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D-type influenza virus fluorescent quantitative PCR (polymerase chain reaction) and LAMP (isothermal amplification) fluorescent quantitative PCR primer pair and application thereof

A technology of influenza virus and type D, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microorganisms, etc., can solve the problems of affecting the detection results, false positives, etc., and achieve good repeatability, strong specificity, and simple and fast detection methods Effect

Inactive Publication Date: 2020-06-26
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

Studies in recent years have shown that influenza C virus can also infect livestock such as pigs and cattle, and the genome composition of influenza C virus is the same as that of influenza D virus, so it may be falsely detected when detecting influenza D virus in herds. Influenza virus, false positive results appear, affecting test results

Method used

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  • D-type influenza virus fluorescent quantitative PCR (polymerase chain reaction) and LAMP (isothermal amplification) fluorescent quantitative PCR primer pair and application thereof
  • D-type influenza virus fluorescent quantitative PCR (polymerase chain reaction) and LAMP (isothermal amplification) fluorescent quantitative PCR primer pair and application thereof
  • D-type influenza virus fluorescent quantitative PCR (polymerase chain reaction) and LAMP (isothermal amplification) fluorescent quantitative PCR primer pair and application thereof

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Embodiment 1

[0037] 1. Routine PCR amplification of the target fragment of IDV genome

[0038] The PCR reaction system is: 24 μL of Gold Mix (Green), 2 μl of upstream and downstream primers (10 μmol / L), 2 μL of DNA template (template plasmid), and a total volume of 30 μL. After the above components were mixed and centrifuged briefly, the following procedures were performed on the PCR machine: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 s, annealing at 52°C for 10 s, and extension at 72°C for 40 s; for 40 cycles, finally take 5 μL of the reaction product for 2% agarose gel electrophoresis, the results are as follows figure 1 shown.

[0039] 2. Preparation of Positive Plasmids

[0040] According to the QIAprep Spin Miniprep kit, a small amount of positive bacterial liquid was extracted and purified, and stored at -20°C for future use.

[0041] 3. Optimization of real-time fluorescent quantitative PCR reaction system and reaction conditions

[0042]Using Oklahoma13...

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Abstract

The invention belongs to the technical field of D-type influenza virus detection and in particular relates to a D-type influenza virus fluorescent quantitative PCR (polymerase chain reaction) and LAMP(isothermal amplification) fluorescent quantitative PCR primer pair and application thereof. D-type influenza viruses mainly affect animals including pigs and cattle, and has certain threats to development of the livestock industry, and provision of stable and reliable detection methods has great significances. The invention provides primer sequences for real-time fluorescent quantitative PCR (polymerase chain reaction) detection on the D-type influenza viruses and a corresponding kit, the primers are designed for conserved sequences in a D-type influenza virus HE gene, are capable of achieving good detection specificity and sensitivity, have sensitivity which is 100 times higher than those of common PCRs and fluorescent quantitative LAMP, detection on massive universal samples can be achieved, rapid and high-sensitivity real-time fluorescent quantitative PCR detection on the D-type influenza viruses can be achieved, and great significances can be achieved for improving detection precision on the D-type influenza viruses.

Description

technical field [0001] The invention belongs to the technical field of detection of type D influenza virus, and in particular relates to a primer for detecting type D influenza virus by fluorescent quantitative PCR and a detection kit containing the primer. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Influenza virus is a single-stranded negative-sense RNA virus belonging to the family Orthomyxoviridae. In the past, influenza viruses were divided into type A virus (Influenza A virus, IAV), type B virus (Influenza B virus, IBV) and type C virus (Influenza C virus, ICV) three influenza viruses, of which influenza A virus is further divided into 18 HA subtypes and 11 NA ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 于志君朱彤吴家强陈为京高月花
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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