African swine fever virus P72 gene fluorescent probe PCR detection primer probe set, kit and method thereof

An African swine fever virus and detection kit technology, applied in the biological field, can solve problems such as insufficient detection sensitivity, and achieve the effects of short detection time, high detection sensitivity and high detection efficiency

Pending Publication Date: 2021-06-01
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection sensitivity of these kits is insufficient; or the primer probes designed based on individual strains can only detect a small number of virus strains

Method used

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  • African swine fever virus P72 gene fluorescent probe PCR detection primer probe set, kit and method thereof
  • African swine fever virus P72 gene fluorescent probe PCR detection primer probe set, kit and method thereof
  • African swine fever virus P72 gene fluorescent probe PCR detection primer probe set, kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Primer Design and Construction of African Swine Fever Virus pUC19-Amplified Fragment Recombinant Plasmid

[0027] Download the p72 gene sequences of 257 strains related to the African swine fever virus genotype II where the Chinese epidemic strains are located on GenBank, as shown in Table 1, screen the conserved region, and the conserved gene nucleotide sequence is shown in SEQ ID NO: 4 If there are inconsistent bases in this region, the IUPAC international standard will be used for degenerate processing. The degenerate nucleotide sequence is shown in SEQID NO: 5. Use the degenerate p72 gene sequence to design primers, and design 20 pairs of primers And supporting probes, the primers do not contain degenerate bases, and the combination with the highest specificity and sensitivity is selected from these 20 sets of primer probes as the components of the kit. Table 2 is the screened forward and reverse primers and The sequence of the probe. Using the designed f...

Embodiment 2

[0043] The fluorescent PCR detection kit of embodiment 2 African swine fever virus P72 gene

[0044] This embodiment provides a kind of African swine fever virus P72 gene fluorescent probe PCR detection kit, said kit includes the primer probe set as described in embodiment 1.

[0045] According to at least one embodiment of the present disclosure, the kit further includes 2×Taq PCR Mix.

[0046] According to at least one embodiment of the present disclosure, the kit also includes a negative control and a positive control.

[0047] According to at least one embodiment of the present disclosure, the negative control is double distilled water.

[0048] According to at least one embodiment of the present disclosure, the positive control is a cloning plasmid containing the P72 gene, and the cloning plasmid is pUC19; the nucleotide sequence of the P72 gene is shown in SEQ ID No.4.

Embodiment 3

[0049] The fluorescent PCR detection method of embodiment 3 African swine fever virus P72 gene

[0050]This embodiment uses the kit of Example 2 to detect the African swine fever virus P72 gene:

[0051] 1. Extraction of sample nucleic acid

[0052] Nucleic acid extraction: Use Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and follow the instructions to extract the genomic DNA of the sample to be tested.

[0053] 2. The configuration of the PCR reaction system: each test sample corresponds to a PCR reaction tube, each reaction component in each PCR reaction tube and the volume added: the total reaction volume of PCR amplification is 25 μl, including 12.5 μl of 2 ×Taq PCRMix, 1.25 μl of 10 μM forward primer and 1.25 μl of 10 μM reverse primer, 0.5ul of 10 μM specific fluorescent probe, 5 μl of template nucleic acid sample, 4.5 μl of ultrapure water.

[0054] Further, in order to avoid the failure or c...

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Abstract

The invention provides an African swine fever virus P72 gene fluorescent probe PCR detection primer probe set, the nucleotide sequence of a forward primer of the primer probe group is shown as SEQ ID NO.1, the nucleotide sequence of a reverse primer of the primer probe group is shown as SEQ ID NO.2, and the nucleotide sequence of a specific fluorescent probe is shown as SEQ ID NO.3, the 5'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3 'end of the specific fluorescent probe is marked with a fluorescent quenching group. The invention further provides the African swine fever virus P72 gene fluorescent probe PCR detection kit and the method thereof. The kit and the detection method thereof disclosed by the invention have the advantages of wide coverage range, short detection time and high detection efficiency, and can be used for detecting the African swine fever virus in samples such as saliva, nasal swabs, blood and tissues of pigs.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular to a fluorescent probe PCR detection primer probe set, kit and method for African swine fever virus P72 gene. Background technique [0002] African swine fever is an acute, virulent, hemorrhagic, highly contagious infectious disease of pigs caused by African swine fever virus. The virulent strain can kill domestic pigs within about 5-14 days after infection, and the lethality rate is close to 100%. Since African swine fever virus has a complex immune evasion mechanism, lacks typical neutralizing antibodies, has no effective vaccines for epidemic prevention, and has no specific therapeutic drugs, the establishment of a rapid and accurate detection method is crucial for the timely and effective prevention and control of African swine fever virus. Pestilence appears to be particularly important. The development of assays that can detect as many strains as possible is essential. [0003]...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2563/107
Inventor 翁长仁邱龙新
Owner LONGYAN UNIV
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