁰A positive recombinant plasmid of the copy.

FIG. 5 is a graph showing the results of sensitivity detection of conventional TaqMan probe fluorescent quantitative PCR of the ASFV B646L gene in the example; wherein 1 is 3.9 × 10 ⁹A copied positive recombinant plasmid; 2 is 3.9X 10 ⁸A copied positive recombinant plasmid; 3 is 3.9 multiplied by 10 ⁷A copied positive recombinant plasmid; 4 is 3.9 multiplied by 10 ⁶A copied positive recombinant plasmid; 5 is 3.9 multiplied by 10 ⁵Sun of copyA sex recombinant plasmid; 6 is 3.9X 10 ⁴A copied positive recombinant plasmid; 7 is 3.9X 10 ³A copied positive recombinant plasmid; 8 is 3.9X 10 ²A copied positive recombinant plasmid; 9 is 3.9 multiplied by 10 ¹A positive recombinant plasmid of the copy.

FIG. 6 is a diagram showing the results of specific detection of the ASFV B646L gene in the examples; wherein, 1 is a positive recombinant plasmid 3.9 multiplied by 10 of African swine fever virus B646L gene ⁶Copying a detection result as a template; 2, taking the nucleic acid of the hog cholera virus as a detection result of the template; 3, the detection result takes the porcine reproductive and respiratory syndrome virus nucleic acid as a template; 4, the detection result of the porcine circovirus type 2 nucleic acid is taken as a template; and 5 is the detection result of the negative control.

FIG. 7 is a positive recombinant plasmid pMD19-T-ASFV-p 723.9X 10 of ASFV B646L gene in example ⁶The result of the repeated detection of the copies.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and the accompanying drawings, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Materials, methods and results

1. Viral strains

Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and porcine circovirus type 2 (PCV2) are all purchased from commercial vaccines on the market, wherein the live classical swine fever virus vaccine and the live porcine reproductive and respiratory syndrome virus vaccine are purchased from Guangdong Yongshu biological pharmacy, Inc., and the inactivated classical swine circovirus type 2 vaccine is purchased from Shanghai Haili biotechnology, Inc.

2. Primary reagents and instruments

2 × Premix Ex Tag (Probe qPCR) (available from TaKaRa, Cat No. RR390). The reagent used in the application is an analytical pure or biochemical reagent, and the experimental water meets the specification of first-grade water in GB/T6682. All reagents were dispensed in containers without dnase contamination. Fluorescence quantitative PCR instruments (LightCycler96), ultraviolet spectrophotometer (BioTek) and the like are all available from the laboratory.

3. Design and screening of primers and probes

Two pairs of specific primers and five specific probes are designed according to the sequence of the B646L gene (p72 protein gene, accession number: MH713612) which is preserved and stable in the genome sequence of African swine fever virus published in GenBank for subsequent experimental screening. Specific sequences of the primers and probes are shown in Table 1. All primers and probes of the present application were synthesized by Biotechnology engineering (Shanghai) Inc.

TABLE 1 fluorescent quantitative PCR primer and Probe sequences

In Table 1, in the ASFV-F1R1-probe1 probe, the bases at positions 7, 11, 13 and 17 are locked nucleic acid modified bases; in the ASFV-F1R1-probe3 probe, the 13 th, 14 th and 15 th bases are locked nucleic acid modified bases; in the ASFV-F2R2-probe1 probe, the 12 th, 13 th and 14 th bases are locked nucleic acid modified bases; in the ASFV-F2R2-probe2 probe, the 6 th, 9 th, 14 th and 17 th bases are locked nucleic acid modified bases. All probes were labeled with FAM at the 5 'end and BHQ1 at the 3' end.

