Multiple fluorescent quantitative PCR detection primer and reagent kit for novel goose astrovirus, goose paramyxovirus and goose parvovirus, and application

A multiplex fluorescence quantitative, goose paramyxovirus technology, applied in the field of molecular biology, to achieve high feasibility and application prospects, good reproducibility, high specificity and specificity

Inactive Publication Date: 2019-07-05
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Description
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At present, there is no record of using multiplex fluorescent quantitative PCR method...

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  • Multiple fluorescent quantitative PCR detection primer and reagent kit for novel goose astrovirus, goose paramyxovirus and goose parvovirus, and application
  • Multiple fluorescent quantitative PCR detection primer and reagent kit for novel goose astrovirus, goose paramyxovirus and goose parvovirus, and application
  • Multiple fluorescent quantitative PCR detection primer and reagent kit for novel goose astrovirus, goose paramyxovirus and goose parvovirus, and application

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Embodiment 1

[0050] 1.1 Primer design and synthesis

[0051] The sequence refers to the sequence of each target gene in the Gene Bank database, and 3 sets of specific primers and probes that can amplify 3 goose pathogens are designed. The primers and probes are designed by ABI's Primer Express Software v2.0 and are designed by BGI Synthesized by the company, store at -20℃ for later use.

[0052] The nucleotide sequences of the three sets of primers and probes are shown in Table 2 below:

[0053] Table 2 Nucleotide sequences of primers and probes

[0054]

[0055] 1.2 Viral nucleic acid extraction and cDNA synthesis

[0056] Use Easy Viral DNA / RNA Kit (spin column type viral DNA / RNA extraction kit) to extract three viral DNA / RNA, and use HiScript cDNA first-strand synthesis kit to reverse transcription of three viral RNAs: 40μL system , Add 16μL of RNA template, 2μL of random primers, 2×RTMIX 20μL, HiScript EnzymeMix 2μL to the EP tube, total volume 40μL, and place in PCR machine for cDNA synthesis...

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Abstract

The invention relates to the technical field of virus detection, in particular to a multiple fluorescent quantitative PCR detection primer pair for novel goose astrovirus, goose paramyxovirus and goose parvovirus, and an application of a specific probe. The established multiple fluorescent quantitative PCR system can accurately detect single or mixed infection of the novel goose astrovirus, the goose paramyxovirus and the goose parvovirus, the multiple fluorescent quantitative PCR system is optimized, the optimal primer concentration and the optimal probe concentration are determined, the detection precision is higher, and the multiple fluorescent quantitative PCR detection primer pair has important advantages in the field of virus identification.

Description

Technical field [0001] The invention relates to a virus detection technology, in particular to a fluorescent quantitative PCR detection primer, probe composition and detection method for identifying new goose astrovirus, goose paramyxovirus, and goose parvovirus, and belongs to molecular biology technology field. Background technique [0002] Astroviruses belong to the astrovirus family (astroviridae), and are divided into two genera according to the host: Mamastrovirus and Avastrovirus. There are currently 3 genera of Avian Astrovirus (Avastrovirus 1-3), including Duck astrovirus, Avian nephritisa strovirus, Turkey astrovirus, etc., Turkey astrovirus Shape virus is its representative species. Astrovirus is a single-stranded, non-enveloped small RNA virus. Its genome is about 6.8kb in length. It contains 4 parts from 5'end to 3'end: one 85-nucleotide 5'end non- Coding region, 3 open reading frames (ORFs) (ORF1a, ORF1b, ORF2), 1 80-nucleotide 3'non-coding region, 1 30-nucleotid...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/70C12Q1/6851C12Q2600/16C12Q2537/143C12Q2531/113C12Q2563/107
Inventor 孟凯王友令于江袁小远张玉霞杨金星李莉黄玉珍冯友涛
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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