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Real-time fluorescent PCR (polymerase chain reaction) detection primer probe set, kit and method for African swine fever virus CD2V gene

An African swine fever virus and real-time fluorescence technology, applied in the field of molecular biology, can solve the problems of biosafety of natural attenuated strains and passaged attenuated strains, and achieve the effect of avoiding cap-opening pollution, simple identification, fast and efficient methods

Active Publication Date: 2020-01-17
南宁壮博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A large number of experimental results show that: natural attenuated strains and passage attenuated strains still have problems in biosafety

Method used

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  • Real-time fluorescent PCR (polymerase chain reaction) detection primer probe set, kit and method for African swine fever virus CD2V gene
  • Real-time fluorescent PCR (polymerase chain reaction) detection primer probe set, kit and method for African swine fever virus CD2V gene
  • Real-time fluorescent PCR (polymerase chain reaction) detection primer probe set, kit and method for African swine fever virus CD2V gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the design of the fluorescent PCR detection primer probe group of African swine fever virus CD2V gene

[0033] In this example, referring to the African swine fever virus CD2V gene sequence published on GenBank, multiple sequences were compared using DNAMAN 6.0 software. A large number of experiments have shown that different primers have a certain impact on the effect and sensitivity of PCR amplification. Therefore, this study preliminarily designed three sets of primer probes for the target gene fragments: CD2V-1, CD2V-2 and CD2V-3 (primer sequences are shown in Table 1), and the sequences of the three sets of primer probes can be compared with African swine fever The corresponding sequence of the viral CD2V gene specifically binds. Wherein, the sizes of the target fragments amplified by the above three sets of primers are 145bp, 106bp, and 164bp respectively.

[0034] Table 1 CD2V-1, CD3V-2 and CD2V-3 sequence information

[0035]

[0036] The 3 g...

Embodiment 2

[0037] Embodiment 2: Fluorescent PCR detection kit and detection method of African swine fever virus CD2V gene

[0038] This embodiment provides a fluorescent PCR detection kit for African swine fever virus CD2V gene, which includes a PCR reaction mixture, a positive control substance and a negative control substance.

[0039] The components of the PCR reaction mixture and the final reaction concentrations of each component are as follows: 1*PCR Buffer, 1mmol / LdNTP, 2.5mmol / L MgCl 2 , 400nmol / L primer mixture, 100nmol / L specific fluorescent probe, 3U DNA polymerase, 0.5U UNG enzyme.

[0040] In the primer mixture described in this embodiment, the base sequence of the forward primer is shown in SEQ ID No.4, and the base sequence of the reverse primer is shown in SEQ ID No.5, wherein the forward primer The molar ratio of primer and reverse primer is 1:1.

[0041] The specific probe base sequence of the African swine fever virus CD2V gene provided in this example is shown in SE...

Embodiment 3

[0055] Embodiment 3: Apply the kit of the present invention to carry out actual sample detection

[0056] In order to verify that the primers, probes, and reagents of the present invention can be used normally in actual conditions, the actual conditions are simulated, and the primers, probes, and reagents of the present invention are used to detect actual samples. The actual samples were provided by the Animal Health Testing and Evaluation Center of Qingdao Agricultural University, a total of 12 samples, of which 4 were ASFV wild strain positive pig serum, 4 were CD2V gene deletion vaccine strain positive pig serum, 4 were normal pig (wild virus Both strain and vaccine strain were negative) serum. The detection result was consistent with the known PCR detection result, and the coincidence rate was 100%.

[0057] The invention has the advantages of rapid, real-time, sensitive and accurate identification of wild ASFV strains and vaccine strains.

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Abstract

The invention belongs to the technical field of molecular biology, and discloses a real-time fluorescent PCR (polymerase chain reaction) detection primer probe set, kit and method for African swine fever virus CD2V gene A nucleotide sequence of a forward primer of the primer probe set is shown as SEQ ID NO. 4, a nucleotide sequence of a reverse primer is shown as SEQ ID NO. 5, and a nucleotide sequence of a specific fluorescent probe is shown as SEQ ID NO. 6, wherein a fluorescent reporter group is marked at the 5' end of the specific fluorescent probe, while a fluorescent quenching group is marked at the 3' end of the specific fluorescent probe. The primer probe set and kit provided by the invention help quickly, conveniently, efficiently and specifically detect whether a sample containsAfrican swine fever virus and whether the sample is identified as CD2V gene deletion, and are suitable for clinical identification detection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a fluorescent PCR detection primer probe set, a kit and a detection method of African swine disease virus CD2V gene nucleic acid. Background technique [0002] African swine fever (ASF) is an acute porcine infectious disease with high morbidity and mortality, which has a huge health and socioeconomic impact on the international trade of pigs and pig products. The epidemiology of ASF is very complex, and it can be transmitted through many routes, such as feed, transportation, insect vectors and so on. The causative agent of the disease, African swine fever virus (ASFV), is the only member of the African swine fever virus family and belongs to Asfivirus. It is a complex enveloped virus with an icosahedral morphology consisting of four concentric layers and a large double-stranded DNA molecule, between 170 and 193 kbp in length. [0003] Vaccination is one of...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2563/107
Inventor 黄震巨李春燕李沛林晓滕丽琼
Owner 南宁壮博生物科技有限公司
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