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Primer, probe and reagent kit for universal influenza A virus nucleic acid detection

A type of influenza A virus and kit technology, applied in biochemical equipment and methods, microorganisms, methods based on microorganisms, etc., can solve problems such as the difficulty in designing primers and probes, and achieve high detection rate, high sensitivity, and simple combined effect

Pending Publication Date: 2020-11-10
江苏派森杰生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using the PCR method to detect viral nucleic acids, one of the difficulties is that the mutation of the virus brings difficulties to the design of primers and probes. In order to accurately identify the influenza A virus in circulation, it is necessary to regularly check the sequence of the circulating strains. Align and analyze, update and optimize primer and probe sequences

Method used

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  • Primer, probe and reagent kit for universal influenza A virus nucleic acid detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Design and synthesis of primers and probes

[0017] One of the keys of the present invention is to design a set of primers and probes that can be used for general nucleic acid detection of influenza A virus. According to the genome sequence of influenza A virus in the GenBank database, use MAFFT software to compare and analyze the consistency of different types of gene sequences, select the relatively conserved regions of the sequence mutation sites of influenza A virus matrix protein M1 and M2 to design genome-wide specificity Amplification primer sequence,

[0018] The designed primers and probe sequences are:

[0019] The nucleotide sequence of the forward primer IFN-UN-F is shown in SEQ ID No: 1, namely: 5'-CAAGACCAATCCTGTCACCTYTR -3'; the nucleotide sequence of the reverse primer IFN-UN-R is shown in SEQ ID No: 2, namely: 5'-TCTACGCTGCAGTCCTCGCTC-3'; and the nucleotide sequence of the probe IFN-UN-P as shown in SEQ ID No: 3, namely: 5'-ACGCTCACCGTGC...

Embodiment 2

[0020] Embodiment 2: the extraction of RNA

[0021] Influenza A virus was cloned and sequenced in accordance with the "Molecular Cloning Experiment Guide (Third Edition)". The throat swabs, sputum, alveolar lavage fluid, tissue exudate, feces and urine collected from 60 patients with cold symptoms were used as samples to extract influenza virus cell cultures and use high-purity viral RNA reagents High Pure Viral RNA Kit (High Pure Viral RNA Kit) was used to extract RNA from influenza virus samples, and the specific extraction steps were performed according to the operating instructions. Among them, sputum, throat swab liquid and feces samples need to be pretreated, and 0.01M PBS, pH7.5, should be added to sputum and throat swab samples in advance; The suspension was centrifuged at 12000×g for 10 min, and the supernatant was taken for RNA extraction, eluted with 50 μl DEPC water, and stored at -80°C.

Embodiment 3

[0022] Embodiment 3: the establishment of Real-time RT-PCR amplification method

[0023] Using the influenza virus RNA extracted from each sample in Example 2 as a template, a real-time RT-PCR reaction was performed (reagents for the reaction were purchased from TaKaRa).

[0024] The fluorescent quantitative RT-PCR reaction system is as follows:

[0025] The 25μl rRT-PCR detection system contains: 2×OneStep RT-PCR Buffer 12.5μl, 10μM forward primer (SEQ ID No:1) 1.5μl, 10μM reverse primer (SEQ ID No:2) 1.5μl, 10μM probe Needle (SEQ ID No:3) 0.5μl, Rox Reference Dye Ⅱ (50×) 0.5μl, 5U / μl Ex Taq HS 0.5μl, 40U / μl PrimeScriptRT enzyme Mix 0.5μl, sample RNA to be tested 5μl, RNase Free dH 2 O 2.5 μl. Put the reaction tube of the above reaction system into a fluorescent quantitative PCR instrument, and set the rRT-PCR reaction conditions as follows: reverse transcription at 50°C for 20 minutes, reaction at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing / extension...

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Abstract

The invention relates to the technical field of biological nucleic acid detection, in particular to a primer, probe and reagent kit for universal influenza A virus nucleic acid detection. The primer and the fluorescent probe are designed according to new mutation sites of influenza A virus matrix protein M1 and M2 sequences, and the primer and the probe for universal nucleic acid detection of influenza A viruses provided by the invention have strong degeneracy and high detection rate for influenza A virus subtypes.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer, a probe and a kit for detection of general-purpose influenza A virus nucleic acid. Background technique [0002] Influenza viruses are referred to as influenza viruses for short, and are divided into three types: A, B, and C. According to the sequence differences of hemagglutinin (HA) and neuraminidase (NA) on the envelope of influenza virus, influenza A virus can be divided into multiple types. Influenza A viruses that can infect humans include H1N1, H3N2, H5N3, H5N2, H7N9 and other types. Not only the reassortment phenomenon among influenza A viruses is very common, but also base mutations are prone to occur during the genome replication process, which further increases the genetic diversity and brings difficulties to clinical diagnosis. [0003] The identification of influenza A virus type by Real-time RT-PCR (rRT-PCR) method can confirm the diagnosis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/156C12Q2521/107C12Q2563/107
Inventor 申红星刘庭君王华
Owner 江苏派森杰生物科技有限公司
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