KR-20260062954-A - Nucleic acid purification method and kit for nucleic acid purification

Abstract

The present invention aims to provide a method for purifying nucleic acid from a sample containing nucleic acid in a short time and with high purity without using a proton-type zeolite, and a kit for such purification. The nucleic acid purification method of the present invention comprises a process of contacting a sample containing nucleic acid with a non-proton-type zeolite.

Inventors

• 나카가와 시미즈 유키

Assignees

• 에이껜 가가꾸 가부시끼가이샤

Dates

Publication Date

20260507

Application Date

20240813

Priority Date

20230901

Claims (15)

1. A method for purifying nucleic acid comprising a process of contacting a sample containing nucleic acid with a non-proton type zeolite.
2. In paragraph 1, A purification method wherein the above sample is derived from at least one specimen selected from the group consisting of blood, bodily fluid, urine, feces, sputum, saliva, nasal discharge, smear fluid, amniotic fluid, and mouthwash.
3. In paragraph 1 or 2, A purification method in which, in the above contact process, at least a portion of the nucleic acid is not adsorbed onto a non-proton type zeolite.
4. In paragraph 1 or 2, A purification method wherein the above-mentioned non-proton type zeolite is at least one selected from the group consisting of a ferrierite type zeolite, a ZSM-5 type zeolite, a Y type zeolite, and a beta type zeolite.
5. In paragraph 4, A purification method wherein the above-mentioned non-proton type zeolite is at least one selected from the group consisting of ferrierite- NH4 type zeolite, ferrierite-K type zeolite, ZSM-5- NH4 type zeolite, Y-Na type zeolite, and beta- NH4 type zeolite.
6. In paragraph 2, A purification method further comprising, prior to the above contact process, a process for obtaining a sample containing the nucleic acid by mixing the sample with a nucleic acid extraction reagent containing a surfactant and/or heat-treating the sample.
7. In paragraph 6, A purification method in which the above surfactant contains an anionic surfactant.
8. In Paragraph 7, A purification method in which the above-mentioned anionic surfactant is dodecyl sulfate.
9. In paragraph 1 or 2, A purification method in which the nucleic acid is RNA and/or DNA.
10. A kit for purifying nucleic acids equipped with a non-proton type zeolite.
11. In Paragraph 10, A kit in which the above-mentioned non-proton type zeolite is at least one selected from the group consisting of a ferrierite type zeolite, a ZSM-5 type zeolite, a Y type zeolite, and a beta type zeolite.
12. In Paragraph 11, A kit in which the above-mentioned non-proton type zeolite is at least one selected from the group consisting of ferrierite- NH4 type zeolite, ferrierite-K type zeolite, ZSM-5- NH4 type zeolite, Y-Na type zeolite, and beta- NH4 type zeolite.
13. In Paragraph 10, The above kit is a kit for purifying nucleic acids derived from at least one specimen selected from the group consisting of blood, fluid, urine, feces, sputum, saliva, nasal discharge, smear fluid, amniotic fluid, and mouthwash, and further comprises a nucleic acid extraction reagent comprising a surfactant.
14. In Paragraph 13, A kit in which the above surfactant contains an anionic surfactant.
15. In Paragraph 14, Kit in which the above-mentioned anionic surfactant is dodecyl sulfate.

Description

Nucleic acid purification method and kit for nucleic acid purification The present invention relates to a nucleic acid purification method and a kit for nucleic acid purification. Currently, nucleic acid amplification technologies, represented by PCR and LAMP methods, have permeated all fields of biology, including molecular biology and medicine, and are widely used in applications such as genetic diagnosis, DNA analysis, food inspection, environmental hygiene inspection, and animal and plant inspection. When amplifying nucleic acids in a sample, it is typically necessary to extract and purify the nucleic acids from the sample. Methods for extracting and purifying nucleic acids from a sample include, for example, using commercially available nucleic acid extraction kits. On the other hand, while using a general commercially available nucleic acid extraction kit (e.g., QIAamp Viral RNA Mini Kit (manufactured by Qiagen)) yields nucleic acids of high purity, it requires multiple devices such as high-speed centrifuges and heating heaters, making it difficult to use the kit unless the operating environment is equipped with such facilities. Furthermore, the process is cumbersome as it involves dozens of steps, requiring approximately one hour for the extraction and purification of nucleic acids (e.g., Non-patent Literatures 1-2). In nucleic acid amplification technology, when the number of test samples is large, it is desirable to prepare samples suitable for nucleic acid amplification in a short time; therefore, extraction and purification methods that involve cumbersome operations are difficult to apply. Furthermore, while commercially available nucleic acid extraction kits (e.g., Kaneka Simple DNA Extraction Kit, Template Prepper for DNA, Nippon Jinsa, ISOSPIN Viral RNA) allow for rapid extraction and purification of nucleic acids, the purity of the extracted nucleic acids may be low. Moreover, even when using such kits, heating operations may be required or the operation process may involve dozens of steps, making it difficult to claim that nucleic acids can be extracted and purified simply and quickly with high purity. In light of these issues, the market demands a method that extracts and purifies nucleic acids with high purity simply and quickly without requiring large equipment. In addition, methods for extracting and purifying nucleic acids from cell lysates, etc., using zeolites are also known. For example, Patent Document 1 discloses a method for preparing a sample for nucleic acid amplification used for amplifying nucleic acids contained in a biological sample, comprising an extraction step of adding a nucleic acid extraction reagent containing an anionic surfactant and/or alkali to a biological sample to obtain a nucleic acid extract, and a step of contacting the nucleic acid extract with a zeolite, wherein the zeolite is a proton-type zeolite and the substance adsorbed to the zeolite is removed. Patent Document 2 discloses a method for isolating nucleic acids from a biological solution (e.g., cell lysate), wherein a pretreatment method of a clarified lysate using a zeolite is disclosed, and it is described that by adding a zeolite to a cell lysate treated with an alkaline reagent such as sodium dodecyl sulfate (SDS), the SDS, etc. is adsorbed, and the cell lysate can be clarified by centrifugation. The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments. [Nucleic Acid Purification Method] The method for purifying nucleic acid according to the present embodiment comprises a process (contact process) of contacting a sample containing nucleic acid with an aproton-type zeolite. As shown in the examples described below, the aproton-type zeolite does not adsorb at least some of the nucleic acid in the sample containing nucleic acid, while adsorbing substances other than nucleic acid. Therefore, by contacting the sample containing nucleic acid with the aproton-type zeolite, substances other than nucleic acid can be removed from the sample containing nucleic acid, allowing nucleic acid to be purified easily, quickly, and with high purity. Samples containing nucleic acid A sample containing nucleic acid is an aqueous solution or aqueous suspension in which nucleic acid and a substance other than nucleic acid are dissolved or suspended. The type of nucleic acid included in the sample is not particularly limited and may be DNA or RNA, and may be single-stranded or double-stranded. The origin of the nucleic acid is also not limited and may be, for example, nucleic acid derived from animals, plants, fungi, bacteria, or viruses. The method of the present invention can particularly purify RNA easily, quickly, and with high purity. The pH of the sample containing nucleic acid is not particularly limited. Substances other than nucleic acids include proteins (e.g., enzymes, glycoproteins, polypeptides, etc.), lipi