CN109679946B

CN109679946B - Blood virus RNA protective agent and blood collection tube

Info

Publication number: CN109679946B
Application number: CN201910006118.8A
Authority: CN
Inventors: 周杰锋; 王德明
Original assignee: Ningbo Ajcore Biotechnology Co ltd
Current assignee: Ningbo Ajcore Biotechnology Co ltd
Priority date: 2019-01-04
Filing date: 2019-01-04
Publication date: 2020-11-13
Anticipated expiration: 2039-01-04
Also published as: CN109679946A

Abstract

The application provides a blood virus RNA protective agent and a blood collection tube. The blood virus RNA protective agent is prepared by dissolving the following components in DEPC water: 100-500mg/ml of glycine, 1-5mg/ml of aprotinin, 1-5mg/ml of wortmannin, 10-40mg/ml of glutathione, 100-500mg/ml of sodium thiocyanate, 1-3mg/ml of anticoagulant and 5-20mg/ml of membrane protective agent. The blood virus RNA protective agent can provide long-time protection for virus RNA in blood at normal temperature, prevent the degradation and pollution of the virus RNA and effectively ensure the accuracy and effectiveness of blood samples.

Description

Blood virus RNA protective agent and blood collection tube

Technical Field

The application belongs to the field of molecular biology detection and the field of medical equipment, and particularly relates to a blood virus RNA protective agent and a blood collection tube.

Background

Viruses are composed of a nucleic acid molecule with or without a protein (e.g., prions). Viruses are very small in size, extremely simple in structure, but highly parasitic, relying entirely on the energy and metabolic systems of the host cell. When it comes into contact with the host cell, the coat of the protein is removed, and its nucleic acid (gene) invades into the host cell, and by means of the latter replication system, a new virus is replicated in accordance with the instruction of the viral gene, and finally the host cell is killed. DNA viruses are few in nature and are basically RNA viruses such as AIDS virus, tobacco mosaic virus, SARS virus, MERS virus, Ebola virus (EBV), Spanish influenza virus, influenza A H1N1 virus, avian influenza virus, etc. After a human body is infected by RNA virus, virus RNA can be detected in human blood along with the mass propagation of the virus, so that whether the human body is infected by the virus can be confirmed by carrying out corresponding virus detection on the blood. Therefore, the detection of blood virus RNA and its application in gene diagnosis and the like are of great significance for the diagnosis and monitoring of RNA viruses and the like. However, the content of the viral RNA in blood is low, the content of the RNase in blood is high, and the viral RNA is easily degraded and disappeared under the unprotected state; in addition, the blood is easy to coagulate during the storage and transportation of the isolated blood, and the cell membrane of the cells in the blood is also easy to be damaged to pollute the viral RNA, so that the development of an effective and feasible protective agent for prolonging the storage life of the viral RNA is necessary.

The whole blood is used as a human body sample which is easy to obtain, and is particularly suitable for laboratories and clinical detection. The high-quality RNA can be used for virus detection, and can meet the research requirements of biological downstream experiments such as RT-PCR, qRT-PCR, Northern blot, gene expression profile, transcriptome sequencing, nuclease protection experiments, array analysis and the like.

Therefore, the method does not use special expensive reagents, can stably store blood at normal temperature for a long time, protects virus RNA in the blood from being polluted and damaged, and has important significance for screening and detecting diseases.

Disclosure of Invention

In view of the above problems, it is a first object of the present invention to provide a blood viral RNA protective agent which not only prevents blood coagulation but also inhibits rnase in blood to protect viral RNA therein. The second purpose of the present invention is to provide a blood collection and blood collection tube comprising the protective agent.

In order to achieve the first object, the invention adopts the following technical scheme:

a blood virus RNA protective agent is prepared by dissolving the following components in DEPC water: 100-500mg/ml of glycine, 1-5mg/ml of aprotinin, 1-5mg/ml of wortmannin, 10-40mg/ml of glutathione, 100-500mg/ml of sodium thiocyanate, 1-3mg/ml of anticoagulant and 5-20mg/ml of membrane protective agent.

As a further improvement, the RNA protective agent comprises the following components dissolved in DEPC water to prepare: 200-400mg/ml of glycine, 2-4mg/ml of aprotinin, 2-4mg/ml of wortmannin, 20-30mg/ml of glutathione, 200-400mg/ml of sodium thiocyanate, 1.5-2.5mg/ml of anticoagulant and 8-15mg/ml of membrane protective agent.

As a further improvement, the anticoagulant is one or more of EDTA-3K and EDDHA salt, and the membrane protective agent is one or more of sorbitol, rhamnose and trehalose.

As a further improvement, the anticoagulant is EDTA-3K, and the membrane protective agent is sorbitol and trehalose. Preferably, the mass ratio of the sorbitol to the trehalose is 1: 2-2: 1.

