Kit for nucleic acid extraction with magnetic bead method, magnetic bead and preparation method of magnetic bead

A technology of kit and magnetic bead method, which is applied in the fields of biochemical equipment and methods, DNA preparation, microbial measurement/inspection, etc., can solve the problems of cross-contamination between samples, nucleic acid loss, damage, etc. the effect of time

Active Publication Date: 2021-06-18
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the magnetic bead method mainly uses lysozyme, proteinase K and other biologically active enzymes to incubate and combine the method of breaking the wall of the lysate for nucleic acid separation, and then uses magnetic beads for nucleic acid purification and other operations; this method can be combined with automatic extraction equipment to realize automatic operation , reduce pollution and operational errors caused by operators, and extract nucleic acids with high yield and good purity; however, the extraction steps are cumbersome and the operation cycle is long. The entire extraction takes 1.5 hours to 3 hours, or even longer. It needs to be extracted overnight, which is very difficult Difficult to meet the needs of rapid detection
[0005] Moreover, the existing magnetic bead method nucleic acid extraction kit also has the following disadvantages: First, in the lysis link, it is usually necessary to use biologically active enzymes such as lysozyme and proteinase K in combination with powerful guanidinium salts such as guanidine hydrochloride or guanidine isothiocyanate. Protein denaturant, incubate at 56°C-65°C to destroy the protein and other components in the sample tissue, release the nucleic acid, and then add a certain proportion of isopropanol or absolute ethanol to precipitate the nucleic acid; the general operating time of this process Incubation for 25 minutes to 1 hour, or even overnight, is difficult to meet the needs of rapid detection or on-site detection
Second, afte

Method used

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  • Kit for nucleic acid extraction with magnetic bead method, magnetic bead and preparation method of magnetic bead
  • Kit for nucleic acid extraction with magnetic bead method, magnetic bead and preparation method of magnetic bead
  • Kit for nucleic acid extraction with magnetic bead method, magnetic bead and preparation method of magnetic bead

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0093] The sample preparation process used in the following examples is as follows:

[0094] In this example, in order to better illustrate the performance advantages of the kit in virus sample extraction, porcine transmissible gastroenteritis virus vaccine was used as the representative of RNA virus, and fowlpox virus vaccine was used as the representative of DNA virus, and prepared respectively to simulate virus infection. Serum samples, and then use the kit of this example, other comparison kits or comparison reagents to extract RNA and DNA respectively. Wherein the preparation steps of the serum sample of simulated virus infection are as follows:

[0095] Operation 1: Take 2.5mL goat serum and add it to the porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus triple live vaccine bottle, blow and mix well, and set aside;

[0096] Operation 2: Take 2.5mL goat serum and add it to the fowlpox virus vaccine bottle, blow and mix well, and set asid...

Embodiment 1

[0107] Example 1 Preliminary optimization of cell lysate formula

[0108] In this example, two formulas of cell lysates were designed for preliminary screening of experimental schemes.

[0109] The formula of Scheme 1 is: 25mM Tris-HCl, 10mM EDTA (pH8.0), 1% SDS, 1% 3-[3-(cholamidopropyl) dimethylamino]-1-propanesulfonic acid, 0.5% N - Sodium lauroyl sarcosinate, 1M NaCl, 0.1% Antifoam204, 10% PEG6000.

[0110] The formula of scheme two is: 25mM Tris-HCl, 10mM EDTA (pH8.0), 4M guanidine isothiocyanate, 5vol% Triton X-100, 10g / L 3-[3-(cholamidopropyl) dimethyl Amino]-1-propanesulfonic acid, 5g / L sodium N-lauroyl sarcosinate, 0.28M NaCl, 0.1vol% Antifoam204, 100g / LPEG 6000.

[0111] The washing liquid formula used in this example is 20mM Tris-HCL, 0.5M NaCL, 100g / LPEG6000.

[0112] The magnetic beads of this embodiment are prepared by the following method:

[0113] a) Preparation of nanospheres: weigh 30g FeCl 3 -6H 2 0. 80g of anhydrous sodium acetate and 20g of PEG2000 w...

Embodiment 2

[0131]Embodiment 2 cell lysate formulation optimization

[0132] In order to further optimize the formulation of the cell lysate, this example is aimed at the guanidine isothiocyanate, TritonX100, PEG6000, 3-[3-(cholamidopropyl) dimethylamino]-1- The concentration of main components such as propanesulfonic acid was optimized. The optimization scheme is shown in Table 3. Formulation 9 in Table 3 will crystallize when placed at room temperature after preparation, and is not suitable for cell lysate.

[0133] Table 3 Cell lysate formulation optimization

[0134] Numbering component 1 component 2 Component 3 Component 4 Component 5 Component 6 Component 7 Component 8 Component 9 Recipe 1 2M 1vol% 0 5 5 0.28M 0.1vol% 10mM 10mM Recipe 2 2M 2vol% 100 10 5 0.28M 0.1vol% 10mM 10mM Recipe 3 2M 5vol% 200 50 5 0.28M 0.1vol% 10mM 10mM Recipe 4 3M 5vol% 0 10 5 0.28M 0.1vol% 25mM 10mM Re...

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Abstract

The invention discloses a kit for nucleic acid extraction with a magnetic bead method, magnetic beads and a preparation method of the magnetic beads. The kit for nucleic acid extraction with the magnetic bead method comprises a cell lysis solution, the magnetic beads and a washing solution, wherein the cell lysis solution contains guanidine isothiocyanate, Triton X-100, 3-[3-(cholamidopropyl) dimethylamino]-1-propanesulfonic acid and N-sodium lauroyl sarcosinate. According to the kit for nucleic acid extraction with the magnetic bead method, the components of the lysis buffer solution are optimized, so that cells can be quickly and fully lysed, proteins can be quickly and fully denatured, the proteins and nucleic acid are effectively separated, and the nucleic acid is released; and the working efficiency of the cell lysis solution is improved, and the nucleic acid extraction time is shortened.

Description

technical field [0001] The present application relates to the field of nucleic acid extraction, in particular to a kit for magnetic bead nucleic acid extraction, magnetic beads and a preparation method thereof. Background technique [0002] Nucleic acid is the substance that carries genetic information in cells, and plays an extremely important role in the heredity, variation and protein biosynthesis of organisms. With the rapid development of molecular biology detection technology, molecular diagnostic technology based on nucleic acid information has become a new clinical method and idea for effective disease diagnosis, especially in the fields of infectious diseases, tumors and genetic diseases. The newly developed molecular diagnostic technology for infectious diseases not only effectively makes up for the shortcomings of traditional methods, but also provides clinicians with information on bacterial, fungal or viral infections, results of drug resistance genes of pathoge...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806C12Q2527/125C12Q2563/143C12Q2563/149Y02A50/30
Inventor 黄超杰鲁津津黄硕路通耿春雨陈芳蒋慧
Owner MGI TECH CO LTD
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