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EMA-ddPCR primer and probe for detecting infectious ASFV and application

A probe and positive technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial determination / inspection, etc., can solve the problem of reducing PCR amplification efficiency and sensitivity, GC content, different base preference conformational characteristics, EMA binding Efficiency is not the same problem, to achieve the effect of shortening the test cycle, good application prospects, and reducing false positives

Active Publication Date: 2021-10-15
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When EMA enters a virus with damaged capsid protein, it will bind to the viral nucleic acid at a certain stoichiometric ratio. In theory, the target gene can be modified by EMA to inhibit the PCR amplification of the target fragment, so the longer target gene amplification fragment It will increase the probability of binding to EMA, thereby improving the inhibitory effect of EMA, but longer amplification fragments may reduce the amplification efficiency and sensitivity of PCR reactions
In addition, different regions and positions in the viral genome have different binding efficiencies with EMA due to their different GC content, base preference, and conformational characteristics.

Method used

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  • EMA-ddPCR primer and probe for detecting infectious ASFV and application
  • EMA-ddPCR primer and probe for detecting infectious ASFV and application
  • EMA-ddPCR primer and probe for detecting infectious ASFV and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Detecting infectious ASFV amplified fragment screening, lead optimization and specificity of test probes:

[0020] 1.1 Design of ASFV infection with the reference primers and probes

[0021] The applicant ASFV genome, the 27 selected target sequence to design primers and probes, designed primer probe combination designated ASFV-AC1 ~ ASFV-AC27; primer and probe sequences were SEQ ID NO.1 ~ SEQ IDNO.81 shown; e.g. ASFV-AC1 the primers shown in SEQ ID NO.1 ~ SEQ ID NO.2, SEQ ID NO.3 probe is shown; the remaining composition and so on.

[0022] 19 are simultaneously selected combination of the detection probe ASFV conventional primers, designated ASFV-ZL1 ~ ASFV-ZL15, wherein:

[0023] ASFV-ZL1 primer probe set derived from the primer and probes of claim CN2020100456541 requirements;

[0024] ASFV-ZL2 primer probe set of primers and probes derived from the P72 gene of claim 4 CN111172321A the requirements;

[0025] ASFV-ZL3 primer probe set of primers and probes derived from g...

Embodiment 2

[0076] EMA ASFV optimization method for detecting infectious: Example 2

[0077] 2.1EMA concentration optimization

[0078] An equal volume were pipetted complete inactivation of the test sample specimen, with different concentrations EMA (final concentration 0,0.25,0.5,1,1.5,2,2.5,3μg / mL) were incubated in the dark at 4 ℃ swirl 30min, the end of incubation after using the PMA-Lite TM Solutions of the sample analyzer of the LED light photolysis 20min. Tiangen using automated nucleic acid extractor to extract nucleic acid, and thus best performed using qRT-PCR reaction system and conditions obtained in Example 1 as a template. Screening can detect the lowest concentration E MA infectious ASFV purposes.

[0079] Table 4 EMA concentration optimization

[0080] EMA final concentration (μg / ml) CT value average ± standard deviation △ CT 0 33.523±0.016 0 0.25 35.923±0.128 2.4 0.5 36.237±0.155 2.714 1 36.923±0.052 3.4 1.5 38.245±0.128 4.722 ...

Embodiment 3

[0090] Example 3: Application of microfluidic chip-based detection kit infectious ASFVddPCR

[0091] This reference to the embodiments of rural China Ministry of Agriculture issued the "outbreak of African swine fever contingency embodiment (second edition 2020)" and the Chinese Academy of Agricultural Sciences released "African swine fever cleaning and disinfection Techniques (Second Edition)" mentioned ASFV standard sterilization, disinfection active substance content, disinfectant concentration and disinfectant action time analog aldehyde (formaldehyde), an alcohol disinfectant (75% ethanol), chlorine-containing disinfectant (84 disinfectant), quaternary ammonium salts disinfectant (benzalkonium chloride), a peroxide-based disinfectant (hydrogen peroxide), potassium hydrogen-based (oxone) inactivated sample (erythrocyte assay was negative after verification disinfectants as inactivated complete group), taking additional viral ASFV erythrocyte adsorption test was positive for th...

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Abstract

The invention belongs to the field of African swine fever virus detection, and provides an EMA-ddPCR primer and a probe for detecting infectious ASFV and application of the EMA-ddPCR primer and the probe for detecting the ASFV, and the ddPCR primer and the probe for detecting the ASFV can well distinguish nucleic acid positive and virus positive of the African swine fever virus and have the advantages of being high in sensitivity and good in repeatability. The primer probe and the detection method are applied to a ddPCR kit for detecting infectious ASFVs, and all experiments can be completed within 2-3 hours. Compared with a conventional infectious ASFV detection test, the method has the advantages that the test period of infectious ASFV detection is greatly shortened, and the requirements on scientific research conditions and equipment are reduced; nucleic acid and infectious virions of virus inactivation caused by virus capsid protein damage are effectively distinguished, the detection accuracy is improved, and the false positive phenomenon occurring in actual detection is reduced.

Description

Technical field [0001] The present invention relates to the field of nucleic acid detection ASFV, more particularly, it relates to EMA-ddPCR primers, probes and Detection of infectious ASFV. Background technique [0002] African swine fever (African Swine Fever, ASF) is an acute swine caused by African swine fever virus (African Swine FeverVirus, ASFV), heat resistance, highly contagious disease, the incidence and mortality rates of up to 100%. World Organization for Animal Health (OIE) to be included in the statutory report animal diseases, my country will be listed as a class of animal diseases. In 1921, ASF was originally discovered in Kenya, the fifties and sixties in Africa, Europe, South America popular last century. Because ASFV infection mechanism complex, the world has no effective preventive vaccine African swine fever, take strict biosecurity measures in the farms ASFV clear existing and prevent exposure to the virus and pigs is currently the only effective prevention ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/159C12Q2563/173
Inventor 金梅林高朔邹维华吕长杰邹忠杨丽孙小美
Owner HUAZHONG AGRI UNIV
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