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African swine fever virus wild virus infection and gene deletion virus strain dual fluorescence quantitative PCR detection composition, method and kit

An African swine fever virus and detection kit technology, applied in the biological field, can solve the problems affecting the early differential diagnosis of African swine fever virus infection, inability to distinguish between the positive of wild virus infection and the positive of vaccine immunity, and achieves good social application prospects, good Consistent, well-specific results

Active Publication Date: 2020-09-18
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, gene deletion vaccines based on MGF360 and MGF505 gene deletions have great social application prospects, but the existing detection methods are all based on the development and establishment of African swine fever wild virus infection, and this type of gene deletion vaccine will be put into use in the future After that, the existing detection methods will not be able to distinguish between positive wild virus infection and positive vaccine immunity, seriously affecting the early differential diagnosis of African swine fever virus infection

Method used

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  • African swine fever virus wild virus infection and gene deletion virus strain dual fluorescence quantitative PCR detection composition, method and kit
  • African swine fever virus wild virus infection and gene deletion virus strain dual fluorescence quantitative PCR detection composition, method and kit
  • African swine fever virus wild virus infection and gene deletion virus strain dual fluorescence quantitative PCR detection composition, method and kit

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Embodiment 1

[0043] Design and synthesis of embodiment 1 primers and probes

[0044] According to the published ASFV genome sequence in NCBI GenBank data, the B646L and MGF505-2R gene sequences of the popular strain Pig / HLJ / 2018 (Accession no.MK333180) in my country were selected (Table 1), and Primer3Plus (http: / / www. primer3plus.com) software for primer and probe design. Primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. Primers and probes were diluted to 10 μmol / L with sterile water and stored at -20°C.

[0045] Table 1 Primer and Probe Information

[0046]

[0047] Further use the DNAstar Megalign software to evaluate the sequence conservation of the primers and probes in the prevailing strains, the selected strains are shown in Table 2, and the results are shown in figure 1 . Depend on figure 1 B646L and MGF505-2R gene sequence comparison analysis shows that the primers and probe sequences designed by the present invention are highly conserved amon...

Embodiment 2

[0050] The selection of positive control in embodiment 2 kit

[0051] The standard plasmids pUC57-B646L and pUC57-MGF505-2R were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and kept in our laboratory. The standard plasmid concentration was determined by NanoDrop One (Thermo Fisher Scientific). The gene copy number calculation formula is as follows: Y (copies / μL) = [concentration of plasmid DNA (ng / μL) × 10 -9 / (length of plasmid DNA bp×660)]×6.02×10 23 . It was determined that the gene copy numbers of the standard plasmids were 2.9×10 9 copies / μL (pUC57-B646L) and 1.5×10 9 copies / μL (pUC57-MGF505-2R).

Embodiment 3

[0052] The establishment of embodiment 3 double fluorescence quantitative PCR detection method

[0053] (1) The composition of the dual fluorescent quantitative PCR detection kit

[0054] Dual fluorescence quantitative PCR detection kit, including probe method fluorescence quantitative detection reagent (probe method qPCR reaction premix reagent Premix Ex Taq); internal reference dye ROX Reference Dye II; upstream primer B646L-F1, downstream primer B646L-R1, probe Needle B646L-P1, upstream primer MGF505-2R-F1, downstream primer MGF505-2R-R1, probe MGF505-2R-P1; positive control: standard plasmid pUC57-B646L and pUC57-MGF505-2; negative control: double distilled water .

[0055] (2) Establishment of standard curve

[0056] After preliminary screening, it was determined that the reaction system for dual fluorescent quantitative PCR was 25 μL, including 2 μL of the above-mentioned primer mixture and 1 μL of the probe mixture (the final concentration of each primer and probe was...

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Abstract

The present invention relates to an African swine fever virus wild virus infection and gene deletion virus strain dual fluorescence quantitative PCR detection composition, method and kit. A TaqMan probe fluorescence quantitative detection method is used, the dual fluorescence quantitative PCR detection method for a B646L gene and a MGF505-2R gene is established, and the method can effectively distinguish a wild virus infection and vaccine strains containing MGF505 gene deletion. The method has good specificity, sensitivity and reproducibility and provides a good technical support for differential diagnosis of African swine fever virus wild viruses and vaccine viruses. Clinical sample testing shows that the method and a Thermo Fisher African swine fever detection kit have relatively good consistent results when detecting the wild virus infection. Besides, the method can distinguish the MGF505 gene deletion strain and has a good social application prospect.

Description

technical field [0001] The invention relates to a double fluorescent quantitative PCR detection composition, method and kit for African swine fever virus wild virus infection and gene deletion strain, and belongs to the field of biotechnology. Background technique [0002] African swine fever (ASF) is a highly contagious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV). rates and high mortality. The World Organization for Animal Health lists it as a legally notifiable animal disease; my country lists it as a first-class animal disease. ASFV was first reported in Kenya in 1921, and then spread widely in most African countries; historically, ASFV was prevalent in Central and Western Europe (France, Spain, Portugal, etc.) and the Americas (Brazil), and was then controlled and purified. In 2007, African swine fever entered Georgia in the Caucasus region, and then became endemic in Eastern European countries and Russia. In August 2018, the Afr...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/101C12Q2545/113C12Q2561/101
Inventor 张改平郭振华金前跃孙亚宁李青梅杨继飞王方雨乔松林郭军庆
Owner HENAN ACAD OF AGRI SCI
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