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PCR primers directed at 5'-UTR gene and used for detecting short beak and dwarfism syndrome-goose parvovirus

A technology for detection of goose parvovirus and gene, applied in the field of primers for detection of short beak dwarf syndrome goose parvovirus, can solve problems such as not seen, achieve small fragments, high specificity, and avoid false positive results of PCR detection

Active Publication Date: 2018-09-04
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no research reports on the design of specific primers for 5'-UTR to detect SBDS-GPV at home and abroad.

Method used

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  • PCR primers directed at 5'-UTR gene and used for detecting short beak and dwarfism syndrome-goose parvovirus
  • PCR primers directed at 5'-UTR gene and used for detecting short beak and dwarfism syndrome-goose parvovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 PCR detection method for detection of goose parvovirus with short beak dwarf syndrome

[0022] 1. Materials:

[0023] Short beak and dwarf syndrome goose parvovirus M15 strain (SBDS-GPV M15), genetically recombined Muscovy duck goose parvovirus PT strain (MDGPV PT), Muscovy duck parvovirus P strain (MDPV P) and classic goose parvovirus FJ strain (C-GPV FJ) were isolated, identified and preserved by the animal virus laboratory of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0024] Two, steps

[0025] 1. Instruments and reagents: T100 gradient PCR instrument was purchased from Bio-Rad; pMD18-T vector and DL2000Marker were purchased from Bao Bio-Engineering (Dalian) Co., Ltd.; Q5 ultra-fidelity 2×Master Mix was purchased from NEB Company ; DNA purification kit E.Z.N.A.TM Gel Extraction Kit and plasmid rapid extraction kit E.Z.N.A.TM PlasmidMini Kit were purchased from Omega Corporation.

[0026] 2. Prime...

Embodiment 2

[0035] Example 2 Specific detection of short beak dwarf syndrome goose parvovirus PCR

[0036] Take the positive samples of duck tissues artificially infected with SBDS-GPV, C-GPV, MDGPV and MDPV, and the negative samples of healthy duck tissues. According to Example 1, the total DNA is extracted by conventional methods, PCR amplification is performed, and the results are detected by gel electrophoresis. Results A specific band of 130 bp was produced in the SBDS-GPV infected disease material, but none of the C-GPV, MDGPV, MDPV infected disease material and healthy duck tissue samples produced a specific band of 130 bp. The detection results are shown in Figure 1. The PCR product was recovered and purified, cloned into pMD18-T vector and sequenced. The results showed that the sequence of the amplified target fragment had the highest homology (95%-100%) with the corresponding sequence of the 5'-UTR of different virus isolates of SBDS-GPV published by GenBank. Therefore, the pri...

Embodiment 3

[0038] Example 3 Sensitivity detection of short beak dwarf syndrome goose parvovirus PCR

[0039] The SBDS-GPV virus DNA was extracted by the conventional method according to Example 1, and the extracted DNA was serially diluted 10 times, and PCR amplification was performed to detect the relative sensitivity of the method. The results of PCR detection are shown in Figure 2. The specific primers SBDS-GPV-F1 (SEQ ID NO: 1) and SBDS-GPV-R1 (SEQ ID NO: 2) can detect the lowest relative DNA concentration sensitivity of 3 pg, and the sensitivity of the primers is good.

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Abstract

The invention discloses a pair of PCR primers directed at 5'-UTR gene and used for detecting the short beak and dwarfism syndrome-goose parvovirus. The primers are composed of an upstream primer and adownstream primer, wherein the upstream primer is SBDS-GPV-F1 and has an oligonucleotide sequence of 5'-GCACGTGACAGGATGTGCGTCA-3'; and the downstream primer is SBDS-GPV-R1 and has an oligonucleotidesequence of 5'-GCGCGCAAAATATTTTCT-3'. The primers provided by the invention have high specificity, small fragment size and high sensitivity; the whole PCR process can be completed within 2 hours; andthe primers can specifically detect the presence of the short beak and dwarfism syndrome-goose parvovirus, and effectively avoid false positive results in PCR detection of the short beak and dwarfismsyndrome-goose parvovirus.

Description

technical field [0001] The invention relates to a primer for detecting goose parvovirus with short beak dwarf syndrome, which belongs to the field of animal husbandry and veterinary detection. Background technique [0002] Short beak and dwarfism syndrome-gooseparvovirus (short beak and dwarfism syndrome-gooseparvovirus, SBDS-GPV) is a member of the Parvoviridae dependent virus genus, is a pan-tissue tropism single-stranded DNA virus, clinically mainly causes muscovy duck and cherry Valley duck is a highly contagious infectious disease characterized by growth disorder, short beak, exposed tongue, and easy bone breakdown. Under artificial infection conditions, it is infectious to different species of waterfowl. The disease was first reported by Villatte D. to be prevalent in French muscovy ducks. In 2009, Palya V. in Hungary isolated and identified the pathogen, and named it SBDS-GPV. In 2015, Chen Shaoying et al. SBDS-GPV infection was detected in the flock, and SBDS-GPV wa...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701
Inventor 王劭陈少莺肖世峰陈仕龙程晓霞林锋强俞博朱小丽
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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