Primer probe combination, kit and detection method for detecting duck adenovirus type 3 based on RAA technology

A technical detection and primer-probe technology, which is applied in the field of primer-probe combination based on RAA technology to detect duck adenovirus type 3, to achieve high-throughput detection, shorten reaction time, and rapid detection

Pending Publication Date: 2021-01-05
山东信达基因科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the virus mainly focuses on morphology, pathogenicity, gene sequence analysis, etc., and there is no accurate and rapid detection method

Method used

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  • Primer probe combination, kit and detection method for detecting duck adenovirus type 3 based on RAA technology
  • Primer probe combination, kit and detection method for detecting duck adenovirus type 3 based on RAA technology
  • Primer probe combination, kit and detection method for detecting duck adenovirus type 3 based on RAA technology

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Embodiment 1

[0032] The present invention also provides a combination of primers and probes for detecting duck adenovirus type 3, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3 , the nucleotide sequence of the probe is shown in SEQID NO:5.

[0033] This specific example provides a detection method for detecting duck adenovirus type 3 virus based on RAA technology, and the primer-probe combination used in the detection method is the above-mentioned primer-probe combination. The detection method specifically includes the following steps:

[0034] (1) Configure the negative control, positive control and RAA reaction system of the sample to be tested. The RAA reaction system is:

[0035]

[0036] RT fluorescent basic reaction unit;

[0037] Sample DNA template to be detected or duck adenovirus type 3 virus DNA template or RNase-removed water 2 μL;

[0038] Total volume 50 μL;

[0...

Embodiment 2

[0044] Design and screening of embodiment 2 duck adenovirus type 3 virus detection primers and probes

[0045] According to duck adenovirus type 3 virus-specific Hexon gene (Hexon gene sequence is shown in SEQ ID NO: 6) as target gene, carry out primer probe design, according to DAdV-3 representative strain (CH-GD-12-2014 Strains, reference sequence GenBank accession number: KR135164) Hexon gene sequence characteristics, design Hexon gene-specific primers and probes.

[0046]Design 2 upstream primers and 2 downstream primers to screen the best primers and probes respectively. The region where the upstream primers are located corresponds to the 1230th base of the reference sequence, and the bases corresponding to this position in the 2636 Hexon gene sequences are C and There are two types of T, so the primer sequence is designed as a degenerate base Y, the 1248th base, in the 2636 Hexon gene sequences, there are two bases corresponding to this position, C and T, so the primer s...

Embodiment 3

[0064] The detection sensitivity of embodiment 3 primer probe

[0065] Extract duck adenovirus type 3 virus DNA according to the instructions of the DNA extraction kit, measure the original concentration of the extracted DNA template, and dilute to 10 7 copy / μL, and then diluted to 10 in a 10-fold gradient 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Copy / μL, 10 copies / μL, take 2 μL respectively as the reaction template, and detect the sensitivity of the primers and probes of Combination 3 according to the detection method of Example 1.

[0066] The result is as figure 2 As shown, 1-6 in the figure correspond to 10 copies / μL, 10 2 copies / μL, 10 3 copies / μL, 10 4 copies / μL, 10 5 copies / μL, 10 6 Copy / μL, the combination of primers and probes of combination 3 designed by the present invention is 10 at the concentration of duck adenovirus type 3 virus DNA template 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Amplification c...

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Abstract

The invention discloses a primer probe combination, a kit and a detection method for detecting duck adenovirus type 3 based on an RAA technology. The primer probe combination is designed according toduck adenovirus type 3 virus specific Hexon gene, and the kit and the detection method utilize the primer probe combination to perform RAA reaction on a to-be-detected sample. Whether the to-be-detected sample contains the duck adenovirus type 3 virus or not is judged according to an amplification reaction result. The kit and the detection method are applied to rapid detection of the duck adenovirus type 3 virus, and accurate, rapid, sensitive and convenient detection of the duck adenovirus type 3 virus is realized.

Description

technical field [0001] The invention relates to the technical field of duck adenovirus detection methods, in particular to a primer-probe combination, a kit and a detection method for detecting duck adenovirus type 3 based on RAA technology. Background technique [0002] Avian adenoviruses are common infectious agents of poultry and wild birds worldwide. Most avian adenoviruses can replicate in healthy poultry with very mild symptoms or no symptoms of infection. In mixed infection, avian adenoviruses can become conditional pathogens. In recent years, a researcher has isolated a new strain of virus from Muscovy ducks in a Muscovy duck farm in Guangdong. The clinical lesions of the infected ducks were yellowing liver, brittle texture, swelling, hemorrhage and necrosis, which were obtained by high-throughput sequencing. The full gene sequence of the virus (GenBank No. KR135164.1, CH-GD-12-2014 strain), the pathogen was initially named duck adenovirus type 3 (DAdV-3) through ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101
Inventor 邹海涛马广斌李阳马丽王学波李朝阳李广森鹿洪伟梁栋
Owner 山东信达基因科技有限公司
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