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Real-time fluorescent quantitative PCR (polymerase chain reaction) detecting reagent kit for orf virus

A technology for real-time fluorescence quantification and oral ulcer virus, which is applied in the field of virus molecular biology detection

Inactive Publication Date: 2012-09-19
GUANGZHOU BONIZZI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, no real-time fluorescent PCR kit for detecting aphthus virus has been developed at home and abroad, so the development of this kit has become a top priority

Method used

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  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detecting reagent kit for orf virus
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detecting reagent kit for orf virus
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detecting reagent kit for orf virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design of primers and Taqman probes for real-time fluorescent quantitative PCR detection of aphthous ulcer virus

[0037] Referring to the full genome sequence of the ORFV NZ2 strain of oropharyngeal virus (GenBank accession No.DQ184476.1) collected by GenBank, select the conserved region of the ORFV 024 gene, which is [ORFV NZ2 strain genome (GenBank accession No.DQ184476.1) 23758bp-23844bp fragment , using ABI Primer Express 3.0 and Primer 5.0 primer design software to design and synthesize real-time fluorescent quantitative PCR primers and probes. At the 5' end of the probe, FAM was selected as the fluorescent labeling reporter luminescent group, and at the 3' end Tamara was used as the quenching group.

[0038] The sequence is as follows:

[0039] Upstream primer (ORFV-Forward primer): 5'-TCTGTGCATGCTCAAGATCCT -3' (SEQ ID NO: 1 in the sequence listing);

[0040] Downstream primer (ORFV-Reverse primer): 5'-GCCAAGTTGCAGGTTTTTCTT -3' (SEQ ID NO: 2 in the ...

Embodiment 2

[0042] Example 2 Real-time fluorescent quantitative PCR detection of aphthous ulcer virus

[0043] 1. Establishment of standard curve

[0044] 1. Construction of pET21b-ORFV024 recombinant plasmid

[0045] (1) Clone the target gene ORFV024 gene of sheep oral disease virus

[0046] Strictly follow the requirements in the P2 laboratory negative pressure biosafety cabinet for the extraction of aphthus virus genomic DNA. The specific steps are as follows: collect the lip scabs of sheep infected with aphthous ulcer virus under aseptic conditions, grind them with a mortar, resuspend the powder in normal saline, take 200 μL of the suspension containing the virus, and use Roche High Pure Viral Nucleic Acid Kit ( Purchased from Roche Company, USA) According to the instructions, viral genomic DNA was extracted.

[0047] The PCR reaction system was 50 μL, and the following components were added in sequence: 5 μL 10×PCR buffer (purchased from Promega, USA), 2.0 mM MgCl 2 , 0....

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Abstract

The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detecting reagent kit for an orf virus and belongs to the technical field of viral molecular biology detection. The real-time fluorescent quantitative PCR detecting reagent kit for the orf virus comprises a pair of primers of nucleotide sequences shown in SEQ ID NO:1-2 and a TaqMan probe of a nucleotide sequence shown in SEQ ID NO:3. The detecting reagent kit can quickly and accurately detect the orf virus from samples and is high in detecting sensitivity and simple and convenient to sample, detecting efficiency is greatly improved, pollution can be effectively prevented, and the detecting reagent kit is of practical significance in the field of inspection and quarantine of import and export animals.

Description

technical field [0001] The invention relates to a molecular biology detection method for viruses in the field of biotechnology, in particular to a real-time fluorescence quantitative PCR detection kit for aphthus virus which can be used for early and rapid diagnosis of aphthus virus. Background technique [0002] Orf, also known as contagious ecthyma, is an acute contagious disease of goats and sheep caused by Orf virus (ORFV). It is characterized by the formation of erythema, papules, abscesses, ulcers, and thick warty scabs on the nose and breasts. ORFV mainly infects goats and sheep, and can also infect humans and other animals. It is a zoonotic disease and is a legally notifiable infectious disease stipulated by the World Organization for Animal Health. Oral sore is widely distributed in many sheep-raising countries, and it is also more common in places where there are more sheep in northern my country and northwest China. It is one of the main diseases of sheep. Due t...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 杜红延罗树红郝文波李明
Owner GUANGZHOU BONIZZI BIOTECH CO LTD
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