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Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method

An isothermal nucleic acid amplification and reaction reagent technology, applied in the field of high-sensitivity nucleic acid amplification technology, can solve the problems of large power consumption, cost and application range limitations, and achieve the effect of simple operation, simple operation and low technical requirements

Active Publication Date: 2012-12-12
JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Conventional nucleic acid in vitro amplification technology (PCR technology), due to the need to use expensive PCR equipment and consume a lot of power, its cost and application range are limited

Method used

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  • Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method
  • Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method
  • Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Detection of foot-and-mouth disease virus (FMDV) antigen VP1 gene transgenic tobacco

[0036] First, the FMDV antigen VP1 gene is inserted into the vector p16APT containing the tobacco chloroplast-specific Prrn promoter and the psbA terminator to form the expression cassette of the target gene; then the complete expression cassette is excised by restriction enzymes and Inserted into the vector pTRV containing homologous fragments rpl2-trnH-psbA and trnK-ORF509A of tobacco chloroplast genome to form a complete plasmid expressed by tobacco chloroplast containing FMDV antigen VPI gene.

[0037]On the other hand, cultivate aseptic tobacco (Nicotiana tabacum), take aseptic tobacco seedlings at the 4-6 leaf stage, select expanded leaves of aseptic tobacco seedlings with a diameter of about 3 cm as explants, and place them on a 9 cm sterile culture dish center. There is a thin layer of MS medium containing 0.2mol / L mannitol and 0.2mol / L sorbitol in the petri dish, w...

Embodiment 2

[0106] Example 2 Detection of Mn-SOD Gene Transgenic Tobacco

[0107] In this example, the superoxide dismutase (Mn-SOD) gene was first inserted into the vector pRTL2 containing the tobacco cell nucleus E35S promoter, TEV Leader and 35S terminator to form the expression cassette of the target gene. Then the complete expression cassette is inserted into the tobacco transformation vector Pzp211 through the action of the restriction endonuclease pstI to form a complete plasmid expressing in the tobacco cell nucleus containing the superoxide dismutase (Mn-SOD) gene.

[0108] On the other hand, cultivating aseptic tobacco seedlings: sow the seeds of tobacco leaves on MS medium under aseptic conditions, and take the young leaves of the aseptic seedlings for transformation after about 6-8 weeks. Tobacco leaf discs were infected with Agrobacterium containing the recombinant plasmid. Sterile tobacco leaf discs need to be pre-cultured on MS1 ​​solid medium for 3 days. After the Agroba...

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Abstract

The invention discloses an isothermal nucleic acid amplification reaction reagent which comprises Tris buffer solutions, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, adenosine triphosphate (ATP), deoxy-ribonucleoside triphosphate (dNTPs), phosphocreatine, creatine kinase, a primer group, single-strand binding (SSD) protein, colon bacillus helicase RecQ protein, UvsY protein and DNA polymerase. The invention further discloses an isothermal nucleic acid amplification method which includes steps of extracting DNA or performing inverse transcription after DNA extracting, and adding the isothermal nucleic acid amplification reaction reagent and reacting the mixture at a temperature in a range between 25 DEG C and 45 DEG C for 10 minutes to 60 minutes to complete the nucleic acid amplification. Compared with traditional polymerase chain reaction (PCR) technologies and according to the technology, nearly no professional instrument is needed, the operation is simple, technical requirements for operators are low, and the reaction time only needs about half an hour.

Description

technical field [0001] The present invention relates to a technology for isothermal nucleic acid amplification, specifically a high-sensitivity nucleic acid amplification that utilizes Escherichia coli helicase RecQ protein, UvsY protein, UvsX protein and recombinant Klewnow exo-polymerase to achieve non-PCR instrument dependence technology. Background technique [0002] The use of polymerase chain reaction (full English name: Polymerase Chain Reaction, referred to as PCR) for nucleic acid amplification in vitro is a revolutionary technology developed since 1983, and has been widely used in modern agriculture, medicine and food industry. field. Through this technology, the efficient amplification of trace amounts of nucleic acid can be achieved, and a very small amount of specific nucleic acid sequence molecules can be amplified to a level that can be detected by the instrument. PCR consists of three basic reaction steps: denaturation-annealing (annealing)-extension: ① Den...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 汤赛君于小兰王秀东应清界王智宏程奇
Owner JIANGSU QITIAN GENE BIOTECHNOLOGY CO LTD
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