Type D influenza virus fluorescent quantitative PCR primer pair and kit

A technology for influenza virus and fluorescence quantification, applied in the field of bioengineering, can solve the problems of unfavorable application and high cost of synthetic probes, and achieve high sensitivity, simple and fast detection method, and good repeatability

Active Publication Date: 2018-09-28
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported abroad that the probe method of RT-PCR was used to detect influenza D virus, but the cost of synthetic probes is too high, which is not conducive to the actual production application.

Method used

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  • Type D influenza virus fluorescent quantitative PCR primer pair and kit
  • Type D influenza virus fluorescent quantitative PCR primer pair and kit
  • Type D influenza virus fluorescent quantitative PCR primer pair and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1, routine PCR amplification of the target fragment of IDV genome.

[0015] Dissolve 4 μg of artificially synthesized template plasmid dry powder in 80 μL H2O. The PCR reaction system is: 12.5 μL of Primix ExTaqTM enzyme, 1 μL of upstream and downstream primers (10 μmol / L), 2 μL of DNA template (template plasmid), plus sterilized three-distilled water to a total volume of 25 μL. After the above components were mixed and centrifuged briefly, the following procedures were performed on the temperature gradient PCR instrument: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds; 34 cycles, and finally extension at 72°C 10min. Take 6 μL of the reaction product for 1% agarose gel electrophoresis, the results are as follows: figure 1 shown.

Embodiment 2

[0016] Example 2, preparation of positive IDV plasmid.

[0017] After electrophoresis of the PCR target product, use TaKaRa MiniBEST Agarose Gel DNA Extraction KitVer.4.0 to perform gel recovery according to the instructions. Using the pMD18-T Vector Ligation Kit from TaKaRa Company, the total volume of the reaction system was 5 μL as follows: 2.25 μL of PCR purified product, 2.5 μL of Ligation Solution I, 0.25 μL of pMD18-T Vector, operated on ice, and reacted at 16°C for 4 hours. Transformation of recombinant plasmids, colony PCR identification, sequence analysis, and a small amount of positive bacterial liquid was extracted and purified according to TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0, and stored at -20°C for future use.

Embodiment 3

[0018] Example 3, optimization of real-time fluorescent quantitative PCR reaction conditions.

[0019] 3.1 Optimization of annealing temperature: Real-time fluorescent quantitative PCR amplification was carried out with the positive plasmid as a template, the optimal annealing temperature was screened, and 7 temperature gradients were set, 54.0°C, 54.3°C, 55.0°C, 56.0°C, 57.2°C, 58.2°C, 58.7°C. The reaction system is 25 μL, and the reaction procedure is shown in Table 1:

[0020] Table 1 Reaction program

[0021]

[0022] With any copy number of positive plasmids, seven different annealing temperature gradients were used for real-time fluorescent quantitative PCR detection, and the results showed that the annealing temperature was the best at 55°C.

[0023] 3.2 Optimization of primer concentration: according to the optimal annealing temperature determined above, real-time fluorescent quantitative PCR amplification was carried out using the positive plasmid as a template, ...

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Abstract

The invention discloses a type D influenza virus fluorescent quantitative PCR primer pair and a kit. The primer pair is designed by intercepting a middle conserved sequence of a type D influenza PB1 sequence published by JQ922306, KM392476, KM392483 and KX768825 on GenBank. The primer pair can be used for preparing the type D influenza virus fluorescent quantitative PCR detection kit, so that virus content of type D influenza virus (IDV) can be detected. The kit comprises the primer pair as prescribed in the claim 1, a positive standard plasma, a TB GreenTM Premix Ex Taq II real-time fluorescent quantitative PCR reagent and ddH2O. The type D influenza virus (IDV) real-time fluorescent quantitative PCR detection kit, which is prepared by the primer pair provided by the invention, is convenient and rapid in detection method, good in repeatability, high in sensitivity and strong in specificity; high-flux samples can be detected; and rapid and high-sensitivity real-time fluorescent quantitative PCR detection can be implemented on the type D influenza viruses.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a primer pair for fluorescent quantitative PCR of type D influenza virus and a kit. Background technique [0002] Influenza is a zoonotic infectious disease caused by influenza virus (Influenza Virus) that seriously endangers public health and the development of animal husbandry. Previously, influenza viruses could be divided into 3 types according to the antigenicity of nucleoprotein: type A, type B and type C influenza virus (IAV, IBV, ICV). In recent years, the existence of a new type of influenza virus has been discovered. In 2011, in Oklahoma, a strain belonging to a putative new genus, not yet recognized by official classification, was isolated from influenza diseased pigs and tentatively identified influenza virus D (IDV). In September 2016, the Executive Committee of the International Committee on Taxonomy of Viruses approved the naming of a new virus—type D infl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2563/107C12Q2531/113
Inventor 李智丽马保华张辉华李文锋曹嫦妤赵云翔黄淑坚郭锦玥梅敏敏黄雯晶李晓文
Owner FOSHAN UNIVERSITY
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