CN114134139B

CN114134139B - Full-automatic nucleic acid extraction kit

Info

Publication number: CN114134139B
Application number: CN202111424363.4A
Authority: CN
Inventors: 彭俊; 苗凤杰; 汪中厅
Original assignee: Wuhan Jiayuan Biomedical Engineering Co ltd
Current assignee: Wuhan Jiayuan Biomedical Engineering Co ltd
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-08-12
Anticipated expiration: 2041-11-26
Also published as: CN114134139A

Abstract

The invention provides a nucleic acid extraction kit, which comprises a deep hole plate, an isolation sleeve, a cracking binding liquid, a washing liquid and an eluent, wherein the deep hole plate comprises an extraction part and a waste liquid collection part, a pipe groove is formed in the extraction part and used for accommodating an extraction system, a through hole is formed in the bottom of the pipe groove, the pipe groove is communicated with the waste liquid collection part through the through hole, and the reagent composition in the pipe groove is further improved. The invention can complete the whole nucleic acid extraction process in one tube groove by optimizing the design of the deep hole plate. Meanwhile, after the reagent composition of the kit is further optimized, the quantity and the purity of the nucleic acid extracted from one tube groove are ensured. Through the improvement of the invention, the flux of the full-automatic nucleic acid extractor is improved to six times of the original flux, and the quality of the extracted DNA is ensured.

Description

Full-automatic nucleic acid extraction kit

Technical Field

The invention relates to the field of applied molecular biology, in particular to a full-automatic nucleic acid extraction kit.

Background

With the development of molecular biology and the development of gene markers, gene detection is increasingly applied to diagnosis, and the molecular diagnosis industry is formed. Nucleic acid extraction is the cornerstone and essential link of the molecular diagnostic industry. The nucleic acid extraction mainly comprises three steps of cell breaking, nucleic acid sedimentation and nucleic acid re-dissolution, and the middle of the three steps is subjected to multiple times of oscillation, stirring and washing, so that the method belongs to a simple procedure with a large amount of repeated labor. The manual extraction not only can generate higher labor cost, but also has low error rate and repeatability, improves the sample pollution probability and is not beneficial to high-throughput samples. The full-automatic nucleic acid extraction instrument gradually becomes one of the necessary instruments of each large medical detection service organization due to standardization and low labor cost, and is operated by a matched nucleic acid extraction kit.

The design of the deep hole plate of the existing full-automatic nucleic acid extraction kit greatly limits the extraction flux of a full-automatic nucleic acid extraction instrument. Therefore, there is a need for a new nucleic acid extraction kit for high-throughput, fully automated nucleic acid extraction.

Disclosure of Invention

In order to solve the problems, the invention provides a nucleic acid extraction kit, which comprises a deep hole plate, an isolation sleeve, a cracking combination liquid, a washing liquid and an eluent, wherein the deep hole plate comprises an extraction part and a waste liquid collection part, a pipe groove is arranged in the extraction part and used for accommodating an extraction system, a through hole is arranged at the bottom of the pipe groove, and the pipe groove is communicated with the waste liquid collection part through the through hole.

In a specific embodiment, the lysis binding solution comprises 50 mmol/L NaCl, 25 mmol/L Tris, 35 mmol/L EDTA, 1.2 mol/L guanidine hydrochloride, 0.5% by mass cetyl trimethyl ammonium bromide, 5% by volume TritonX-100, 15 g/L BSA and 35 g/L magnetic beads. Preferably, the pH of the lysis binding solution is between 8.0 and 8.5. The invention optimizes the components of the lysis binding solution, so that the lysis binding solution not only can better preserve heparin magnetic beads, but also can efficiently lyse cell samples, such as blood samples, throat swab samples and the like.

In a specific embodiment, the wash solution comprises wash solution I, wash solution II, and wash solution III. Three washing steps are sequentially performed by setting three gradients of washing solutions to gradually wash away ions and proteins contained in nucleic acids.

In a specific embodiment, the washing solution I comprises 100 mmol/L NaCl, 30 mmol/L sodium dodecyl sulfate, 5% volume fraction Triton X-100, and 85% volume fraction ethanol.

In a specific embodiment, the washing solution II contains 50 mmol/L NaCl, 3% volume fraction TritonX-100, and 80% volume fraction ethanol.

In a specific embodiment, the washing III contains 30 mmol/L NaCl, 5% PEG4000 by mass fraction and 75% ethanol by volume fraction.

In a specific embodiment, the eluent is nuclease-free water.

The invention can complete the whole nucleic acid extraction process in one tube groove by optimizing the design of the deep hole plate. Meanwhile, after the reagent composition of the kit is further optimized, the quantity and the purity of the nucleic acid extracted from one tube groove are ensured. Through the improvement of the invention, the flux of the full-automatic nucleic acid extractor is improved to six times of the original flux, and the quality of the extracted DNA is ensured.

