Primer pair, kit and method for detecting goose astrovirus and goose calicivirus

An astrovirus, goose embedded cup technology, applied in the field of molecular biology, to achieve good specificity

Active Publication Date: 2021-06-11
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

Although the RT-PCR method has been widely used, the single-plex RT-PCR method can only detect one virus in one reaction, and cannot perform combined RT-PCR detection of multiple virus types

Method used

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  • Primer pair, kit and method for detecting goose astrovirus and goose calicivirus
  • Primer pair, kit and method for detecting goose astrovirus and goose calicivirus
  • Primer pair, kit and method for detecting goose astrovirus and goose calicivirus

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The design and synthesis of embodiment 1 primer

[0039] The invention is a pair of primers for detecting goose astrovirus and goose calicivirus. According to the whole genome sequence of goose astrovirus and goose calicivirus in GenBank, primers are designed in their relatively conserved regions by using Oligo 6.0 software; including GoAstV Upstream primer, GoAstV downstream primer, GoCV upstream primer and GoCV downstream primer, the nucleotide sequence of the GoAstV upstream primer is as described in SEQ ID NO: 1, the nucleotide sequence of the GoAstV downstream primer is as in SEQ ID NO: 2 Said, the nucleotide sequence of the GoCV upstream primer is as described in SEQ ID NO: 3, and the nucleotide sequence of the GoCV downstream primer is as described in SEQ ID NO: 4. The designed two pairs of specific primers (see the next paragraph for specific sequences) were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0040] Specifically, GoAstV upstream primer ...

Embodiment 2

[0046] This embodiment is a test kit for detecting goose astrovirus and goose calicivirus, which contains 80 μL of the primer pair for detecting goose astrovirus and goose calicivirus described in Example 1 (each primer concentration Both are 10 μmol / L) and reaction reagents for double one-step RT-PCR, specifically 100 μL DL2000, 200 μL RT-PCR one-step enzyme, 250 μL enzyme buffer, 300 μL RNase Free dH 2 O, 20 μL positive control and 20 μL negative control.

Embodiment 3

[0047] Example 3 Double one-step RT-PCR detection of goose astrovirus and goose calicivirus

[0048] 1. Preparation of samples to be tested

[0049] refer to According to the instruction manual of the Viral DNA / RNA Kit kit, extract goose paramyxovirus, muscovy duck reovirus, duck new type reovirus, type Ⅰ duck hepatitis virus, duck Tembusu virus, goose astrovirus and goose calicivirus RNA, Muscovy duck parvovirus, goose parvovirus, duck adenovirus DNA, and using SmartSpec TM After the Plus Nucleic Acid Protein Detector measures their nucleic acid concentration and purity, store them at -70°C for later use.

[0050] 2. Establishment of double one-step RT-PCR reaction

[0051] Use the FastKing one-step RT-PCR kit to perform double one-step RT-PCR, through the amount of primer pair GoAstV upstream primer / GoAstV downstream primer, primer pair GoCV upstream primer / F4 used in the reaction solution and the annealing temperature of the double RT-PCR Optimum concentration of prim...

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Abstract

The invention relates to a primer pair, a kit and a method for detecting goose astrovirus and goose calicovirus. The primer pair disclosed by the invention comprises a GoAstV upstream primer, a GoAstV downstream primer, a GoCV upstream primer and a GoCV downstream primer which are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. The kit disclosed by the invention comprises the primer pair. The method comprises the following steps: S1, extracting RNA (Ribonucleic Acid) of a sample; S2, adopting the primer pair, amplifying by using a dual one-step RT-PCR reaction program, and then detecting; if a strip appears at 452 bp, indicating that the goose astrovirus is positive, and if a strip appears at 711 bp, indicating the goose calico virus is positive. The scheme provided by the invention is used for molecular epidemiological detection of goose astrovirus and goose calico virus, and is good in specificity and reliable in result.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer pair, a kit and a method for detecting goose astrovirus and goose calicivirus. Background technique [0002] Goose astrovirus (GoAstV) is a non-enveloped, single-stranded positive-sense RNA virus. GoAstV particles have a 20-hedron symmetric structure with a diameter of 28nm-30nm and a genome length of 7148-7299bp. GoAstV is one of the main pathogens causing fatal gout in geese. The main clinical manifestations are mental depression, sleepiness on the ground, and reduced food intake. Huge economic loss is one of the important diseases that endanger the goose industry in the world at present. The virus widely exists in the breeding geese in my country, which has seriously affected the economic benefits of the goose industry. [0003] Goose calicivirus (GoCV) is a newly emerged waterfowl calicivirus, which is a member of the family Caliciviridae. It is a non-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2521/107C12Q2537/143
Inventor 王劭林甦陈仕龙江丹丹郑敏陈少莺
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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