CN113186037B

CN113186037B - Nucleic acid scavenger

Info

Publication number: CN113186037B
Application number: CN202110442978.3A
Authority: CN
Inventors: 胡晓飞; 李�杰; 董雯; 李志明; 刘海龙
Original assignee: Shandong Bohong Gene Technology Co ltd
Current assignee: Shandong Bopeng Biotechnology Co ltd
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2022-12-13
Anticipated expiration: 2041-04-23
Also published as: CN113186037A

Abstract

The invention provides a nucleic acid scavenger, and relates to the field of disinfection and cleaning. The nucleic acid scavenger comprises a citric acid buffer solution, peroxide, a metal ion salt, a cracking agent, EDTA and a surfactant, can effectively scavenge nucleic acid pollution in equipment consumables and the environment, avoids false positive of an experiment, is non-toxic, harmless, efficient and convenient, and ensures the safety of laboratory staff and the environment.

Description

Nucleic acid scavenger

Technical Field

The invention relates to a nucleic acid scavenger, and belongs to the field of disinfection and cleaning.

Background

PCR, known as Polymerase chain reaction (English name: polymerase chain reaction), is a molecular biology technique for amplifying a large number of specific DNA fragments in vitro by using the principle of DNA double-strand replication, and can rapidly amplify a large number of copies by using a small amount of DNA as a template. The PCR technology is widely applied to more fields of biology and related disciplines, and has remarkable effect in the fields of medical research, criminal evidence collection, molecular archaeology and the like.

PCR is a process that mimics DNA replication in vitro based on the principle of half-retained replication of DNA paired with base complementarity. The method mainly comprises the following three steps: denaturation-annealing-extension. Denaturation: utilizing the characteristics of DNA to form single strand by unwinding at the high temperature of 95 ℃; annealing: when the temperature is lowered, the primer will be combined with the single-stranded DNA, and the DNA polymerase is combined with the primer; delay pipeStretching: when the temperature reaches 72 ℃ which is the optimum temperature for DNA polymerase, the DNA polymerase will synthesize a complementary strand along the 5'-3' direction of the DNA strand. As the PCR proceeds, DNA is used as a template and is initially exponentially amplified, and theoretically, a specific fragment can be amplified to 2 over N cycles ⁿ ^-1 It can amplify millions of times of target nucleic acid in a very short time to obtain a great amount of specific target gene segments and obtain very high detection sensitivity. But its most major drawback is its extreme susceptibility to contamination.

Nucleic acid laboratory contamination mainly includes several types: sample contamination, reagent contamination, environmental contamination, equipment contamination, and other contamination, and the like. Sample and reagent pollution is eliminated comparatively simply, and removable sample and reagent or send other laboratories to verify, and environmental pollution and equipment pollution then need carry out thorough disinfection and cleaning to equipment and environment. The traditional method for removing the environmental and equipment pollution mainly comprises the steps of wiping by using isopropanol, alcohol or depc and irradiating and disinfecting by using an ultraviolet lamp. However, organic solvents such as isopropanol and alcohol are harmful to human bodies, depc belongs to a strong carcinogen, and sterilization by adopting an ultraviolet lamp wastes time and labor. In order to ensure the safety of the experiment of the experimenter and save the precious time of the experimenter, a non-toxic, harmless, time-saving and labor-saving reagent is needed to be invented to help eliminate the nucleic acid pollution in a laboratory and improve the accuracy of the experiment.

Disclosure of Invention

The invention provides a nucleic acid scavenger which is non-toxic, harmless, time-saving, labor-saving, efficient and convenient.

In order to solve the technical problems, the invention provides the following technical scheme:

a nucleic acid scavenger comprising the following components and amounts:

further, the peroxide is one or more of hydrogen peroxide, sodium peroxide and peroxyacetic acid.

Further, the metal ion salt is Cu ²⁺ 、Ni ²⁺ 、Zn ²⁺ One or more of them.

Further, the cracking agent is guanidine thiocyanate.

Further, the surfactant is one or more of coco glucoside and lauryl glucoside.

Compared with the prior art, the invention has the following beneficial effects:

the nucleic acid scavenger provided by the invention can effectively remove nucleic acid pollution in equipment consumables and the environment, avoids false positive of an experiment, is non-toxic, harmless, efficient and convenient, and ensures the safety of laboratory workers and the environment.

Drawings

FIG. 1 is a graph showing the results of a PCR experiment of nucleic acid scavenger on the nucleic acid scavenging ability;

FIG. 2 is a graph of the results of qPCR experiments with nucleic acid scavengers for their ability to scavenge nucleic acids;

FIG. 3 is a graph of the results of a nucleic acid scavenger stability experiment.