Drawings

FIG. 1 is a top view of a deep well plate in a commercially available full-automatic nucleic acid extraction kit, and one nucleic acid extraction unit is outlined by a dotted line.

FIG. 2 is a top view of a deep well plate in the fully automatic nucleic acid extraction kit according to examples 1 and 2 of the present invention, and a nucleic acid extraction unit is outlined by a dotted line.

FIG. 3 is a schematic view showing the structure of a groove and a waste liquid collecting chamber of a deep well plate in the fully automatic nucleic acid extraction kit according to examples 1 and 2 of the present invention.

Wherein the reference numerals represent the following: 11. tube groove 111, through hole 21, waste liquid collecting cavity 22 and plug body.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The main structural components of the current commonly used full-automatic nucleic acid extraction instrument are a magnetic rod and two displacement components which respectively provide magnetic force and displacement, and the magnetic rod is fixedly connected with a displacement part. Therefore, the extraction process is mainly limited by the design of nucleic acid extraction kits. Commonly used fully automated nucleic acid extraction kits typically include: the device comprises a deep hole plate, an isolation sleeve, a magnetic bead suspension, a lysis solution, a washing solution and an eluent. As shown in FIG. 1, each extraction unit comprises six wells, one for holding the sample-lysate mixture, one for holding the magnetic beads, three for holding the wash solution, and one for holding the eluent. In the extraction process, firstly, the isolation sleeve is arranged on the other displacement part, the isolation sleeve is moved to the magnetic bead hole to mix the magnetic beads uniformly, the magnetic rod is inserted into the isolation sleeve to transfer the magnetic beads to the sample-lysate mixing system, the magnetic beads are adsorbed after mixing uniformly, the magnetic beads are sequentially transferred to three washing holes to be washed, and then elution is carried out.

The method is limited by the design of a deep-hole plate of a nucleic acid extraction kit, the flux of the existing full-automatic nucleic acid extractor is not high, and the sample which can be processed at one time is limited.

To solve the above problems, we slightly modified the nucleic acid extractor, adding a washing solution storage box and an eluent storage box, and a pump and a pipeline, so as to add a washing solution/eluent to the corresponding extraction tube well or remove a liquid from the tube well at an appropriate time.

Example 1

This embodiment has reformed transform the deep hole board, and the deep hole board after the transformation is shown as fig. 2 and 3, including extraction portion and waste liquid collection portion, extraction portion 1 includes a plurality of chase 11, chase 11 bottom is provided with through-hole 111, waste liquid collection portion 2 is provided with waste liquid collection chamber 21, and with the position of through-hole 111 is provided with cock body 22, cock body 22 with through-hole 111 cooperates, can stopper and seal through-hole 111. The waste liquid collecting part 2 is detachably connected with the extracting part 1. The nucleic acid isolation instrument is provided with a mechanical arm capable of lifting and pressing an extraction part, so that the plug body 22 plugs and closes the through hole 111 when the extraction part is pressed, and the plug body 22 is separated from the through hole 111 when the extraction part is lifted.

When nucleic acid extraction is performed using the deep well plate, the plug body 22 is separated from the through-hole 111 by lifting up the extraction part 1 after completion of the cleavage or washing reaction, and the waste liquid is introduced into the waste liquid collection part. Then the extraction part 1 is pressed down again to make the tube groove 11 in a closed state again, and then the next washing solution is added through a pipeline for washing, or the eluent is added for elution after all the washing processes are finished.

Through the transformation of the deep hole plate, on the basis of the existing full-automatic nucleic acid extractor, the flux which is 6 times higher than that of the full-automatic nucleic acid extractor with the same volume can be realized only by making some modularized small changes, such as the addition of a magnetic rod, a mechanical arm structure and a pipeline structure, and the change of the position of a heating module.

After modification, other reagents in a commercial full-automatic nucleic acid extraction kit are used, the deep-well plate and the extraction method are used for extracting DNA of a blood sample, and the result shows that compared with the commercial kit, the kit after the design of the deep-well plate is improvedThe concentration of the extract is slightly reduced, the purity is greatly reduced, and OD is reduced ₂₆₀ /OD ₂₈₀ The ratio of (A) is only 1.51. + -. 0.01. Therefore, due to the modification of the deep-well plate and the change of the extraction method, other reagents of the original kit are not applicable to the kit.

