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Taqman probe fluorescent quantitative PCR kit and method for simultaneously detecting three viruses

A fluorescence quantification and kit technology, which is used in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., to achieve good specificity and stability.

Active Publication Date: 2019-12-13
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no published literature on the simultaneous detection of three viruses (African swine fever, African horse fever, and West Nile virus) using the fluorescent quantitative PCR method with Taqman probes

Method used

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  • Taqman probe fluorescent quantitative PCR kit and method for simultaneously detecting three viruses
  • Taqman probe fluorescent quantitative PCR kit and method for simultaneously detecting three viruses
  • Taqman probe fluorescent quantitative PCR kit and method for simultaneously detecting three viruses

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Effect test

Embodiment 1

[0051] Embodiment 1 (set up the Taqman fluorescent quantitative PCR detection system that detects 3 kinds of viruses simultaneously)

[0052] According to the sequences of the three viruses registered in NCBI, the sequences were compared by DNAstar software to find out the specific conserved sequences of the three pathogenic gene sequences. Using the PrimerQuest Tool tool in Integrated DNA Technologies (https: / / sg.idtdna.com / site), design specific primers and probe sequences for the specific conserved sequences of the three viral genes, and after comparison and screening, finally determine An optimal set of primers and MGB probe sequences such as Figure 1-Figure 6 Shown:

[0053] The primer pair and probe nucleotide sequence for detecting African swine fever are SEQ ID NO.1-3:

[0054] PF ASFV : 5'-CTACTCACCACGCAGAGATAAG-3';

[0055] PR ASFV : 5'-ACCATGAGCAGTTACGGAAA-3';

[0056] Probe ASFV : FAM-5'-ACAGCTCTTCCAGACGCATGTTCA-3'-TAMRA;

[0057] The primer pair and probe...

Embodiment 2

[0072] Embodiment 2 (for detecting 3 kinds of viruses Taqman fluorescent quantitative PCR kit and detection method)

[0073] The three viruses are: African swine fever, African horse sickness and West Nile virus;

[0074] The above method includes preparing a fluorescent quantitative PCR reaction system. The above reaction system includes three reaction systems, and the total amount of each PCR reaction system is 20 μL:

[0075] System 1 includes PF ASFV 0.6 μL, PR ASFV 0.6 μL, Probe ASFV 0.3 μL, the volume of Probe qPCR Mix is ​​10 μL, the volume of DNA template is 2 μL, ddH 2 The amount of O is 6.5 μL;

[0076] System II includes PF AHSV 0.6 μL, PR AHSV 0.6 μL, Probe AHSV 0.3 μL, the volume of Probe qPCR Mix is ​​10 μL, the volume of DNA template is 2 μL, ddH 2 The amount of O is 6.5 μL;

[0077] System III includes PF WNV 0.6 μL, PR WNV 0.6 μL, Probe WNV 0.3 μL, the volume of Probe qPCR Mix is ​​10 μL, the volume of DNA template is 2 μL, ddH 2 The amou...

Embodiment 3

[0079] Embodiment 3 (stability)

[0080] The samples were placed at three temperatures, respectively 4°C and 37°C, at room temperature for 6 months, and the control group used samples placed at -20°C. The results at the three temperatures were consistent with the control group. It shows that the detection method has good stability.

[0081]

[0082] The present invention determines primer pairs and probes that can simultaneously detect three viruses through research, and the PCR kit that utilizes the primer pairs and probes to make mainly includes primer pairs and fluorescent quantitative PCR probes for the three viruses. The kit can quickly and accurately detect African swine fever, African horse fever, and West Nile virus, and the sensitivity reaches 10 1 The copy number and the specificity of bovine respiratory syncytial virus, bovine coronavirus and bovine infectious rhinotracheitis virus are good, and its stability is still good after being placed at 4°C, 37°C, and r...

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Abstract

The invention discloses a Taqman probe fluorescent quantitative PCR kit and method for simultaneously detecting three viruses, and relates to the technical field of molecular biology. The three viruses are African swine fever, African horse sickness and West Nile virus; the kit comprises a PCR reaction solution, wherein the PCR reaction solution comprises three groups of primer pairs and three groups of probes. The kit can rapidly and accurately detect African swine fever, African horse sickness and West Nile virus, has sensitivity reaching 101 copies, has good specificity with bovine respiratory syncytial virus, bovine coronavirus and infectious bovine rhinotracheitis virus, and has good stability after being placed at the temperature of 4 DEG C, 37 DEG C and room temperature for 6 months.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a Taqman probe fluorescent quantitative PCR kit and method for simultaneously detecting three viruses. Background technique [0002] African swine fever (ASF) is an acute, hemorrhagic, severe infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and various wild boars (African wild boar, European wild boar, etc.). The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 100%, clinical manifestations are fever (up to 40-42 ℃), rapid heartbeat, dyspnea, partial cough, serous or mucous purulence in eyes and nose Secretion, cyanosis of the skin, obvious bleeding of lymph nodes, kidneys, and gastroint...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101Y02A50/30
Inventor 格日勒图黄天鹏包伟东
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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