African swine fever virus fluorescent type RAA detecting kit

A technology of African swine fever virus and detection kit, which is applied in the field of bioengineering, can solve the problems of complex operation and long time consumption, and achieve the effect of simple operation

Pending Publication Date: 2019-10-22
ZHENGZHOU ZHONGDAO BIOTECHNOLOGY CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological detection methods such as HAD, ELISA, and IFA recommended by OIE are time-consuming and complicated to operate, and are not suitable for rapid detection of African swine fever virus

Method used

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  • African swine fever virus fluorescent type RAA detecting kit
  • African swine fever virus fluorescent type RAA detecting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 The design of a kind of African swine fever fluorescent type RAA detection kit primer and probe

[0032] RAA nucleic acid amplification technology has a certain difference in primer design and conventional PCR primer design. There are two oligonucleotides to form a pair of primers, which specifically recognize the upstream and downstream nucleotide sequences of a nucleic acid target; the length is 30- Between 35 nucleotides (nt), there are no palindromic sequences, continuous single-base repeat sequences, and internal secondary structure regions in the sequence; the Tm value of the primers is not the main consideration in the design; the optimal primer pair needs to be optimized and screened through experiments out. The probe design sequence does not overlap with the specific primer recognition site, the length is 46-52nt, the sequence avoids palindromic sequences, internal secondary structures and continuous repeated bases; there are four modification site...

Embodiment 2

[0040] Embodiment 2 Construction of a recombinant vector of an African swine fever fluorescent type RAA detection kit

[0041] The full-length p72 gene of the ASFV reference gene type II Chinese isolate (accession number: MH713612.1) registered on GenBank was artificially synthesized and cloned into the pUC57 vector as a positive control.

Embodiment 3

[0042]Example 3 Establishment of a reaction system for African swine fever fluorescent type RAA detection reagent

[0043] The pUC57-p72 plasmid constructed in Example 2 was used as a template for constant temperature amplification reaction. Multiple sets of primer-probe combinations were screened, and finally the primer-probe combination described in Example 1 was used as the best combination. The reaction system in the test stage was: add 42.5 μl of hydrolysis buffer to the RAA reaction unit, add 1 μl of 20 μmol / L ASFV-F / ASFV-R, 0.5 μl of 20 μmol / L ASFV-P, and then add 2 μl of pUC57-p72 plasmid DNA as For the template, add 2.5 μL magnesium acetate solution (280 mM) to the reaction tube to start the reaction, cover the lid and centrifuge, mix up and down, centrifuge for 10 seconds, and detect immediately. The reaction program is: 40 seconds at 39°C; 40 cycles at 39°C for 30 seconds, where fluorescence signals are collected.

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Abstract

The invention discloses an African swine fever virus fluorescent type RAA detecting kit which comprises specific primers ASFV-F and ASFV-R and an RAA-exo probe ASFV-P. The basic groups in a target amplification subsequence are substituted by tetrahydrofuran (THF residues), dT-fluorophore and dT-quenching groups, a marked reported fluorescent dye is FAM, a fluorescent quenching group is BHQ-1, splitting decomposition can be conducted on the probe at the THF site by exonuclease in an agent, the fluorescent group can be separated from the quenching groups, and fluorescent signals are generated. By means of the kit, at the constant temperature of 37-42 DEG C, the African swine fever virus is detected within 20 minutes, the specificity is 100%, and the detection sensitivity is 10 copies per microliter. Thus, the kit has the advantages of being simple, rapid and sensitive in operation and provides effective technological means for on-site rapid detecting and screening of the African swine fever virus.

Description

technical field [0001] The invention relates to a fluorescent type RAA detection kit for African swine fever virus, which belongs to the technical field of bioengineering. Background technique [0002] African swine fever (ASF) is a severe, hemorrhagic infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV). The acute type is characterized by high fever, reticuloendothelial hemorrhage, and high lethality, with a case fatality rate close to 100%. The World Organization for Animal Health (OIE) lists it as a class of disease that must be declared, which is more harmful to the pig industry. In August 2018, the African swine fever epidemic was reported for the first time in Liaoning Province of my country, which marked the introduction of African swine fever into my country and caused huge losses to our pig industry. Since there is no effective vaccine for prevention and control, a rapid and accurate detection method was established becomes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/507C12Q2522/101C12Q2537/1376C12Q2563/107
Inventor 赵林萍娄亚坤杨楠付燕峰梁小红任宝红崔兴涛曾小宇
Owner ZHENGZHOU ZHONGDAO BIOTECHNOLOGY CO LTD
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