Color-based loop-mediated reverse-transcription isothermal amplification technique for deteching human infected A H7N9 influenza virus genes

An influenza virus, isothermal amplification technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve the problem of human-to-human transmission, and achieve short detection time, high sensitivity, and high specificity Effect

Inactive Publication Date: 2014-11-19
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transmission route of this virus is mainly through the respiratory tract, and it can also be infected by contact with the secretions or excretions of infected poultry, and can also be infected by direct contact with the virus, but there is no definite evidence of human-to-human transmission.

Method used

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  • Color-based loop-mediated reverse-transcription isothermal amplification technique for deteching human infected A H7N9 influenza virus genes
  • Color-based loop-mediated reverse-transcription isothermal amplification technique for deteching human infected A H7N9 influenza virus genes
  • Color-based loop-mediated reverse-transcription isothermal amplification technique for deteching human infected A H7N9 influenza virus genes

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Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0015] Embodiment 1: RT-LAMP detects the specificity of people's infection with type A H7N9 influenza based on color

[0016] Influenza virus RNA from different sources was extracted according to Qiagen kit instructions. Establish the following amplification reaction system: 1 μL RNA sample, 1 μL Bst2.0 DNA polymerase (8 U / μL), 1 μL MV reverse transcriptase (10 U / μL), 10 μL (2×) reaction solution (Guangzhou Diao Biotechnology Co., Ltd.), 1 μL of HNB (3 mmol / L), 0.8 μL of each of the six primers corresponding to the HA gene [primer concentrations are F3 and B3 (5 pmol / μL), BIP and FIP (40 pmol / μL), Loop-1 and Loop-2 (20 pmol / μL )], supplemented with dH 2 0 to 20 μL. Mix well, react in a water bath or a turbidimeter (Eiken, Japan) at 63°C for 1.5h, and stop the reaction at 85°C for 2min, observe the color change and white precipitate. Only the human influenza A (H7N9) virus circulating this time had specific amplification.

Embodiment approach 2

[0017] Embodiment 2: Sensitivity of color-based RT-LAMP detection of human infection with influenza A (H7N9) virus

[0018] 10-fold serial dilution of human influenza A (H7N9) RNA extracted from samples to 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 and 10 8 Dilution, RT-LAMP detection system is the same as embodiment 1. The sensitivity of HA gene detection can reach 10 7 Dilution RNA, NA gene detection up to 10 5 Dilution RNA.

Embodiment approach 3

[0019] Embodiment 3: RT-LAMP detection based on color The result of human infection with influenza A (H7N9) virus can be judged by naked eyes (observing color change and white precipitation) or colorimetric value

[0020] After the RT-LAMP reaction is completed, observe the color change and the white precipitate at the bottom of the tube with the naked eye. You can also use 20 μL to measure the absorbance at 650 nm. In the positive reaction tube, a white precipitate can be observed at the bottom of the tube with the naked eye, and the color inside the tube changes from purple before the reaction to sky blue after the reaction, and the absorbance at 650nm is ≥0.31.

[0021]

[0022]

[0023]

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Abstract

The invention belongs to the field of application of biotechnology, and relates to application of all levels of disease prevention and control mechanisms, influenza detection network laboratories, sentinel hospitals and the like in detecting and monitoring human A H7N9 influenza virus HA gene and NA gene of fever patient throat swabs and other specimens, particularly application in color-based loop-mediated reverse-transcription isothermal amplification technique (RT-LAMP).Computer software is utilized to analyze conserved sequences of human A H7N9 influenza virus H7 gene and N9 gene to respectively design six primers which are matched with eight bond zones in identified gene target sequence; an HNB (hydroxynaphthol blue) dye is added before the reaction, so that 1.5 hours of amplification can be completed by one step in a 63 DEG C isothermal environment; and finally, the color change and white precipitate on the tube bottom are observed by the naked eyes to determine the detection result. Compared with the traditional RT-PCR (reverse transcription-polymerase chain reaction) process, the method provided by the invention has the advantages of higher safety, higher specificity and higher sensitivity and is more convenient.

Description

field of invention [0001] The invention uses the circle-mediated reverse transcription isothermal amplification technology based on the HNB color to detect the H7 gene and N9 gene of human infection with influenza A (H7N9) virus. It is used for the detection and monitoring of human infection with influenza A (H7N9) virus by disease prevention and control institutions at all levels, influenza detection network laboratories, and sentinel hospitals. Background of the invention [0002] H7N9 influenza virus is a new type of recombinant influenza A virus. Its gene is a genetic reassortment type from wild birds in East Asia and chicken flocks in Shanghai, Zhejiang, and Jiangsu in China. The virus was only found in birds before, but the world's first reported cases of human infection with H7N9 influenza virus were in Shanghai and Anhui at the end of March 2013. The transmission route of this virus is mainly through the respiratory tract, and it can also be infected through contact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/6844
Inventor 马学军舒跃龙聂凯王大燕丁雄
Owner 中国疾病预防控制中心病毒病预防控制所
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