Example 2

On the basis of example 1, we have searched for other reagents and found that the major problems occurred in washing, which was not thorough enough and was high in protein and lipid due to all processes performed in one tube. Through multiple experiments, the reagent composition of the kit is improved. The kit comprises the following reagents:

1) and (3) a lysis binding solution which comprises 50 mmol/L NaCl, 25 mmol/L Tris, 35 mmol/L EDTA, 1.2 mol/L guanidine hydrochloride, 0.5% by mass of hexadecyl trimethyl ammonium bromide, 5% by volume of Triton X-100, 15 g/L BSA and 35 g/L magnetic beads. Preferably, the pH of the lysis binding solution is between 8.0 and 8.5.

2) The washing liquid comprises washing liquid I, washing liquid II and washing liquid III, wherein,

washing solution I contains 100 mmol/L NaCl, 30 mmol/L sodium dodecyl sulfate, 5% volume fraction TritonX-100, and 85% volume fraction ethanol.

Wash II contained 50 mmol/L NaCl, 3% volume fraction TritonX-100, and 80% volume fraction ethanol.

Wash III contained 30 mmol/L NaCl and a volume fraction of 75% ethanol.

3) Eluent, nuclease-free water.

In the lysis conjugate, we combined the magnetic beads and the lysate components. At the same time, we improve the composition of the lysis solution to make the lysis more sufficient.

In the washing liquid, we tested various washing liquid compositions, and the effect is not particularly ideal. The effect was improved upon the addition of the sodium dodecyl sulfate and TritonX-100 combination, but the residue of both in the eluate affected the extracted DNA for PCR amplification. Thus, we continued to modify the washing solution, and performed three times of washing by designing different washing solutions, to obtain the kit of this example. After tests, the quantity and the purity of the extracted DNA are found to meet the requirements, and can meet the requirements of a nucleic acid extractor and an extraction kit sold in the market, but the extraction flux is 6 times of that of the nucleic acid extractor and the extraction kit sold in the market.

The nucleic acid extraction kit is compared with a commercially available extraction kit, nucleic acid extraction is carried out on blood and throat swab samples, and the extracted DNA is used for PCR amplification for quality control verification.

The method of using the kit of the present invention is as follows: 1) mixing the blood sample with the lysis binding solution, and adding the mixture into a tube groove 11 of an extraction part of the deep-hole plate; 2) installing the deep hole plate in a nucleic acid extractor, installing an isolation sleeve, and performing oscillation incubation at a specific temperature; 3) after the cracking reaction is finished, inserting the magnetic rod into the isolation sleeve, and standing to enable the magnetic beads to be adsorbed on the outer surface of the isolation sleeve; 4) the mechanical arm draws the isolation sleeve and the magnetic rod out of the deep hole plate together, the extraction part 1 is lifted by another mechanical arm, the plug body 22 is separated from the through hole 111, the pipe groove 11 is communicated with the waste liquid collection part 2, liquid in the pipe groove 11 enters the waste liquid collection part, then the extraction part is pressed down, and the plug body 22 seals the through hole 111; 5) inserting the isolation sleeve and the magnetic rod into the extraction part 11, then extracting the magnetic rod, adding a washing solution I into the tube groove 11, carrying out oscillation washing, discharging waste liquid in the above mode after washing, then sequentially washing II and III, and adding eluent to elute after washing to obtain a DNA solution.

As shown in Table 1, the kit of this example was used to obtain a product having a good extraction yield and purity, while having a throughput 6 times that of the commercially available kit.

TABLE 1 comparison of the kits of the invention with commercially available kits

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A nucleic acid extraction kit is characterized by comprising a deep hole plate, an isolation sleeve, a cracking binding liquid, a washing liquid and an eluent, wherein the deep hole plate comprises an extraction part and a waste liquid collection part, a pipe groove is formed in the extraction part and used for accommodating an extraction system, a through hole is formed in the bottom of the pipe groove, the pipe groove is communicated with the waste liquid collection part through the through hole, a plug body is arranged in the waste liquid collection part at a position corresponding to the through hole, and the plug body is matched with the through hole and can plug and seal the through hole;

the lysis binding solution comprises 50 mmol/L NaCl, 25 mmol/L Tris, 35 mmol/L EDTA, 1.2 mol/L guanidine hydrochloride, 0.5% by mass fraction of hexadecyl trimethyl ammonium bromide, 5% by volume fraction of Triton X-100, 15 g/L BSA and 35 g/L magnetic beads, and the pH of the lysis binding solution is 8.0-8.5;

the washing solution comprises a washing solution I, a washing solution II and a washing solution III; the washing solution I comprises 100 mmol/L NaCl, 30 mmol/L sodium dodecyl sulfate, 5% by volume of TritonX-100 and 85% by volume of ethanol; the washing solution II comprises 50 mmol/L NaCl, 3% of TritonX-100 in volume fraction and 80% of ethanol in volume fraction; the washing solution III contains 30 mmol/L NaCl, 5% of PEG4000 by mass fraction and 75% of ethanol by volume fraction;

the eluent is nuclease-free water.