Amplifying system and method for detecting H1N1 virus RNA

An amplification system and virus technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as being unsuitable for grassroots units and on-site detection, false positives, etc., to achieve real-time monitoring, methods Simple, easy to collect effects

Inactive Publication Date: 2017-12-29
JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RT-LAMP technology requires multiple pairs of primers and high requirements for target genes, which will lead to false positives, and has higher requirements for operating environment and operating skills. These methods are not suitable for application in grassroots units and on-site detection

Method used

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  • Amplifying system and method for detecting H1N1 virus RNA
  • Amplifying system and method for detecting H1N1 virus RNA
  • Amplifying system and method for detecting H1N1 virus RNA

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preparation example Construction

[0057] The preparation method of the amplification system is not particularly limited in the present invention, and it is prepared by a preparation method well known to those skilled in the art. If the amplification system is a freeze-dried powder, the present invention preferably adopts a negative pressure freeze-drying method to prepare the amplification system powder. In the present invention, the preferred freeze-drying pressure is 0.005-0.035KPa, more preferably 0.01-0.02KPa; the freeze-drying temperature is preferably -50-20°C, more preferably -35-10°C; the freeze-drying time is preferably It is 6.5~20h, more preferably 11~18h.

[0058] In the present invention, when the amplification system is used, the raw materials of the amplification system components in freeze-dried powder state are dissolved, and the solvent used for dissolving the raw materials of the amplification system is a reaction buffer. Preferably, the reaction buffer is polyethylene glycol aqueous soluti...

Embodiment 1

[0068]Preparation of Amplification System for Detection of H1N1 Virus RNA

[0069] Prepare an isothermal nucleic acid amplification system in a 200 μL centrifuge tube according to the following ratio, with a volume of 50 μL.

[0070]

[0071]

[0072] Wherein, the sequences of forward primer, reverse primer and fluorescent probe are as follows:

[0073] Forward primer sequence:

[0074] 5'-ATGCGAACAATTCAACAGACACTGTAGACACAG-3',

[0075] Reverse primer sequence:

[0076] 5'-CCTGGGTAACACGTTCCATTGTCTGAACTAGGTG-3';

[0077] The fluorescent probe sequence is:

[0078] 5'-AGAATGTAACAGTAACACTCTGTTAACCTTCTAGAAGACAAGCATAACG-3';

[0079] The above primers and probe sequences were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0080] The amplification system prepared above was freeze-dried in a freeze dryer under negative pressure to form a powder amplification system. The vacuum freeze-drying pressure was 0.02KPa, the freeze-drying temperature was -35°C, and the ...

Embodiment 2

[0082] Preparation of Amplification System for Detection of H1N1 Virus RNA

[0083] Prepare an isothermal nucleic acid amplification system in a 200 μL centrifuge tube according to the following ratio, with a volume of 100 μL.

[0084]

[0085]

[0086] Wherein, the sequences of forward primer, reverse primer and fluorescent probe are as follows:

[0087] Forward primer sequence:

[0088] 5'-ATGCGAACAATTCAACAGACACTGTAGACACAG-3',

[0089] Reverse primer sequence:

[0090] 5'-CCTGGGTAACACGTTCCATTGTCTGAACTAGGTG-3';

[0091] The fluorescent probe sequence is:

[0092] 5'-AGAATGTAACAGTAACACTCTGTTAACCTTCTAGAAGACAAGCATAACG-3';

[0093] The above primers and probe sequences were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0094] The above-prepared amplification system was freeze-dried in a freeze dryer under negative pressure to form a powder amplification system. The vacuum freeze-drying pressure was 0.015KPa, the freeze-drying temperature was -18°C, and t...

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Abstract

The invention relates to the field of molecular biological detection and discloses an amplifying system and method for detecting H1N1 virus RNA. The amplifying system comprises Tris buffer liquid, magnesium acetate, potassium chloride, dithiothreitol, polyethylene glycol, ATP, dNTPs, phosphocreatine, recombinase, singe-strand binding protein, UvsY protein, exonuclease, fluorescent probe, DNA polymerase, revertase, RNA enzyme inhibitor and H1N1 virus specificity primer. The method includes: mixing the amplifying system with H1N1 virus RNA, performing equal-temperature amplifying, and using a fluorescent probe to detect an amplifying product in real time. The method is simple to operate, instruments are minimized and convenient to carry, and the amplifying system is short in amplifying time, high in specificity and sensitivity and suitable for basic-level and on-site detection.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to an amplification system and method for detecting H1N1 virus RNA. Background technique [0002] The so-called "H1N1" is the abbreviation of the virus name, its "H" refers to hemagglutinin (Hemagglutinin), and "N" refers to neuraminidase (Neuraminidase), both of which are antigen names on the virus. It means: a virus with "hemagglutinin (Hemagglutinin) type 1, neuraminidase (Neuraminidase) type 1". There are 1-16 types of hemagglutinin and 1-9 types of neuraminidase. The same series as H1N1 are H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H3N8, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, H12N5, H7N9, H10N8 and so on. Since H1N1 can directly infect humans and cause human illness or death, its public health significance is becoming more and more significant. Strengthening the research and detection of H1N1 has far-reaching significance in reducing world economic los...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 郑伟陈淑丹郑乐怡吕沁风吴忠华郭利川汤赛君王智宏应清界
Owner JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD
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