Through a large amount of comparison and screening, the detection range of the combination of the upstream primer ASFV-F1 of the sequence shown in Seq ID No.1, the downstream primer ASFV-R1 of the sequence shown in Seq ID No.2 and the probe ASFV-F1R1-probe1 of the sequence shown in Seq ID No.3 on positive plasmids is 3.9 x 10 ⁰～3.9×10 ⁹copies/uL, and the other three combinations (combination of the upstream primer ASFV-F1 having the sequence shown in Seq ID No.1, the downstream primer ASFV-R1 having the sequence shown in Seq ID No.2, and the probe ASFV-F1R1-probe3 having the sequence shown in Seq ID No.5, the upstream primer ASFV-F2 having the sequence shown in Seq ID No.6, and the downstream primer ASFV-F2 having the sequence shown in Seq ID No.7Combination of primer ASFV-R2 and probe ASFV-F2R2-probe1 having sequence shown in Seq ID No.8, combination of upstream primer ASFV-F2 having sequence shown in Seq ID No.6, downstream primer ASFV-R2 having sequence shown in Seq ID No.7 and probe ASFV-F2R2-probe2 having sequence shown in Seq ID No. 9) for positive plasmid detection range of 3.9X 10 ¹～3.9×10 ⁹copies/uL. In this example, the upstream primer ASFV-F1 having the sequence shown in Seq ID No.1, the downstream primer ASFV-R1 having the sequence shown in Seq ID No.2, and the probe ASFV-F1R1-probe1 having the sequence shown in Seq ID No.3 were finally selected from the primer probes shown in Table 1, and the combination of the primers and the probes was used for efficient detection of African swine fever virus.

4. Preparation of Positive control in kit

A partial sequence (primer ASFV-F1:5 '-CTTTGGTGCGGCTTGTGCAA-3', primer ASFV-R1:5 '-TGACTGGATATAAGCACTTGGTTGGC-3') of a main structural protein gene B646L gene (p72 protein gene, accession number: MH713612) of African swine fever virus published in NCBI GenBank is sent to a synthetic gene fragment of Shanghai biological engineering (Shanghai) GmbH, and the synthetic gene fragment is cloned into a pMD19-T vector and named as pMD19-T-ASFV-p 72. And (3) feeding a positive control plasmid pMD19-T-ASFV-p72 for sequencing, comparing a sequencing result with a GenBank sequence, and determining the concentration and purity of the plasmid by using a full-automatic ultraviolet spectrophotometer.

5. Viral nucleic acid extraction

Viral genomes of CSFV, PRRSV and PCV2 were extracted as templates according to the protocol of the viral nucleic acid extraction kit (magenta (Meiji) organism, R4410-03).

Optimization of PCR reaction conditions

A20 uL reaction system is adopted, the annealing temperature is set to be 58 ℃, 58.5 ℃, 59 ℃, 59.5 ℃, 60 ℃, 60.5 ℃, 61 ℃ and 61.5 ℃ for 8 temperature gradients to carry out amplification, an amplification curve is obtained, and the annealing temperature is optimized.

The concentrations of the upstream and downstream primers were amplified in 5 reaction systems at 0.3, 0.4, 0.5, 0.6, and 0.7umol/L to obtain amplification curves, and the concentrations of the upstream and downstream primers were optimized.

The probe concentration is amplified according to 5 concentration gradient reaction systems of 0.05umol/L, 0.1umol/L, 0.15umol/L, 0.2umol/L and 0.25umol/L to obtain an amplification curve and optimize the probe concentration.

The reaction conditions are as follows: pre-denaturation at 95 deg.C for 10 min; amplification was carried out at 95 ℃ for 45 cycles, 15s, 60 ℃ for 30 s. After the amplification is finished, the specificity of the reaction system and the primer thereof is verified.

TABLE 2 Real-time PCR reaction System

As a result of comparison of Ct values of amplification curves at different annealing temperatures (Tm), the Ct value at 59 ℃ was 17.26, which is the minimum value among 8 annealing temperatures, as shown in FIG. 1. Therefore, the pMD19-T-ASFV-p72 positive control plasmid has the highest amplification efficiency at the annealing temperature of 59 ℃.

Amplification curves are obtained by amplifying reaction systems of 5 upstream and downstream primers with different concentrations of 200, 300, 400, 500 and 600nmol/L, and the concentrations of the upstream and downstream primers are optimized. As shown in FIG. 2, the optimal concentration of the upstream and downstream primers was 0.3. mu. mol/L, since the amplification efficiency of the pMD19-T-ASFV-p72 positive control plasmid was the highest and the Ct value was the lowest, at a concentration of 0.3. mu. mol/L. The optimal concentration of the probe was 0.15umol/L depending on the lowest Ct value, as shown in FIG. 3.

