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Amplification system and method for detecting RNA of Japanese encephalitis virus

A technology of Japanese encephalitis virus and amplification system, which is applied in the field of molecular biology detection, can solve the problems of false positives, unsuitable for grassroots units and on-site detection, etc., and achieves the effect of simple method, real-time monitoring and simple operation

Active Publication Date: 2017-12-26
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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  • Amplification system and method for detecting RNA of Japanese encephalitis virus
  • Amplification system and method for detecting RNA of Japanese encephalitis virus
  • Amplification system and method for detecting RNA of Japanese encephalitis virus

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preparation example Construction

[0056] The present invention does not specifically limit the preparation method of the amplification system, and the preparation method is well-known to those skilled in the art for preparation. If the amplification system is a freeze-dried powder, the present invention preferably adopts negative pressure freeze-drying to prepare the amplification system powder. In the present invention, the pressure of freeze drying is preferably 0.005 to 0.035KPa, more preferably 0.01 to 0.02KPa; the temperature of freeze drying is preferably -50 to 20°C, more preferably -35 to 10°C; the time of freeze drying is preferably It is 6.5 to 20 hours, more preferably 11 to 18 hours.

[0057] In the present invention, when the amplification system is in use, the amplification system component raw materials in a freeze-dried powder state are dissolved, and the solvent used to dissolve the amplification system raw materials is a reaction buffer. Preferably, the reaction buffer is an aqueous polyethylen...

Embodiment 1

[0067] Preparation of amplification system for detecting Japanese encephalitis virus RNA

[0068] Prepare the isothermal nucleic acid amplification system in a 200μL centrifuge tube according to the following ratio, with a volume of 50μL.

[0069]

[0070]

[0071] The sequences of the forward primer, reverse primer and fluorescent probe are as follows:

[0072] Forward primer sequence: 5’-ATCCTYCTGCTGTTGGTCGCTCCGGCTTA-3’,

[0073] Reverse primer sequence: 5'-ATCATRCGGACRTCYAATGTTGGTTTGTCG-3';

[0074] The fluorescent probe sequence is:

[0075] 5’-AAGGAGCYAGTGGAGCYACTTGGGTGGACYTGGTGYTAGAAGGAGA-3’;

[0076] The above primer and probe sequences were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.

[0077] 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 freeze-drying time was 10h. Use reaction b...

Embodiment 2

[0079] Preparation of amplification system for detection of Japanese encephalitis virus RNA

[0080] Prepare the isothermal nucleic acid amplification system in a 200μL centrifuge tube according to the following ratio, with a volume of 100μL.

[0081]

[0082]

[0083] The sequences of the forward primer, reverse primer and fluorescent probe are as follows:

[0084] Forward primer sequence: 5’-ATCCTYCTGCTGTTGGTCGCTCCGGCTTA-3’,

[0085] Reverse primer sequence: 5'-ATCATRCGGACRTCYAATGTTGGTTTGTCG-3';

[0086] The fluorescent probe sequence is:

[0087] 5’-AAGGAGCYAGTGGAGCYACTTGGGTGGACYTGGTGYTAGAAGGAGA-3’;

[0088] The above primer and probe sequences were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.

[0089] The amplification system prepared above was freeze-dried in a freeze dryer under negative pressure to become a powder amplification system. The vacuum freeze-drying pressure was 0.015KPa, the freeze-drying temperature was -18°C, and the freeze-drying time was 14h. Use rea...

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Abstract

The invention relates to the molecular biological detection field and discloses an amplification system and a method for detecting RNA of Japanese encephalitis virus. The amplification system contains the following component: a Tris buffer solution, magnesium acetate, potassium chloride, dithiothreitol, polyethylene glycol, ATP, dNTPs, creatine phosphate, recombinase, single stranded binding protein, UvsY protein, exonuclease, a fluorescent probe, DNA polymerase, reverse transcriptase, an RNA enzyme inhibitor and a Japanese encephalitis virus specific primer. The amplification system and RNA of the Japanese encephalitis virus are mixed and are subjected to isothermal amplification, and an amplification product is detected in real time by virtue of a fluorescence probe method. The method provided by the invention has the beneficial effects that the operation is simple, an instrument is miniaturized and convenient to carry, the amplification time is short, and the method is high in specificity and sensitivity and suitable for basic level and field 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 Japanese encephalitis virus RNA. Background technique [0002] Japanese encephalitis virus is spherical, 40nm in diameter, with a core composed of capsid protein (C) and nucleic acid inside, covered with a lipid-containing envelope, and the surface has envelope glycoprotein (E) thorns Proud, that is, viral hemagglutinin, there is an inner membrane protein (M) in the envelope, which participates in the assembly of the virus. The viral genome is a single-stranded positive-stranded RNA with a full length of 11 kb. It encodes structural proteins C, M, E and non-structural proteins NS1-NS5 from 5'to 3'. The viral RNA directly functions as mRNA in the cytoplasm and translates Structural proteins and non-structural proteins assemble and mature in the rough endoplasmic reticulum of the cytoplasm, and then bud and release. If a human...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2527/125C12Q2522/101C12Q2521/507C12Q2521/319C12Q2563/107
Inventor 陈淑丹郑伟吕沁风郑乐怡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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