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Nucleic acid extraction-free fluorescent isothermal amplification detection kit for African swine fever viruses

An African swine fever virus and isothermal amplification technology, which is applied in the field of thermal amplification detection technology and complete kits, can solve the problems of poor accuracy and strong subjectivity, and achieve the effects of easy and accurate judgment, fast speed and simplified detection operation

Active Publication Date: 2019-09-13
BEIJING SENKANG BIOTECHNOLOGY DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing nucleic acid isothermal amplification method mainly relies on the naked eye to observe the color change of the reaction solution to determine the result, which is highly subjective and poor in accuracy.

Method used

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  • Nucleic acid extraction-free fluorescent isothermal amplification detection kit for African swine fever viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Implementation Example 1: Primer Design and Construction of African Swine Fever Virus pMD20-T-VP72 Gene Recombination Plasmid

[0030] 1. Primer design: According to the p72 gene sequences of 24 genotype reference strains of African swine fever virus included in GenBank (see Table 1), the conserved regions were screened, and 8 pairs of 16 primers were designed, compared and screened.

[0031] Table 1 African swine fever reference strains

[0032]

[0033] The nucleotide sequence of the final determined isothermal amplification primer is as follows:

[0034]ASFB3: 5'-GTAGACGCAATATACGCTTTA-3'; SEQ ID NO.1;

[0035] ASFF3: 5'-GCCATTTAAGAGCAGACATT-3'; SEQ ID NO.2;

[0036] ASFBIP: 5'-GTGTATTTCAGGGGTTACAAACAGGTTTTGGAGTCATTAATGAAATCTCGC-3'; SEQ ID NO. 3;

[0037] ASFFIP: 5'-GTAAAACGCGTTCGATTTTCCCTTTTGTGGTGGTTATTGTTGGTGT-3'; SEQ ID NO.4;

[0038] ASFLB: 5'-GATGTAAAGTTCATTATTCGTG-3'; SEQ ID NO.5;

[0039] ASFLF: 5'-TGATACGTGTCCATA-3'; SEQ ID NO.6.

[0040] 2. Construct...

Embodiment 2

[0041] Embodiment 2 Establishment of African swine fever virus fluorescence isothermal amplification reaction system

[0042] By comparing and analyzing the VP72 gene sequence of African swine fever virus, designing and screening primers, and preliminarily establishing a fluorescent isothermal amplification detection method for African swine fever virus, and the primer concentration, Mg 2+ Concentration, amount of SYBR GreenI fluorescent dye, amount of Bst DNA polymerase, reaction temperature and other reaction conditions were optimized to determine the optimum conditions for the detection method:

[0043] The reaction system is 25 μL in total, including three parts: fluorescent isothermal amplification reaction solution, Bst DNA polymerase, and template. Among them, a detection reaction solution consists of 2.5 μL of 10 × Thermopolbuffer, 5.5 μL of primer solution, 4 μL of 5 mol / L betaine solution, 3.5 μL of dNTP Mix (10 mmol / L), 1.5 μL of MgSO 4 (100 mmol / L), 0.5 μL of SYBR...

Embodiment 3

[0045] Example 3 Assembly of African swine fever virus nucleic acid extraction-free fluorescence isothermal amplification detection kit

[0046] 1. Prepare the components of the kit as follows:

[0047] (1) Primer solution preparation: Dilute the artificially synthesized primers ASFF3, ASFB3, ASFFIP, and ASFBIP to 20 μmol / L with DEPC-treated water, and dilute the artificially synthesized primers ASFLF and ASFLB to 40 μmol / L with DEPC-treated water, according to ASFF3:ASFB3: Mix the diluted primers at the ratio of ASFFIP:ASFBIP:ASFLF:ASFLB=0.25:0.25:2:2:0.5:0.5.

[0048] (2) Preparation of positive control: the p72 gene of African swine fever virus was cloned by conventional methods, the pMD20-T-VP72 recombinant plasmid was constructed, and the p72 gene in the plasmid was sequenced. After the sequencing result is correct, the recombinant plasmid bacteria obtained by transforming Escherichia coli is expanded and cultured to extract the recombinant plasmid. Use the Nanodrop2000...

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Abstract

The invention selects a highly conserved p72 gene of African swine fever viruses as a detection target gene to design specific primers, establish a fluorescent isothermal amplification detection method, and develop a nucleic acid extraction-free detection kit for African swine fever viruses. A fluorescent dye is used for indicating an amplification reaction result, thereby facilitating accurate determination; a nucleic acid extraction-free reaction solution is used for processing samples without extracting the DNA of the samples, thereby simplifying the test operation, reducing the cross contamination and lowering the cost; the whole detection process is fast and only requires 50 min; multiple samples can be detected at one time; a used fluorescent isothermal amplification instrument has low cost; and the kit provided by the invention has the advantages of high amplification efficiency and good specificity, and is suitable for use in on-site and grass-roots units for pig breeding, slaughtering, quarantining and monitoring.

Description

technical field [0001] The invention relates to an African swine fever virus nucleic acid fluorescence isothermal amplification detection technology and a complete kit, belonging to the technical field of microbial detection. Background technique [0002] African swine fever (ASF) is a severe infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and various wild boars. After infection with a strong virus strain, it can lead to 100% mortality of animals. The disease has a great impact on the global pig industry. cause huge threats and economic losses. The disease is listed as an animal disease that must be reported by the World Organization for Animal Health (OIE), and it is also a type of animal disease that our country focuses on prevention. On August 3, 2018, the first African swine fever epidemic was confirmed in China, and the disease has occurred in most provinces of my country since then. Since a vaccine that can effectively prevent Af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 杨鹏倪建强王传彬杨林周德刚丁凯刘洋黄思斯吴子函
Owner BEIJING SENKANG BIOTECHNOLOGY DEV CO LTD
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