Isothermal amplication rapid detection method of H7N9 avian influenza virus

A bird flu virus, technical detection technology, applied in the field of virus inspection, can solve the problem that cannot meet the increasing import and export goods

Inactive Publication Date: 2013-09-18
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] In order to improve the efficiency of food detection, save time, and solve the problem that traditional detection methods cannot meet the growing status of import and export goods, the present invention has finally explored a method for detecting influenza virus using the loop-mediated isothermal amplification method through experimental testing. The method has the characteristics of rapid detection, convenience, low cost, and is suitable for application in field sites and grassroots departments.

Method used

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  • Isothermal amplication rapid detection method of H7N9 avian influenza virus
  • Isothermal amplication rapid detection method of H7N9 avian influenza virus
  • Isothermal amplication rapid detection method of H7N9 avian influenza virus

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preparation example Construction

[0041] 1.3 RNA extraction and cDNA preparation

[0042]Take the EP tube treated with DEPC water, add 1mL TRIzol and 200μL inactivated virus solution, mix well and place at room temperature for 5min; add 200μL chloroform, shake vigorously for 30s, let stand at room temperature for 3min, and centrifuge at 4°C for 15min to obtain a layered solution; Transfer the supernatant to a clean EP tube, add 500 μL of isopropanol, let stand at -20°C for 30 minutes, and centrifuge at 4°C for 15 minutes; remove the supernatant; add 1 mL of 75% DEPC ethanol, vortex, and centrifuge at 4°C for 10 minutes; The supernatant was dried in air for 5 min; 50 μL of DEPC water was added to the test tube. Reverse transcription was performed according to the M-MuLV reverse transcriptase process to obtain cDNA, which was stored at -20°C for later use.

[0043] 1.4 Construction of positive recombinant plasmids

[0044] Using specific primers for H7N9 avian influenza HA gene and NA gene, the cDNA was amplif...

Embodiment 1

[0059] Amplification of HA Gene and NA Gene of H7N9 Subtype Avian Influenza Virus

[0060] Take the inactivated virus liquid, extract the virus genome, and carry out reverse transcription according to the M-MuLV reverse transcriptase process to obtain cDNA. Use specific primers to amplify the HA gene and NA gene of the H7N9 subtype avian influenza virus. The results are as follows: figure 1 As shown, 1683bp of HA gene and 1398bp of NA gene were obtained after amplification, which was consistent with the theoretical amplification value.

Embodiment 2

[0062] Identification of positive recombinant plasmids

[0063] The amplified HA and NA genes were cloned into the pGEM-T easy vector and transformed into Escherichia coli TOP10 competent cells, the plasmid was extracted from the purified and amplified recombinant bacteria, and identified by enzyme digestion. The HA recombinant plasmid was used for EcoR I, NotI double cutting, NA recombinant plasmid single cutting with sfi I, the result is as follows figure 2 As shown, the constructed HA and NA recombinant plasmids were digested to obtain the target bands consistent with the theory. The constructed positive plasmid was sequenced, and the sequencing results showed that the amplified sequence was 100% consistent with the H7N9 sequence registered in the GISAID database, indicating that the constructed plasmid did contain the target gene fragment and could be used as a positive plasmid.

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Abstract

The invention discloses a method for detecting H7N9 subtype avian influenza virus through loop-mediated isothermal amplification (LAMP). The method is used for detecting avian influenza virus in the fields of foods and raw materials, environmental samples, medical samples and health and disease prevention. According to the technical scheme, the method is characterized in that a specific primer is designed with regional gene sequences respectively coded as HA and NA in an avian influenza genome serving as target sequences, and a reaction system is optimized, so as to perform target gene specific amplification. The method only needs a constant-temperature device, does not need the thermal denaturation and long-term temperature cycle of a template, realizes the direct observation of the results, and has the characteristics of being low in cost, high in efficiency and simple in operation. The LAMP detection method of the H7N9 subtype avian influenza virus nucleic acid, created by the invention, has the characteristics of being high in specificity, high in sensitivity and convenient and quick, can be carried out in a grass-roots level or a small test site, and brings a novel technology and method for detecting the H7N9 subtype avian influenza virus.

Description

technical field [0001] The invention relates to a virus detection technology, in particular to a method for detecting influenza virus, especially H7N9 avian influenza virus, by using a loop-mediated isothermal amplification method. Background technique [0002] H7N9 avian influenza is a poultry disease caused by the H7N9 subtype avian influenza virus. Across species, human patients infected with the H7N9 virus were found in Shanghai, Jiangsu, and several provinces in China. This is the first time the virus has infected humans in the world. As of May 11, a total of 130 confirmed cases of human infection with H7N9 bird flu have been reported in mainland China, including 33 deaths. H7N9 bird flu has become a new test for the public health mechanism of my country and neighboring countries and regions. At the same time, due to the outbreak of H7N9 bird flu, the domestic poultry breeding industry has been greatly affected. Neighboring countries Vietnam and Indonesia have banned t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 王乃福赵祥平张立怀吴冬雪王玉玲董志珍郑文杰黄晨王建华陈本龙
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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