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Detection primer, kit and detection method of infectious bovine rhinotracheitis virus

A rhinotracheitis virus and detection kit technology, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as cumbersome operation, difficulty in detecting latent infection in vivo, and restrictions on promotion.

Inactive Publication Date: 2015-06-24
HENAN UNIV OF ANIMAL HUSBANDRY & ECONOMY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Serological methods mainly include serum neutralization test, agar diffusion test and enzyme-linked immunosorbent assay, etc., which can be used for IBRv antibody detection, but they are often cumbersome to operate and cannot detect pathogens, making it difficult to detect latent infections in vivo
Etiological detection technology mainly includes virus isolation and identification, nucleic acid detection (PCR, qPCR and nucleic acid probe technology). Although this method can directly detect viruses, it often requires certain experimental equipment and technical requirements. Room promotion has certain restrictions

Method used

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Experimental program
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Effect test

Embodiment 1

[0035] According to the IBRV virulence gene gE sequence, the present invention comprehensively considers its conserved region, whether it forms a secondary structure, G+C content, base random distribution, length and many other factors, and undergoes necessary modifications and a large number of The experiment verified that LAMP primers were designed and synthesized. The primers for the rapid detection of bovine infectious rhinotracheitis virus include a pair of outer primers and a pair of inner primers. The outer primers are F3: 5′-GTGCGTCTGCAGTCTGAG-3′, B3: 5′ -GCTGTACACGGTCTCGGAG-3', internal primers are FIP: 5'-ATGCGGATGAGCGCGCAGTCTTTTTCGACGAGGCTCCCT-3', BIP: 5'-ACGAGACGTGCATCTTCCACCGTACGGCGACGCGAAG-3'.

Embodiment 2

[0037] A detection kit for bovine infectious rhinotracheitis virus, comprising an amplification reaction system, consisting of the following raw materials:

[0038] 2×LAMP Mix 10μL,

[0039] FIP final concentration 0.8μmol / L,

[0040] BIP final concentration 0.8μmol / L,

[0041] F3 final concentration 0.2μmol / L,

[0042] B3 final concentration 0.2μmol / L,

[0043] 25mmol / L of MgCl 2 3 μL,

[0044] 8U / μL Bst DNA polymerase 1.5μL,

[0045] 1 μL of LAMP visible light dye (3mmol / L hydroxynaphthol blue), and distilled water as the rest to make up to 19 μL.

Embodiment 3

[0047] A detection method for bovine infectious rhinotracheitis virus, comprising the following steps:

[0048] (1) Select a commercial viral DNA extraction kit (Shanghai Sangon Bioengineering Co., Ltd.) to extract viral genomic DNA from the tissue to be tested;

[0049] (2) Add 1 μL of the obtained genomic DNA (net content 1ng) into the amplification reaction system: 2×LAMP Mix 10 μL, FIP final concentration 0.8 μmol / L, BIP final concentration 0.8 μmol / L, F3 final concentration 0.2 μmol / L, B3 final concentration Concentration 0.2μmol / L, 25mmol / L MgCl 2 3 μL, 1.5 μL of 8 U / μL Bst DNA polymerase, 1 μL of LAMP visible light dye (hydroxynaphthol blue, 3 mmol / L), make up to 20 μL with distilled water;

[0050] (3) LAMP amplification reaction: Mix all reactants in the system, place at 60°C for 2 hours, and then inactivate at 80°C for 10 minutes;

[0051] (4) Test result: direct observation, if the tissue to be tested carries bovine infectious rhinotracheitis virus, LAMP amplific...

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Abstract

The invention relates to a detection primer, a kit and a detection method of an infectious bovine rhinotracheitis virus. The detection primer comprises a pair of external primers and a pair of internal primers, wherein the external primers are F3:5'-GTGCGTCTGCAGTCTGAG-3', and B3:5'-GCTGTACACACGGTCTCGGAG-3'; and the internal primers are FIP:5'-TGCGGATGAGCGCGCAGTCTTTTTCGACGAGGCTCCCT-3', and BIP:5'-ACGAGACGTGCATCTTCCACCGTACGGCGACGCGAAG-3'. The invention further designs a kit and a loop-mediated isothermal amplification detection method aiming at the detection primer. The detection primer is good in rapid primer detection specificity; and the detection method is fast, simple, convenient, accurate and cheap, and is especially suitable for basic clinical veterinarians.

Description

technical field [0001] The invention belongs to the field of animal inspection and quarantine, and in particular relates to a detection primer, a kit and a detection method for bovine infectious rhinotracheitis virus, and is especially suitable for use by grass-roots clinical veterinarians. Background technique [0002] In recent years, with the increase in the number of dairy cows and the increase in breeding density, the incidence of bovine infectious rhinotracheitis (Infectious Bovine Rhinotracheitis, IBR) is increasing. IBR is an acute, febrile, and contagious infectious disease of cattle, which is listed as a B-type animal infectious disease by the World Organization for Animal Health. The disease is caused by Infectious Bovine Rhinotracheitis virus (IBRv). The clinical manifestations of cattle are inflammation of the upper respiratory tract and tracheal mucosa, dyspnea, runny nose, reproductive tract infection, abortion, mastitis and meningitis. Reports have shown tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/70C12Q1/6844
Inventor 皇甫和平石冬梅许文博
Owner HENAN UNIV OF ANIMAL HUSBANDRY & ECONOMY
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