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RT-LAMP primer combination and kit for detecting HA gene and NA gene of H7N9 virus

A technology of RT-LAMP and primer combination, applied in the field of medical and health inspection, can solve the problem of no H7N9 virus, etc., and achieve the effects of simple result interpretation, efficient amplification and high specificity

Inactive Publication Date: 2016-08-17
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no RT-LAMP kits for the detection of H7N9 virus and reports on its application in the detection of H7N9 virus at home and abroad

Method used

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  • RT-LAMP primer combination and kit for detecting HA gene and NA gene of H7N9 virus
  • RT-LAMP primer combination and kit for detecting HA gene and NA gene of H7N9 virus
  • RT-LAMP primer combination and kit for detecting HA gene and NA gene of H7N9 virus

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preparation example Construction

[0039] The present invention is used to detect the preparation method of the RT-LAMP kit of H7N9 virus HA gene and NA gene, comprises the following steps:

[0040] 1) Obtain the HA and NA gene sequences from the gene database, perform homology analysis by BLAST software, and obtain the specific conserved target sequences of the HA and NA genes;

[0041] 2) According to the conserved target sequence obtained in step 1), design RT-LAMP primers;

[0042] 3) Configure the RT-LAMP reaction solution according to the RT-LAMP primers obtained in step 2) to form a detection system.

Embodiment 1

[0043] Embodiment 1, design is used to detect the specific RT-LAMP primer combination of H7N9 virus HA gene and NA gene

[0044] First, the H7N9 virus HA gene (GenBank number: KC885956.1) and NA gene (GenBank number: KC885958.1) sequences were retrieved from the American gene database GenBank, and the homology analysis was performed by BLAST software to find specific conserved target sequences. The nucleotide sequence of the HA gene-specific conservative target sequence is shown in sequence 13 in the sequence listing, the nucleotide sequence of the NA gene-specific conservative target sequence is shown in sequence 14 in the sequence listing, and then use LAMP primers according to the conservative target sequence Design software Primer design V4 (http: / / primerexplorer.jp / e / ) was used to design RT-LAMP primers.

[0045] Specifically, the specific RT-LAMP primer combination used to detect the HA gene and NA gene of the H7N9 virus in the present invention includes 6 primers for de...

Embodiment 2

[0052] Embodiment 2, the establishment of the RT-LAMP detection method of H7N9 virus HA gene and NA gene

[0053] 1. Extract the RNA of the sample to be tested

[0054] Use the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) commercial RNA extraction kit to extract the nucleic acid in the sample to be tested. The specific extraction method includes the following steps:

[0055] 1.1 Pipette 560 μL of the prepared buffer AVL (containing carrier RNA, see the QIAamp viral RNA mini kit commercial RNA extraction kit manual) into a 1.5mL centrifuge tube. Adjust the buffer AVL-carrier RNA proportionally according to the actual amount of the sample (see the QIAamp viral RNA mini kit commercial RNA extraction kit manual for details)

[0056] 1.2 Add 140 μL of plasma, serum, urine, cultured cell supernatant or cell-free body fluid to the centrifuge tube containing buffer AVL-carrier RNA, mediate for 15 seconds, and mix well.

[0057] 1.3 Place at room temperature for 10 minutes.

...

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Abstract

The invention discloses an RT-LAMP primer combination and a kit for detecting the HA gene and NA gene of the H7N9 virus. By using the combined primer and kit, the HA gene and NA gene of the H7N9 virus can be quickly, conveniently, efficiently, specifically and sensitively detected under an isothermal condition without complex instruments, sample detection on H7N9 can be better met, and a technical platform is provided to quick screening of the H7N9 virus.

Description

[0001] The present invention is a divisional application of a patent application with an application date of October 15, 2013, application number 201310481406.1, and the title of the invention is "RT-LAMP primer combination and kit for detecting H7N9 virus HA gene and NA gene". technical field [0002] The invention belongs to the field of medical and sanitation testing, and relates to a primer combination and a kit for detecting H7N9 virus HA gene and NA gene, and also relates to an RT-LAMP kit and a detection method for detecting H7N9 virus HA and NA genes and apply. Background technique [0003] In March 2013, three patients with severe lower respiratory tract infection were found in Shanghai and Anhui provinces, respectively. The Chinese Center for Disease Control and Prevention confirmed that they were infected with a new type of avian influenza virus (H7N9). As of April 20, 2013 96 cases of H7N9 infection have been confirmed across the country, including 18 deaths, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 袁静黄留玉刘威杨展崔茜
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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