7. Establishment of Standard Curve and sensitivity test

Respectively diluting the positive recombinant plasmid pMD19-T-ASFV-p72 with the concentration and purity determined in the step 4 by 10 times to obtain 3.9 multiplied by 10 ⁰～3.9×10 ⁹Recombinant plasmids were used as standard templates at 10 dilutions of copies/uL, with 3 replicates per template concentration. And (3) performing LNATaqMan probe fluorescent quantitative PCR (an upstream primer shown in SEQ ID No.1, a downstream primer shown in SEQ ID No.2 and a probe shown in SEQ ID No. 3) amplification and conventional TaqMan probe fluorescent quantitative PCR (an upstream primer shown in SEQ ID No.1, a downstream primer shown in SEQ ID No.2 and a probe shown in SEQ ID No. 4) amplification according to the reaction system and the reaction parameters of the LNA TaqMan probe fluorescent quantitative PCR established in the step 6 to obtain a fluorescent amplification curve and draw a standard curve. Determined by observing the amplification curveAnd detecting the lowest copy number of the recombinant plasmid, and finally establishing a standard curve by taking the Ct value as a vertical coordinate and the logarithm of the copy number as a horizontal coordinate to evaluate the sensitivity of the whole PCR system.

The result showed that pMD19-T-ASFV-p72 positive control plasmid was diluted 10-fold to 3.9X 10 ⁰～3.9×10 ⁹The copies/uL are diluted by 10 times, LNA TaqMan probe fluorescent quantitative PCR amplification is carried out, an amplification curve is obtained, as shown in figure 4, the concentration of a standard curve is 3.9 multiplied by 10 ⁰～3.9×10 ⁹Has good correlation in the range of copies/uL, and the linear equation of y is-2.9481 x +40.651, R ²0.9913, drawing a standard curve; performing conventional TaqMan probe fluorescent quantitative PCR amplification to obtain an amplification curve, as shown in FIG. 5, the concentration of the standard curve is 3.9X 10 ¹～3.9×10 ⁹Has good correlation in the range of copies/uL, and the linear equation of y is-3.0843 x +42.572, R ²The standard curve was plotted at 0.9957.

The detection result of taking the recombinant plasmids with different copy numbers as a template shows that the amplification curve of the gene presents a typical S shape and the intervals of all the curves are uniform. The lowest detection quantity of the fluorescent quantitative PCR method based on the B646L gene (p72 protein gene) LNA TaqMan probe is 3.9 copies, while the lowest detection quantity of the conventional TaqMan probe fluorescent quantitative PCR method based on the B646L gene (p72 protein gene) is 39 copies, and the sensitivity of the LNA TaqMan probe fluorescent quantitative PCR method is 10 times higher than that of the conventional TaqMan probe fluorescent quantitative PCR method.

8. Specificity detection

Adopting the LNA TaqMan probe fluorescent quantitative PCR reaction system and reaction parameters established in the step 6 to be 3.9 multiplied by 10 ⁶The copied positive plasmid pMD19-T-ASFV-p72 served as a standard positive control. CSFV, PRRSV and PCV2 genome template is used as the template of other strains, and sterile water is used as a negative control; the specificity of the established method was verified by performing locked nucleic acid probe fluorescent quantitative PCR amplification using a roche LightCycler480 fluorescent quantitative PCR instrument.

As shown in FIG. 6, only pMD19-T-ASFV-p72 positive recombinant plasmid can generate a specific fluorescence curve, and the others are negative, thus proving that the method has better specificity.

9. Repeatability detection

Using 3.9X 10 ⁶And (3) taking the copied positive plasmid pMD19-T-ASFV-p72 as a template, performing locked nucleic acid probe fluorescence quantitative PCR test according to the step 6, and repeatedly detecting for three times to analyze the stability of the locked nucleic acid probe fluorescence quantitative PCR test. The results are shown in FIG. 7, the detection results of the curves 1 to 3 are basically consistent, and corresponding fluorescence curves can be observed at the same position, which indicates that the locked nucleic acid probe fluorescence quantitative PCR method has good repeatability.

10. Detection of clinical samples

The screened primer pairs ASFV-F1, ASFV-R1 and probe ASFV-F1R1-probe1 are adopted to detect the samples to be detected, and the real-time fluorescent PCR method (AFRICAN SWINE F EVER, OIE Terrietal Manual 2012) recommended by OIE is used to detect the samples to be detected.

The detection result of the clinical sample shows that when the screened primer pair ASFV-F1, ASFV-R1 and the probe ASFV-F1R1-probe1 are used for detecting the sample to be detected, no amplification curve appears in 100 samples, the detection result is negative, and the detection result is the same as the detection result of the real-time fluorescent PCR method recommended by OIE.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> institute of animal health of academy of agricultural sciences of Guangdong province

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