Detailed Description

To make the technical problems, technical solutions and advantages of the present invention more apparent, the following description is given with reference to specific embodiments.

Example 1:

the reagent composition of this example is mainly:

example 2:

the reagent composition of this example is mainly:

example 3:

the reagent of the embodiment mainly comprises the following components:

to verify the beneficial effects of the examples, the following experimental verification was performed:

1. experiment on nucleic acid scavenging ability

1. Verification of PCR experiment

Taking eight connecting tubes for experiments, dividing the experiments into four groups, repeating the experiments for three times, and setting a reaction system of a control group as follows: adding 2.5uL of an upstream primer, 2.5uL of a downstream primer, 2uL of a template, 12.5uL of Tap enzyme and 5.5uL of sterile water into the eight-connected tube; the example 1, example 2 and example 3 sets of reaction systems were: in the octal tube, 2.5uL of the upstream primer, 2.5uL of the downstream primer, 2uL of the template, 12.5uL of the Tap enzyme, 3.5uL of sterile water, and 2uL of the nucleic acid scavenger were added, followed by transient centrifugation.

PCR amplification procedure:

2. verification of qPCR experiment

qPCR amplification procedure:

3. experimental verification of nucleic acid clearance capability

After the PCR amplification experiment is finished, the experiment result is verified through agarose gel electrophoresis, the result is shown in figure 1, the serial numbers 1-12 at the upper part in the figure 1 are hole sites, wherein no nucleic acid scavenger is added into the hole sites 1-3, and a bright strip is formed, which indicates that the nucleic acid is not cleared and the gene can be normally expressed; addition of nucleic acid scavengers at sites 4-6 example 1, addition of nucleic acid scavengers at sites 7-9 example 2, and addition of nucleic acid scavengers at sites 10-12 example 3. The results showed that no band was observed at any of the 4-12 th positions, indicating that the nucleic acid was degraded and the gene was not normally expressed. The experimental results demonstrate that nucleic acid scavengers are effective in scavenging nucleic acids.

After the qPCR experiment was completed, the results of the amplification curves were analyzed, and as shown in fig. 2, the amplification curves were not detected in any of examples 1, 2, and 3 in which the nucleic acid scavenger was added, as compared with the control group in which the nucleic acid scavenger was not added, indicating that the nucleic acid was degraded and the expression of the gene was not detected. The experimental results demonstrate that nucleic acid scavengers are effective in scavenging nucleic acids.

2. Stability test

The nucleic acid scavengers were formulated on a time-based schedule and subjected to six-month stability testing (same procedure as for PCR testing, 1, 2 for nucleic acid scavenging capacity testing). The experimental result of the stability of the nucleic acid scavenger is shown in figure 3, numbers 1-3 are control groups, the nucleic acid scavenger is not added, and an obvious band can be seen through agarose gel, which indicates that the nucleic acid is not degraded and the gene can be normally expressed; the results of the experiment with the nucleic acid scavengers of example 1, example 2 and example 3 placed for one month are shown as nos. 4 to 6, the results of the experiment with the nucleic acid scavengers of example 1, example 2 and example 3 placed for two months are shown as nos. 7 to 9, the results of the experiment with the nucleic acid scavengers of example 1, example 2 and example 3 placed for three months are shown as nos. 10 to 12, the results of the experiment with the nucleic acid scavengers of example 1, example 2 and example 3 placed for four months are shown as nos. 13 to 15, the results of the experiment with the nucleic acid scavengers of example 1, example 2 and example 3 placed for four months are shown as nos. 16 to 18, the results of the experiment with the nucleic acid scavengers of example 1, example 2 and example 3 placed for five months are shown as nos. 19 to 21. No obvious band was observed on the agarose gel, indicating that the nucleic acid was degraded and no gene expression was detected. The experimental result shows that the nucleic acid scavenger can be stored for six months at the temperature of 2-8 ℃ in a dark place and still has the capability of degrading nucleic acid.

Claims

1. A nucleic acid scavenger, characterized in that the components and amounts of the nucleic acid scavenger are as follows:

the peroxide is one or more of hydrogen peroxide, sodium peroxide and peroxyacetic acid; the metal ion salt is Cu ²⁺ 、Ni ²⁺ 、Zn ²⁺ One or more of the above;

the cracking agent is guanidine thiocyanate;

the surfactant is one or more of coco glucoside and lauryl glucoside.