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

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...

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, after the magnetic beads are adsorbed, it is usually necessary to use two kinds of washing liquids for two-step washing. First, wash with a high-salt reagent washing liquid containing a certain proportion of absolute ethanol or isopropanol and other alcohols to remove the particles adsorbed on the magnetic beads. The non-specific impurities of the protein, and then wash the second step with the washing solution of high-proportion absolute ethanol and low-salt reagent to remove the salt ions of the magnetic beads; and, after the washing is completed, due to the use of PCR inhibitors such as ethanol , will affect the subsequent PCR amplification. Therefore, it is often necessary to open the cover for 5 to 10 minutes before elution to remove the ethanol. This operation not only prolongs the extraction operation time, but also directly opens the cover and places it in the air. It is easy to cause cross-contamination between samples

Third, the existing magnetic bead method to extract nucleic acid usually needs to elute the nucleic acid adsorbed by the magnetic bead, then absorb the supernatant, remove the magnetic bead, and then use the nucleic acid dissolved in the supernatant to carry out PCR amplification. It will inevitably cause loss of nucleic acid, especially during the transfer of supernatant and eluted nucleic acid solution, some nucleic acid will be discarded, which is not conducive to the extraction and detection of nucleic acid in trace samples, and indirectly reduces the detection sensitivity

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