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Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis viruses

A primer composition and the technology of Mycoplasma bovis, which are applied in the field of primer compositions for detecting Mycoplasma bovis and bovine infectious rhinotracheitis virus, can solve the problems of indeterminate results, complex instruments, and low detection sensitivity, and achieve suppression of false positive results, High specificity, good specific effect

Inactive Publication Date: 2020-03-13
GUANGXI VETERINARY RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the multiple LAMP methods in China have certain limitations, and it is impossible to determine which positive reaction is the result, and it is not a differential diagnosis in the true sense.
Patent application CN107447056A discloses a method for the identification and detection of Mycoplasma bovis and bovine infectious rhinotracheitis, but the sensitivity of this detection method is low, 100 copies / reaction, and the result can only be seen after the reaction product is subjected to electrophoresis, which takes an hour , and it is very easy to contaminate the laboratory
Patent application CN106868215A also discloses a method for the identification and detection of Mycoplasma bovis and bovine infectious rhinotracheitis. The detection sensitivity is also low, 10,000 copies / reaction, and the operation is complicated, and the instruments used are also complicated, which must be completed in a laboratory detection

Method used

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  • Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis viruses
  • Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis viruses
  • Primer composition, kit and method for detecting mycoplasma bovis and infectious bovine rhinotracheitis viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the design of primer composition

[0025] Download the tk gene of IBRV and the opp D / F gene sequence of MB registered on Genebank, use MEGA5.0 for conservation and homology analysis, select a well-conserved region, and use primer5.0 and Primer explore V4 software Two sets of LAMP-specific primers were designed to form the primer composition. Each set includes 4 primers and 1 probe: outer primers F3 and B3, inner primers FIP and BIP, probe Probe. The two probes Probe are respectively labeled with different fluorescent groups: MB-probe is labeled with CY 5.5 fluorescence at the 5' end of the nucleotide sequence shown in SEQ ID No: 5 in the sequence listing, and BHQ3 quencher group at the 3' end , it is bright red at a wavelength of 694nm; IBRV-probe is marked with FITC fluorescence at the 5' end of the nucleotide sequence shown in SEQ ID No: 10 in the sequence listing, and the 3' end is marked with a BHQ1 quencher group, at a wavelength of 520nm It is yell...

Embodiment 2

[0028] Example 2, Verification Test of Dual Fluorescent LAMP Detection Method for Mycoplasma bovis and Bovine Infectious Rhinotracheitis

[0029] 1. Extraction of RNA / DNA template

[0030] According to the instructions of the whole-type gold RNA / DNA co-extraction kit, the total DNA of MB and IBRV was extracted, and the total RNA of the tested strains BVDV, FMDV, VSV, BTV, BRV, and PPRV was extracted at the same time. The extracted total RNA was extracted with whole-type gold Reverse transcription kit Reverse transcription into cDNA. The reverse transcription reaction system is 10 μL: 5×RT buffer 2 μL, 50 μmol / L Random 9mer 0.5 μL, 5U AMV reverse transcriptase 0.5 μL, RNA inhibitor 0.5 μL, 10 μmol / L dNTPs 0.5 μL, RNA template 2 μL.

[0031] 2. Dual fluorescence LAMP reaction system and reaction conditions

[0032] The optimal reaction system for dual fluorescent LAMP is: 1μL DNA template, 2.5μL 10×buffer, 15U of BstDNA 3.0 polymerase, internal primers (MB-FIP, MB-BIP, IBRV-FI...

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Abstract

The invention discloses a primer composition, a kit and a method for detecting mycoplasma bovis and infectious bovine rhinotracheitis viruses. The primer composition designed and screened in the invention comprises two groups of specific primers and probes. Under the condition of an optimized reaction system and under optimized reaction conditions, mycoplasma bovis and infectious bovine rhinotracheitis viruses can be simultaneously identified and detected in the same reaction tube; and the primer composition, the kit and the method have the characteristics of good specificity, high sensitivity, interference resistance, no pollution, convenience and quickness in operation, low cost and the like.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a primer composition, a kit and a method for detecting mycoplasma bovis and bovine infectious rhinotracheitis virus Background technique [0002] Infectious bovine rhinotracheitis virus (IBRV) and Mycoplasma bovis (MB) are the two main common pathogens of bovine respiratory syndrome, and are one of the most serious diseases that endanger the cattle industry. The development of the cattle industry. Infectious Bovine Rhinotracheitis virus (IBRV) mainly causes high fever, rhinitis, dyspnea and abortion in cattle. MB (Mycoplasma bovis, MB) belongs to the Mycoplasma family and the genus Mycoplasma. It mainly causes calf pneumonia, mastitis, keratitis, arthritis and other diseases, and respiratory system diseases are the most common. Because MB has the characteristics of persistent existence and conditional pathogenicity in cattle, the diseases caused by it have been lacking ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/35C12R1/93
CPCC12Q1/6844C12Q1/689C12Q1/705C12Q2531/119C12Q2563/107
Inventor 谢芝勋范晴谢志勤谢丽基熊文婕黄娇玲张艳芳曾婷婷王盛罗思思邓显文刘加波
Owner GUANGXI VETERINARY RES INST
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