Nucleic acid releasing agent, nucleic acid PCR (polymerase chain reaction) amplification method and PCR amplification kit

A nucleic acid release agent and nucleic acid technology, applied in the field of molecular biology, can solve problems such as instability, and achieve the effects of preventing degradation, good repeatability, and high detection sensitivity

Active Publication Date: 2019-03-01
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the structure of RNA is a single-stranded structure, which is unstable and easy to degrade, the requirements in the sample processing process are very high, and more complicated methods are required for pretreatment and nucleic acid extraction and purification of the RNA sample to be amplified to obtain pure After the nucleic acid is removed, the test can be carried out to obtain stable results

Method used

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  • Nucleic acid releasing agent, nucleic acid PCR (polymerase chain reaction) amplification method and PCR amplification kit
  • Nucleic acid releasing agent, nucleic acid PCR (polymerase chain reaction) amplification method and PCR amplification kit
  • Nucleic acid releasing agent, nucleic acid PCR (polymerase chain reaction) amplification method and PCR amplification kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～25

[0076] Embodiments 1-25: Prepare 25 parts of enterovirus throat swab samples (virus preservation solution matrix), take 100 μ L samples respectively and centrifuge at 12000 rpm / min for 10 min, discard the supernatant and add 50 μ L nucleic acid release agent, let stand for 10 min, and then Mix 10 μL with 40 μL PCR reaction solution for real-time fluorescent quantitative PCR amplification. The amplification curve is as follows: figure 1 shown. The nucleic acid release agents used in Examples 1-25 include Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, betaine, bovine serum albumin, Proteinase K, Lithium Lauryl Sulfate and Sodium Hydroxide. Wherein, the molar concentration of Tris-HCl is 0.5mM, the molar concentration of sodium chloride is 500mM, the volume percentage of Tween 20 is 0.1%, the volume percentage of Triton X-100 is 3%, ethylphenyl polyethylene glycol The volume percentage of diol is 0.1%, the mass concentrat...

Embodiment 26～50

[0088] Examples 26-50: Prepare 25 respiratory tract sputum samples (normal saline matrix), take 5 μL samples respectively, add 5 μL nucleic acid release agent, let stand for 10 minutes, then add 40 μL PCR reaction solution and mix for real-time fluorescence quantitative PCR amplification, amplification curve like figure 2 shown. The nucleic acid release agents used include Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, betaine, bovine serum albumin, proteinase K, dodecyl Lithium Alkyl Sulfate and Sodium Hydroxide. Wherein, the molar concentration of Tris-HCl is 500mM, the molar concentration of sodium chloride is 20mM, the volume percentage of Tween 20 is 2%, the volume percentage of Triton X-100 is 0.1%, ethylphenyl polyethylene glycol The volume percentage of alcohol is 3%, the mass concentration of potassium chloride is 5mg / mL, the mass concentration of sodium hydroxide is 50mg / mL, the mass concentration of betaine...

Embodiment 51～90

[0100] Examples 51-90: Prepare 20 HCV serum samples, take 15 μL samples respectively, add 5 μL nucleic acid release agent, let stand for 10 minutes, then mix with 30 μL PCR reaction solution for real-time fluorescent quantitative PCR amplification, the amplification curve is as follows image 3 shown. The nucleic acid releasing agents used include Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethylphenyl polyethylene glycol, betaine, bovine serum albumin and sodium hydroxide. Wherein, the molar concentration of Tris-HCl is 200mM, the molar concentration of sodium chloride is 250mM, the volume percentage of Tween 20 is 1%, the volume percentage of Triton X-100 is 2%, ethylphenyl polyethylene glycol The volume percentage of alcohol is 2%, the mass concentration of potassium chloride is 9mg / mL, the mass concentration of sodium hydroxide is 25mg / mL, the mass concentration of betaine is 10mg / mL, and the mass concentration of bovine serum albumin is 60mg / mL....

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Abstract

The invention relates to a nucleic acid releasing agent, a nucleic acid PCR (polymerase chain reaction) amplification method and a PCR amplification kit. The nucleic acid releasing agent comprises Tris-HCl, sodium chloride, potassium chloride, Tween 20, Triton X-100, Nonidet P-40 and strong base; the molar concentration of the Tris-HCl is 0.5-500mM, the molar concentration of the sodium chloride is 20-500mM, the volume percentage of the Tween 20 is 0.1-2%, the volume percentage of the Triton X-100 is 0.1-3%, the volume percentage of the Nonidet P-40 is 0.1-3%, the mass concentration of the potassium chloride is 5-8mg / mL, and the mass concentration of the strong base is 2-50mg / mL. The nucleic acid releasing agent has the advantages that RNA-containing samples can directly release RNAs at the room temperature, and can be directly mixed with PCR reaction liquid to achieve PCR amplification, so that amplification can be completed without complex nucleic acid extraction and purification processes.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a nucleic acid releasing agent, a nucleic acid PCR amplification method and a PCR amplification kit. Background technique [0002] PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) is a molecular biology technique used to amplify specific nucleic acid fragments. The purpose of detecting trace amounts of nucleic acid. Among them, the commonly used PCR method in medical diagnosis is mainly the real-time fluorescent quantitative PCR (qPCR) method based on dual fluorescent probes. The targets for in vitro diagnosis using qPCR method mainly include human genomic DNA, DNA viruses, bacteria, fungi and RNA viruses, etc. . However, since the structure of RNA is a single-stranded structure, which is unstable and easy to degrade, the requirements in the sample processing process are very high, and more complicated methods are required for pretreatment and nucleic acid extraction ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q1/6806C12Q1/6846C12Q2527/125C12Q2527/137C12Q2527/119C12Q2563/137C12N9/58C12Q1/6848C12Y304/21014
Inventor 纪博知张文曲邓中平戴立忠
Owner SANSURE BIOTECH INC
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