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Cell lysis solution directly used for polymerase chain amplification reaction

A technology of cell lysate and chain amplification, which is applied in the determination/inspection of microorganisms, DNA preparation, recombinant DNA technology, etc. It can solve the problems affecting the amplification, achieve high extraction efficiency, short time consumption, and broaden the application field effect

Pending Publication Date: 2021-12-21
SHANGHAI CRIMINAL SCI TECH RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these compounds are not contained in conventional commercially available PCR buffers, so they may affect the amplification of crude nucleic acid samples after sodium hydroxide lysis in conventional commercially available PCR buffers

Method used

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  • Cell lysis solution directly used for polymerase chain amplification reaction
  • Cell lysis solution directly used for polymerase chain amplification reaction

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Experimental program
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Effect test

Embodiment 1

[0020] Preparation of deep eutectic solvent: first accurately weigh 6.8 grams of imidazole, then add it to a beaker containing 23.2 grams of hexanoic acid, then use a magnetic stirrer to stir at a temperature of 80 ° C until a clear and transparent liquid is formed, and then mix it with the Add 3.5 milliliters of deionized water to the solution, stir for 10 minutes, and set aside.

[0021] Dilute human blood 500 times with normal saline, take 1 microliter of the diluted solution and drop it on a glass slide, let it dry naturally, wipe the blood spot with a cotton swab or a flocking swab, cut off the wiped cotton swab, and put it in Add 100 microliters of imidazole / caproic acid deep eutectic solvent into a 2.0ml centrifuge tube, completely immerse the sample, then heat, incubate and shake at 50°C for 20 minutes, draw 2 microliters of the lysate, and directly add STR- Amplification was carried out in a PCR system, and the total volume of amplification was 10 microliters. At the...

Embodiment 2

[0024] Preparation of deep eutectic solvent: First, accurately weigh 13.6 grams of imidazole, then add it to a beaker containing 28.8 grams of octanoic acid, stir on a magnetic stirrer at 60°C until a clear and transparent liquid is formed, and then add 9.0 milliliters of deionized water, after stirring for 10 minutes, set aside.

[0025] Dilute human blood 500 times with normal saline, take 1 microliter of the diluted solution and drop it on a glass slide, let it dry naturally, stick the blood spot with scotch tape, cut off part of the sticky tape, and put it into 2.0ml Add 100 microliters of imidazole / octanoic acid deep eutectic solvent to the centrifuge tube, completely immerse the sample, then heat and incubate and shake at 60°C for 10 minutes, absorb 3 microliters of the lysate, and directly add it to the STR-PCR system Amplify with a total volume of 10 microliters. At the same time, take 1 microliter of the diluted blood for DNA magnetic bead reagent cleavage and purifi...

Embodiment 3

[0028] Preparation of deep eutectic solvent: first accurately weigh 136 grams of imidazole, then add it into a beaker containing 88 grams of butyric acid, stir on a magnetic stirrer at 70°C until a clear and transparent liquid is formed, and then add 30 milliliters to the system to Ionized water, after stirring for 10 minutes, set aside.

[0029] Dilute human blood 500 times with normal saline, take 1 microliter of the diluted solution and drop it on a glass slide, let it dry naturally, stick the blood spot with scotch tape, cut off part of the sticky tape, and put it into 2.0ml Add 100 microliters of imidazole / butyric acid deep eutectic solvent to the centrifuge tube, completely immerse the sample, then heat, incubate and shake at 55°C for 15 minutes, absorb 1 microliter of the lysate, and directly add it to the STR-PCR system Amplification was performed in a total volume of 10 microliters. At the same time, take 1 microliter of the diluted blood for DNA magnetic bead reagen...

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Abstract

The invention discloses a cell lysis solution directly used for polymerase chain amplification reaction. The cell lysis solution is characterized by comprising the following steps: firstly preparing a deep eutectic solvent, then adding a trace quantity of biological detection material into the deep eutectic solvent, performing incubating at the temperature of 50-60 DEG C, performing shaking for 10-20 minutes, and directly adding the trace quantity of deep eutectic solvent into a PCR amplification system, wherein the quantity of the eutectic solvent does not exceed 30% of the total amplification volume, and the mass ratio of the detection material to the ionic liquid is (1: 99)-(1: 50). According to a method for extracting the DNA by cracking cells through the deepeutectic solvent, a deep eutectic solvent preparation method has the advantages of being easy to operate, short in consumed time, low in cost and high in extraction efficiency, the application field of the deep eutectic solvent is widened, PCR detection can be performed through a trace amount of lysate of a biological sample, and the method used for the invention is equivalent to or slightly superior to a magnetic bead reagent cracking purification method under certain conditions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell lysate directly used in a polymerase chain amplification reaction. Background technique [0002] DNA is an important genetic information substance of organisms. Its detection and analysis have been widely used in various fields, especially the most widely used polymerase chain amplification reaction (PCR) in clinical disease diagnosis, food safety, environmental governance, animal epidemics, etc. , especially in forensic identification has become an indispensable technical means. [0003] Biological samples in the field of criminal investigation have their particularity, and the samples collected on the spot are often trace amounts or even trace amounts. Although in theory a DNA template can complete the PCR reaction, in actual work a certain amount of DNA templates and good quality are required to complete the PCR process. In particular, STR typing technology is often used ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/686
CPCC12N15/1003C12N15/1013C12Q1/686
Inventor 刘亚楠孟宪江张静施嘉骏
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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