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RT-PCR primer for rapidly detecting Ferlavirus and application of RT-PCR primer

A RT-PCR, virus technology, applied in the field of animal bacteriology and molecular biology, can solve the problem of insufficient sensitivity, and achieve the effect of high sensitivity and accurate detection results

Inactive Publication Date: 2018-07-20
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are primers designed according to the target genes of Ferlavirus F, HN, U, etc. for RT-PCR detection, but since there are at least three subgroups of Ferlavirus, most of these methods are limited to a certain subgroup, and the sensitivity still exists. insufficient

Method used

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  • RT-PCR primer for rapidly detecting Ferlavirus and application of RT-PCR primer
  • RT-PCR primer for rapidly detecting Ferlavirus and application of RT-PCR primer
  • RT-PCR primer for rapidly detecting Ferlavirus and application of RT-PCR primer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 RT-PCR rapid detection method construction

[0044] 1.1 Materials and methods

[0045] 1.1.1 Materials

[0046] 1.1.1.1 Strains

[0047] The strains of three different subgroups (A, B, C) of Ferlavirus virus are preserved by the pharmacology laboratory of the College of Animal Science and Technology of Guangxi University. Avian influenza, canine parvovirus, and canine distemper virus are commercially available vaccines. Newcastle disease virus is produced by Guangxi The poultry disease laboratory of the College of Animal Science and Technology of the University provided, and the reovirus was provided by the Guangxi Veterinary Research Institute.

[0048] 1.1.1.2 Main instruments and reagents

[0049] Gradient PCR instrument from TaKaRa Company in Japan, gel imaging system from Alpha Inotech Company in the United States, and micro-volume UV spectrophotometer from Thermo Company in the United States. Viral genome RNA extraction kits, PCR reagents, DNA Mar...

Embodiment 2

[0078] Embodiment 2 Experimental results

[0079] 2.1 Amplification, cloning and sequencing of the target gene

[0080] Use the designed specific primers to carry out RT-PCR amplification on the RNA of the three subgroups (A, B, C) strains of Ferlavirus virus, such as figure 1 As shown, after electrophoresis of RT-PCR products, fragments with the same length as expected amplified appeared. After the RT-PCR product was electrophoresed and recovered by agarose gel, it was connected with the pUC-T vector overnight and transformed into Escherichia coli DH5α. The obtained positive plasmid was identified by PCR and double enzyme digestion, and then sent to Beijing Huada Gene Sequencing Co., Ltd. for sequencing. The sequencing result was completely consistent with the reference sequence, confirming that the primers can specifically amplify the L gene of the Ferlavirus virus.

[0081] 2.2 Optimization of RT-PCR reaction conditions

[0082] Such as figure 2 As shown, the RT-PCR an...

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Abstract

The invention discloses an RT-PCR primer for rapidly detecting Ferlavirus and application of the RT-PCR primer, and belongs to the technical field of animal bacteriology and molecular biology. The RT-PCR primer is composed of an upstream primer having a nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2; and the primer is used toprepare an RT-PCR kit for detecting Ferlavirus. An RT-PCR detection method provided by the kit has high specificity and sensitivity, high repeatability, and high credibility, detection results are quickly and accurately acquired, and the method is low in cost and simple in operation, overcomes low separation rate, time and labor consumption and other deficiencies of a traditional identification method, is suitable for use at a base level, and can be used as a rapid, accurate and simple detection tool for laboratorial rapid detection and large-scale epidemiological investigation of Ferlavirus.

Description

technical field [0001] The invention relates to the technical fields of animal bacteriology and molecular biology, in particular to a fast, simple and low-cost RT-PCR primer for detecting Ferlavirus virus and its application. Background technique [0002] In 1972, a spearhead snake (Ferdelance) in a snake exhibition hall in Switzerland had dyspnea, lethargy, and neurological symptoms, and the mortality rate reached 30%. After that, D.W. Folsch isolated a strain of paramyxovirus from a spearhead snake, which was the first The first report of a paramyxovirus isolated from a reptile. Because it was isolated from the spearhead snake, the virus strain was named Fer-de-Lance (FDLV). In 2011, the International Committee on Taxonomy of Viruses agreed to add a new paramyxovirus genus, Ferlavirus, to the representative strain represented by FDLV. [0003] Ferlavirus mainly causes respiratory system diseases in snakes. The clinical features mainly manifest different degrees of dyspne...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2521/107
Inventor 曾芸苏接瑜李睿君
Owner GUANGXI UNIV
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