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A set of primers for detection of influenza C virus lamp and its application

A technology of influenza virus and RT-LAMP, which is applied in the direction of recombinant DNA technology, microorganisms, and methods based on microorganisms, can solve the problems of long separation time, high cost of reagents and instruments, and difficulty in virus separation, achieving high sensitivity and responsiveness. Stable and reliable, easy to observe and judge the effect of the result

Active Publication Date: 2020-10-09
郑州博睿医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Influenza C virus can be isolated from chicken embryos, canine kidney cells (MDCK cells), and human malignant melanoma cell lines (HMV-II cells), but virus isolation is difficult and takes a long time, which is far from being practical High-throughput and high-efficiency requirements in work
The cost of reagents and instruments required for common nucleic acid and antibody detection methods is relatively high

Method used

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  • A set of primers for detection of influenza C virus lamp and its application
  • A set of primers for detection of influenza C virus lamp and its application
  • A set of primers for detection of influenza C virus lamp and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] With the aid of AlignX and the software PrimerExplorer 4.0 (http: / / primerexplorer.jp / e / ), the inventors selected the relatively conserved region of the MP gene of influenza C virus (GenBank: D26546.1), and designed four primers, including two The outer primers (INF-C F3, INF-C B3) and the two inner primers (INF-C FIP, INF-C BIP) are used as primers for influenza C virus LAMP detection. The primer sequence is shown in SEQ ID NO. 1~4, the details are as follows:

[0083] INF-CF3: 5'-CCAAATAATGGAAATGGTTGAAG-3',

[0084] INF-CB3: 5'- TCTTCTCCAAGGCCAGTA-3';

[0085] IFN-C FIP: 5'- TCTCAACCAAGCTGTGATTGTTC-TACGATCACCCAAACGACTA-3',

[0086] IFN-C BIP: 5'-GAGTAATGTCTCCAGAAAGTGGAAGA-TCGTTCATTGCTGTGCTG-3'.

Embodiment 2

[0088] Utilize the influenza C virus LAMP detection primers designed in Example 1 to prepare a RT-LAMP detection kit, and the ratio of each component of 25 doses is designed as follows:

[0089] (1) RNA extraction solution: TRIZol, 25mL, RNase inhibitor 25μL;

[0090] (2) Reaction mixture:

[0091] Bst DNA polymerase buffer, (10×), 62.5 μL;

[0092] Bst DNA polymerase, 8U / μL, 25μL;

[0093] AMV reverse transcriptase, 10U / μL, 25μL;

[0094] dNTPs, 10 mM, 62.5 μL;

[0095] Betaine (betaine), 25mM, 25μL;

[0096] MgCl 2 Solution, 150mM, 25μL;

[0097] INF-CF3 primer, 5 μm, 25 μL;

[0098] INF-CB3 primer, 5 μm, 25 μL;

[0099] INF-C FIP primer, 10 μm, 25 μL;

[0100] INF-C BIP primer, 10 μm, 25 μL;

[0101] DEPC treated water, 500 μL;

[0102] (3) Staining agent: staining agent HNB (hydroxynaphthol blue), 5mM, 25μL;

[0103] (4) Positive control sample: recombinant plasmid INF-CPMD, 0.1ng / μL, 20μL;

[0104] (5) DEPC treated water, 1mL.

[0105] It should be explained...

Embodiment 3

[0131] The RT-LAMP detection method of influenza C virus using the RT-LAMP detection kit prepared in Example 2 specifically includes the following steps.

[0132] (1) Obtain the RNA sample of the sample to be tested. The specific operation is: immerse the sample to be tested in 300 μL sterile saline, shake and squeeze; add 1 mL of TRIZol to the obtained liquid, and mix well;

[0133] Then add 200 μL of chloroform, mix well and let it stand for 5 minutes, then centrifuge at 10,000 rpm for 5 minutes; take the supernatant and place it in an equal volume of ice-cold (about 0°C) isopropanol, place it at -20°C for 1 hour, and then centrifuge at 10,000 rpm for 5 minutes to obtain the precipitate ;

[0134] The precipitate was washed once with absolute ethanol, dried in the shade, and then dissolved in 10 μL of DEPC-treated water for later use;

[0135] (2) RT-LAMP reaction for detection,

[0136] The volume ratio reference design of 25 μL reaction solution is as follows:

[0137] ...

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Abstract

The invention belongs to the technical field of influenza C virus detection and particularly relates to a patent application of a primer sequence adopted for detecting the influenza C virus by use of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology. A group of primers for LAMP detection of influenza C virus comprises an outer primer pair INF-CF3 / INF-CB3 and an inner primer pair IFN-C FIP / IFN-C BIP; a kit for RT-LAMP detection can be prepared by use of the primer group. In general, based on the principle of existing RT-LAMP technology, the RT-LAMP technology is applied to the influenza C virus detection for the first time; the RT-LAMP technology is widely applicable for both people and livestock and has the advantages of high consistency of operation process, stable and reliable detection result and the like and is of a very important application value to establishing an influenza C virus detection and prevention system.

Description

technical field [0001] The application belongs to the technical field of detection of influenza C virus, and specifically relates to a patent application for a primer sequence used for detection of influenza C virus by using loop-mediated reverse transcription isothermal amplification technology (RT-LAMP). Background technique [0002] The principle of RT-LAMP technology is: design two pairs of primers (a pair of inner primers and a pair of outer primers), specifically recognize six binding regions in the target sequence, Bst Under the catalysis of DNA polymerase and reverse transcriptase AMV, the RNA template can be rapidly, specifically and sensitively amplified through cyclic displacement reaction in a specific constant temperature environment, so as to achieve the purpose of virus detection. [0003] The RT-LAMP amplification is mainly divided into two stages. The first stage is the formation of the dumbbell-shaped amplification initiator: In addition to being complemen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/707C12Q2531/119C12Q2521/107
Inventor 彭夫望吕明张海倩
Owner 郑州博睿医学检验实验室有限公司
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