PCR primer, kit and method for detecting African swine fever virus MGF-505-1R gene

An African swine fever virus and kit technology, which is applied in the field of PCR primers for detecting African swine fever virus MGF-505-1R gene, can solve complex immune escape mechanisms, large differences in biological characteristics, poor cross-immune protection ability, etc. problem, to achieve the effect of high amplification efficiency and high specificity

Pending Publication Date: 2021-05-25
LONGYAN UNIV
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Problems solved by technology

[0003] The African swine fever genome is relatively large, with 24 genotypes, the biological characteristics of different genotypes ar

Method used

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  • PCR primer, kit and method for detecting African swine fever virus MGF-505-1R gene
  • PCR primer, kit and method for detecting African swine fever virus MGF-505-1R gene

Examples

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Embodiment 1

[0025]Example 1 Screening of PCR primers

[0026]This embodiment finds a sequence of China's PIG / HLJ / 2018 (Gene II) MGF-505-1R gene from NCBI, and the nucleotide sequence, such as SEQ ID NO.5, using this sequence in the NCBI BLAST 20 similar to the poisonous strains, the BLAST results are shown in Table 1, and a simple MGF-505-1R gene sequence is found. The simple nucleotide sequence is shown as SEQ IDNO.6, and this sequence is a target sequence. Using Primer5 to set up the downstream primers MGF550-1R_F and MGF550-1R_R, as shown in Table 2, the pair primer amplify the MGF-505-1R gene, and 224 base pairs can be obtained.

[0027]Table 1 BLAST results

[0028] serial number Strain information Mn172368.1: 27747-29342 ASFV / PIG / China / CAS19-01 / 2019 MK628478.1: 27744-29339 ASFV / LT14 / 1490 LR722600.1: 28707-30302 ASFV Czechrepublic Jan-17 LR722599.1: 28710-30305 ASFV Moldova Jan-17 MK543947.1: 27792-29387 Belgium / ETALLE / WB / 2018 MK645909.1: 27751-29346 ASFV-WBBS01 LR53...

Embodiment 2

[0032]Example 2 African swine fever virus MGF-505-1R gene PCR detection kit

[0033]This embodiment provides a kit for detecting an African swine fever virus MGF-505-1R gene, comprising: a PCR mixture, a enzyme mixture, a negative control, and a positive control comprising a primer, the primer The primer 1 or primer 2 of Example 1.

[0034]The PCR mixture of this embodiment includes: TAKARA EX TAQ reaction buffer, 10 mm DNTPS, a pair of primers 1 or primer 1 or primer 2, sterilized ultrapure water.

[0035]The 10x Takara Ex TAQ Reaction Buffer, the DNTPS, the primer, and the sterilized ultrapure ultrapotrophic water solution volume ratio is 2.5: 2.5: 2.5: 11.5.

[0036]The enzyme mixture is a 10X Takara Ex TAQ enzyme having a concentration of 5 U / μL, which contains UDGase.

[0037]The positive control of the present embodiment is a cloned plasmid of a primer 1 or primer 2 amplification fragment, and the cloned plasmid is African swine fever China epidemic strain PIG / CN / HLJ / 2018.

[0038]The n...

Embodiment 3

[0039]Example 3 African swine fever virus MGF-505-1R gene PCR detection method

[0040]In this embodiment, the kit of Example 2 was used to detect the African swine fever virus MGF-505-1R gene:

[0041]1, sample nucleic acid extraction

[0042]Nucleic acid extraction: The sample genomic DNA to be tested is extracted in accordance with the instructions in accordance with the instructions.

[0043]2, the configuration of the PCR reaction system: Each detection sample corresponds to a PCR reaction tube, each of the reaction components and the added volume of each PCR reaction tube, and the upper and lower proderation of 5 μm each each 2.5 μL, 10X TAKARA EX Taq reaction buffer 2.5 μL of DNTPS 2.5 μl of DNTPS 2.5 μL, 1 mm DUTPS 2.5 μl, 10 x Takara EX Taq containing UDG enzymes, 5 μl of sterilized ultrapure water, 5 μl of sample nucleic acid extract, total volume 25 μL.

[0044]Further, in the method established in this embodiment, in order to avoid failing or contamination of the reagent used, there is...

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Abstract

The invention provides a PCR (Polymerase Chain Reaction) primer for detecting an African swine fever virus MGF-505-1R gene. The PCR primer is selected from any one of the following two pairs of primers: a primer 1 consisting of nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2; a primer 2 is composed of nucleotide sequences as shown in SEQ ID No. 3 and SEQ ID No. 4. The invention also provides a kit for detecting the African swine fever virus MGF-505-1R gene, the kit comprises a PCR mixed solution, an enzyme mixed solution, a negative control and a positive control, the PCR mixed solution comprises the primer, and the primer is the primer 1 or the primer 2. The kit provided by the invention has the advantages of high amplification efficiency, high detection sensitivity and high specificity, and can be used for detecting the African swine fever virus MGF-505-1R gene in samples such as saliva, nasal swab, blood, tissue and the like of pigs.

Description

Technical field[0001]The present disclosure relates to the field of biotechnology, and more particularly to PCR primers, kits and methods for detecting African swine fever viruses MGF-505-1R genes.Background technique[0002]African swine fever is an acute, vigorous, hemorrhagic, highly exposed to infectious disease caused by African swine fever virus, and a strong toxic strain can cause death pigs within about 5-14 days after infection, and the death rate is close to 100%. Since the African swine fever virus has a complex immune escape mechanism, lack of typical neutralizing antibodies, no effective vaccines for epidemic prevention, no effects of treatment. According to my country's national conditions and existing viral prevention and control experience, the vaccine is the most effective and economical method of preventing control of African swine fever, developing effective, safe vaccines is very urgent.[0003]The African swine fever genome is large, there are 24 genotypes, and the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848
Inventor 翁长仁林炜明
Owner LONGYAN UNIV
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