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Primer group for detecting gosling plague virus, goose paramyxovirus and duck plague virus as well as kit and detection method of primer group

A technology of goose paramyxovirus and goose plague virus, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of misdiagnosis, laborious and laborious, etc., and achieve good results and high detection accuracy Effect

Pending Publication Date: 2021-11-19
赣州市畜牧水产研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the three viruses of GPV, APMV, and DPV are detected by conventional PCR method, which requires multiple tests, which is not only laborious, but also may cause misdiagnosis

Method used

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  • Primer group for detecting gosling plague virus, goose paramyxovirus and duck plague virus as well as kit and detection method of primer group
  • Primer group for detecting gosling plague virus, goose paramyxovirus and duck plague virus as well as kit and detection method of primer group
  • Primer group for detecting gosling plague virus, goose paramyxovirus and duck plague virus as well as kit and detection method of primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Design and synthesis of specific primers

[0036]The whole genome sequences of Goose Plague Virus (GenBank: KU641558.1), Goose Paramyxovirus (GenBank: MK124761.1) and Duck Plague Virus (GenBank: JQ673560.1) published on GenBank were respectively passed on the online software BLAST. Carry out comparative analysis, design three pairs of specific primers respectively in the selected virus-specific conserved region, the nucleotide sequence of the multiplex PCR detection primer set provided by the present invention is as follows:

[0037] The nucleotide sequences of the detection primers for gosling plague virus are as follows:

[0038] GPV-F989: 5'-GACCGTGACTGCAGCATACA-3',

[0039] GPV-R1289: 5'-GCTGTCTCGGTTCTCAGTGAG-3';

[0040] The nucleotide sequence of the detection primer pair of goose paramyxovirus is as follows:

[0041] APMV-F33: 5'-CGAGATCGTACGGGTAGAAG-3',

[0042] APMV-R425: 5'-ACCATCGATCTCAAGAACAG-3';

[0043] The detection primer pair nucleotide s...

Embodiment 2

[0047] Example 2 Viral nucleic acid extraction and cDNA synthesis

[0048] Use Easypure Viral DNA / RNA kit (spin column virus DNA / RNA extraction kit) to extract goose plague virus (GPV) DNA, duck plague virus (DPV) DNA, and avian influenza virus subtype H9 (AIV-H9) RNA , Goose Astrovirus (GAstV) RNA, Goose Reovirus (GRV) RNA, Goose Paramyxovirus (APMV) RNA. Use the HiScript cDNA first-strand synthesis kit to perform reverse transcription of RNA: use a 20 μL reaction system, add 8 μL of RNA template, 1 μL of random primers, 10 μL of 2×RTMIX, and 1 μL of HiScript Enzyme Mix to the EP tube in sequence, and the total volume is 20 μL. Put it in a PCR machine for cDNA synthesis, and perform a cycle with the program of 25°C for 5 minutes, 50°C for 45 minutes, 85°C for 5 minutes, and 4°C for 5 minutes. After the cDNA is synthesized, store it at -20°C for later use.

Embodiment 3

[0049] The establishment of embodiment 3 single-fold PCR method

[0050] Firstly, through single-plex specific PCR, the reaction system and reaction conditions for each pair of primer sets to amplify the corresponding gene fragments were preliminarily explored. Using the DNA of goose plague virus (GPV), duck plague virus (DPV) and goose paramyxovirus (APMV) obtained in Example 2 as the amplification templates of the respective primer sets, it was found through multiple experiments that each pair The primer sets can well amplify the corresponding gene fragments under the following reaction systems and conditions.

[0051] Gosling plague virus (GPV) single-plex specific PCR reaction system and conditions are as follows:

[0052] PCR reaction system (20 μL): 2×Go Taq Master Green Mix 10 μL, 10 μmol / L GPV-F989 0.4 μL, 10 μmol / L GPV-R1289 0.4 μL, DNA template 2 μL, add sterilized deionized water to a total volume of 20 μL.

[0053] PCR reaction conditions: pre-denaturation at 95°...

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Abstract

The invention discloses a primer group for detecting gosling plague virus, goose paramyxovirus and duck plague virus as well as a kit and a detection method of the primer group. The primer group comprises a goose parvovirus detection primer, a goose paramyxovirus detection primer and a duck plague virus detection primer. The kit comprises the primer group. The multiple PCR detection method established by the invention can accurately detect single or mixed infection of the gosling plague virus, the goose paramyxovirus and the duck plague virus, is high in detection precision, and has important advantages in the field of virus identification.

Description

Background technique [0001] Gosling plague (GP) is an acute or subacute septic infectious disease caused by Goose parvovirus (GPV), which mainly affects goslings aged 4 to 20 days. , neurological symptoms (neck torsion, etc.), necropsy showed hepatomegaly, exudative enteritis and intestinal embolism as the main features. Goose paramyxovirus disease (GMP) is an acute viral infection caused by avian paramyxovirus (APMV), characterized by intractable diarrhea, dyspnea, neurological symptoms, hemorrhage and necrosis of gastrointestinal mucosa The disease can occur in geese of various ages. The younger the age, the higher the morbidity and mortality rate. The morbidity and mortality rate of goslings within 2 weeks of age can reach 100%. The sick geese appear in the later stage of the disease course. nervous symptoms. Duck plague (DP), also known as duck viral enteritis, is an acute, contagious, and fatal disease of ducks and geese caused by duck plague virus (DPV). The detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143
Inventor 钟敏李建军钟云平郭礼荣刘瑞平邱光忠
Owner 赣州市畜牧水产研究所
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