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Kit for detecting H7N9 avian influenza virus on basis of isothermal amplification technique and application thereof

A bird flu virus and technical detection technology, applied in the field of kits for detection of H7N9 bird flu virus by warm amplification technology, can solve the problems of long time consumption, achieve the effect of shortening the detection time and convenient reading of results

Active Publication Date: 2020-03-31
湖北省疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Conventional RT-PCR and real-time quantitative fluorescent RT-PCR can only be tested in laboratories equipped with expensive fluorescent quantitative PCR instruments, and it takes a long time. It takes at least one day, which is not good for on-site rapid diagnosis in grassroots and remote areas

Method used

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  • Kit for detecting H7N9 avian influenza virus on basis of isothermal amplification technique and application thereof
  • Kit for detecting H7N9 avian influenza virus on basis of isothermal amplification technique and application thereof
  • Kit for detecting H7N9 avian influenza virus on basis of isothermal amplification technique and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The design and optimization of embodiment 1 primer

[0046] The key to RPA amplification lies in the design of primers. The length of primers used in conventional PCR is generally 18-25 bp, and there are a variety of mature commercial software available for primer design. The RPA primer length is generally 30-35bp, and the probe length is 46-52bp (generally at least 30bp from the 5' end to the THF site, and at least 15bp from the THF site to the 3' end), because the length of the primers and probes is relatively long. Also consider the GC content of the primer probe, as well as the secondary structure and primer dimer that may affect the experimental effect, and there is no software for RPA primer design at present. Therefore, the design and screening of the RPA primer probe are helpful higher requirements.

[0047] In addition, when designing RPA primers, the selection of the target region is also very critical. The present invention compares the HA and NA gene seque...

Embodiment 2

[0056] Embodiment 2RT-RPA reaction temperature optimization

[0057] Adopt primer pair H2 (H7-FP2 / H7-RP2) and N4 (N9-FP8 / N9-RP8) to carry out RPA detection to H7N9 avian influenza virus, concrete operation step is generally consistent with embodiment 1, difference is: the reaction The system was placed in a constant temperature metal bath at 20°C to 50°C for 20 minutes. After the reaction, the reaction product was added with sample buffer (10 mM Tris salt buffer containing 0.05% Tween-20) and diluted 20 times, and the lateral flow test strip was placed in 200 μL of the diluted product to react for 5-10 min. Take it out and the result can be read directly by naked eye.

[0058] The result is as figure 2 As shown, the RT-RPA reaction can occur when the reaction temperature is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, and the reaction efficiency is the highest when the reaction temperature is 40°C. Under the optimized reaction conditions of the present invention, reverse ...

Embodiment 3

[0059] Example 3 specificity verification

[0060] In order to verify the specificity of the detection kit of the present invention and the detection method thereof, the new type A H1N1pdm09 influenza virus, human seasonal H3N2 influenza virus, type B influenza virus Victoria strain, type B influenza virus Yamagata strain, H5N6 avian influenza virus, The RNA of H7N9 avian influenza virus and H9N2 avian influenza virus was used as a template for RT-RPA reaction, and the reaction products were read with lateral flow test strips. Experimental results such as image 3 , Figure 4 As shown, only the H7N9 avian influenza virus is positive, and the others are all negative, indicating that the RPA primers and detection probes of the present invention have good specificity and do not cross-react with other related viruses.

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Abstract

The invention belongs to the technical field of biology, and specifically relates to a kit for detecting H7N9 avian influenza virus by adopting a recombinase-mediated isothermal amplification technique and application thereof. The kit for detecting the H7N9 avian influenza virus by adopting the recombinase-mediated isothermal amplification technique comprises a reaction buffer solution, a recombinase polymerase amplification (PRA) primer pair, a probe, PRA lyophilized powder, a magnesium acetate solution, sterile water, and a positive control; and the PRA primer pair and the probe are used forperforming amplification detection on an HA gene segment having a sequence shown as SEQ ID NO. 1 and an NA gene segment having a sequence shown as SEQ ID NO. 2. According to the kit for detecting theH7N9 avian influenza virus by adopting the recombinase-mediated isothermal amplification technique, two genetic markers used for detecting the H7N9 avian influenza virus are provided for establishingan RT-RPA amplification detection method of which the minimum detection amount is 200 copies / reaction; and thus, the established RT-RPA amplification detection method are better than conventional RT-RPA in both specificity and sensitivity. Moreover, the whole amplification reaction is carried out at constant temperature of 20-50 DEG C, so that the reaction can be completed in 20 minutes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting H7N9 avian influenza virus by adopting a recombinase-mediated isothermal amplification technique and an application thereof. Background technique [0002] The H7N9 avian influenza virus belongs to the genus Influenza A of the Orthomyxoviridae family. Virus particles are pleomorphic, with a spherical diameter of 80-120 nm and an envelope. The genome is segmented single-stranded negative-strand RNA. H7N9 avian influenza virus is a new type of reassortant virus. In March 2013, the Chinese Center for Disease Control and Prevention confirmed for the first time that H7N9 avian influenza virus can infect humans. Since then, the virus has spread rapidly in mainland China, Hong Kong, Macao and Taiwan, and has shown a seasonal epidemic trend. In November 2013, the Chinese Center for Disease Control and Prevention officially included it in the category of Class B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2537/1376C12Q2521/507C12Q2565/625Y02A50/30
Inventor 李翔方斌余晓刘琳琳叶国军徐军强宋毅
Owner 湖北省疾病预防控制中心