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Method for extracting nucleic acids from biological sample

A technology for extracting nucleic acids from biological samples, applied in biochemical equipment and methods, dissolution of microorganisms, measurement/inspection of microorganisms, etc., can solve problems such as recovery rate less than 1%, low purification rate, recovery rate less than 0.1%

Active Publication Date: 2018-10-23
CT BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, MoBio Laboratories Inc. (Carlsbad, USA) proposed a method for flocculating impurities by using trivalent cations (such as aluminum), and an important feature of concentrating / purifying DNA by using Boom technology (Boomtechnology) is that by using Al 3+ and NH 4 3+ etc. to precipitate impurities, which is very effective for complex samples such as feces or soil and shows the highest efficiency among existing techniques, but its recovery rate is only less than 1%
Qiagen, Inc. (Hilden, Germany) proposed a method for adsorbing and removing materials that inhibit polymerase chain reaction (PCR) by using a carbohydrate-based adsorption matrix, but this method is not suitable for purifying complex samples such as stool samples. ) is very effective, but its recovery rate is less than 0.1%
In addition, ZymoResearch Corp. (Irvine, USA) proposed a method for extracting nucleic acids present in feces by applying physical force using 500 μm beads, but there were other problems due to failure to remove various impurities present in feces samples. The limitation of low purification rate

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  • Method for extracting nucleic acids from biological sample
  • Method for extracting nucleic acids from biological sample
  • Method for extracting nucleic acids from biological sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Isolation and purification of stool samples

[0053] 400 μl of 10 mM tris-hydrochloric acid (pH 8 or greater) as a buffer solution and sodium dodecyl sulfate (SDS, AMRESCO) as a surfactant were added to a 200 mg stool sample at a concentration of 6% by volume based on the buffer solution ( v / v), in order to break the stool sample and the target to be tested and measured, 0.4 g of glass beads (DAIHAN Scientific Co., Ltd.) with a size of 100 μm were put into the mixture, followed by bead beating at 50 Hz (Scientific Industries) 5 minutes. Thereafter, centrifugation (LABOGENE) was performed at 8,000 rpm for 1 minute, the supernatant was transferred to a tube, and 2.5 M sodium sulfate (Na2) was added to the separated supernatant. 2 SO 4 , Sigma-Aldrich) solution, the resulting mixture was stirred to be uniformly mixed, and then centrifuged again at 8,000 rpm for 1 minute. After 1 minute, when impurities are lifted to the top of the liquid, transfer only the so...

Embodiment 2

[0054] Example 2. Determination of Bacterial and Nucleic Acid Recovery from Fecal Samples as a Function of Surfactant Concentration

[0055] 2.1. Isolation and purification of stool samples by varying the concentration of surfactants

[0056] To determine bacterial and nucleic acid recoveries from fecal samples according to SDS concentration, the entire procedure was performed as in Example 1, except that 10 6 Salmonella (salmonella, ATCC) prepared at a concentration of cfu / ml or at 10 6 Extracted Staphylococcus aureus (ATCC) DNA at a concentration of cfu / ml was added to a 200 mg stool sample, and SDS was added so that their respective concentrations became 1.2%, 3%, 4%, 5%, 6% based on the volume of buffer solution %, 10% and 20% (see figure 1 D).

[0057] 2.2. Measurement of total bacterial mass, bacterial recovery and nucleic acid recovery from isolated samples

[0058] By extracting nucleic acids using the Boom technique, in order to measure the total bacterial mass, t...

Embodiment 3

[0060] Example 3. Determination of bacterial recovery from fecal samples as a function of sodium sulfate solution concentration

[0061] 3.1. Isolation and purification of stool samples by varying the concentration of sodium sulfate solution

[0062] To determine the amount of sodium sulfate (Na2SO4) that can extract nucleic acids while efficiently isolating fecal samples 2 SO 4 ) solution, the entire procedure was carried out as in Example 1, except that the concentration of sodium sulfate solution (0.25M (0.1×), 1.25M (0.5×) and 2.5M (1×)) was changed to to which sodium sulfate solution is added (see figure 2 B).

[0063] 3.2. Measurement of total bacterial mass from isolated samples

[0064] As a result of measuring total bacterial recovery by the method described in Example 2.2, the highest total bacterial recovery was shown when a 2.5M (final concentration 0.7M) sodium sulfate solution was used (see figure 2 A), and only the 2.5M concentration showed the result tha...

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Abstract

The present invention relates to a method for extracting nucleic acids from a biological sample, wherein the extraction method presents a novel method for effectively extracting nucleic acids. When nucleic acids are extracted from conventional biological samples, the purification rate is low since various impurities present in the biological samples have not been properly removed. However, according to the present invention, provided is a method for extracting nucleic acids from a biological sample of which the recovery rates of bacteria and nucleic acids are enhanced, by adding a surfactant and a sodium sulfate (Na2SO4) solution in a biological sample disruption step and a purification step, thereby enabling pathogens to be detected with greater sensitivity and accuracy.

Description

technical field [0001] The present invention relates to a method for extracting nucleic acid from a biological sample, and more particularly, to a method for extracting nucleic acid by effectively using a biological sample different from existing commercial methods for extracting nucleic acid Improve nucleic acid recovery by efficiently removing impurities from complex environments and biological samples. Background technique [0002] Recently, various methods for sensitive and accurate detection of pathogens have been developed, such as selective medium methods, specific reaction methods using specific antibodies or antigens, or nucleic acid amplification methods. The detection limit of the above methods has been rapidly developed by introducing amplification of the detection signal using several biological or chemical materials such as nanoparticles, enzymes, chemiluminescent reagents or liposomes, and among them, several methods have been commercialize. [0003] However...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10
CPCC12N15/1017C12N15/1003C12Q1/6806C12Q2523/32C12Q2523/305C12N1/066C12N13/00C12N2509/10C12N2523/00
Inventor 闵畯泓白昶润
Owner CT BIO CO LTD
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