As a specific embodiment, the RNA protective agent is prepared by dissolving the following components in DEPC water: 150mg/ml of glycine, 1mg/ml of aprotinin, 1mg/ml of wortmannin, 20mg/ml of glutathione, 100mg/ml of sodium thiocyanate, 2.0 mg/ml of EDTA-3K, 6 mg/ml of sorbitol and 6 mg/ml of trehalose.

As a further improvement, the anticoagulant is EDTA-3K and EDDHA-Na, and the membrane protective agent is rhamnose. Preferably, the mass ratio of EDTA-3K to EDDHA-Na is 1: 1-4: 1.

As a specific embodiment, the RNA protective agent is prepared by dissolving the following components in DEPC water: 400mg/ml of glycine, 5mg/ml of aprotinin, 5mg/ml of wortmannin, 40mg/ml of glutathione, 300mg/ml of sodium thiocyanate, 1.5 mg/ml of EDTA-3K, 0.8 mg/ml of EDDHA-Na and 10mg/ml of rhamnose.

In another aspect, the invention provides a blood collection tube comprising a protective agent for viral RNA of blood.

The blood virus RNA protective agent can provide long-time protection for virus RNA in blood at normal temperature, prevent the degradation and pollution of the virus RNA and effectively ensure the accuracy and effectiveness of blood samples.

It will be understood by those skilled in the art that the preservatives, antioxidants and nuclease inhibitors herein may be selected not only from the above-described classes, but that other preservatives, antioxidants and nuclease inhibitors known in the art, including but not limited to p-hydroxybenzoic acid, imidazolidinyl urea, ethylparaben, vitamin E, carotene, SOD, σ -lipoic acid, ATA, mercaptoethanol, and the like, are well within the scope of the present disclosure as reasonably selected and verified by the ordinary skill in the art.

It will also be understood by those skilled in the art that the present application may also include other agents such as pH adjusting agents, isotonic agents, cryoprotectants, and the like, the particular species of which may be reasonably selected and verified by those skilled in the art with routine knowledge.

Those skilled in the art will also appreciate that the present application is also applicable to various types of blood collection and collection tubes, including but not limited to glass/plastic materials/vacuum tubes, blood collection bags, blood specimen storage cassettes, and container components in blood testing instruments.

Because potassium ions have a better cytoprotective effect than sodium ions, we speculate that better preservation may be achieved if EDDHA-K (no existing product currently on the market) is used.

By adopting the technical scheme, the applicant takes the selection of a new anticoagulant and a membrane protective agent as key points to research the blood virus RNA protective agent and prevent virus RNA from degrading and being polluted.

Based on the unique chelation performance provided by multiple valence bonds in EDDHA and the good compatibility with other components in blood/reagents, the applicant replaces the common anticoagulant EDTA-3K with partial EDDHA-Na; based on the good tension and surfactant performance of rhamnose and trehalose on cells under low toxicity and high salt conditions, a membrane protective agent of sorbitol, trehalose and rhamnose is selected.

Experiments prove that the novel blood virus RNA protective agent prepared by the strategy can realize excellent virus RNA protective effect, the protective performance of a blood collection tube using the blood virus RNA protective agent is superior to that of a domestic heparin sodium vacuum blood collection tube commonly used in the market and an imported vacuum blood collection tube with high price such as a Streck noninvasive detection blood collection tube, the protective time of approximately 1 week can be provided for virus RNA at normal temperature, and the virus RNA protective agent can be stored for a long time at the temperature of minus 20 ℃ to minus 80 ℃.

In terms of cost, although the EDDHA-Na and rhamnose costs are slightly higher than those of EDTA, heparin and trehalose, the product cost is not significantly influenced because the dosage/cost ratio of each reagent in the blood preservation reagent is very low.

Detailed Description

Reagents and instruments used:

EDTA-3K, rhamnose, xylose, sorbitol, glycine, aprotinin, wortmannin, glutathione, sodium thiocyanate, produced by SIGMA;

EDDHA-Na is produced by TRC;

extracting nucleic acid by using a Roche nucleic acid extraction column, and amplifying and detecting by using an HCV fluorescent quantitative PCR detection kit of Shenzhen P.K. company and an Opticon fluorescent quantitative PCR detector of American Research company;

the blood collection tube containing heparin lithium is produced by Jiangsu Kangjie;

streck non-invasive vacuum blood collection tubes are produced by Streck (anticoagulants, protective agents unknown, presumed to be EDTA and sugars, etc.);

the other reagents and instruments are all in conventional domestic or imported models.

Most of the reagents in the research stage are known imported reagents, and those skilled in the art can understand that the reagents can be replaced by similar/higher-quality domestic/imported reagents for reasons of price/acquisition convenience and the like, and the achieved effect is the same or similar.

EXAMPLE 1 formulation of reagents

The formulations were divided into three groups according to different anticoagulants and membrane protectants and different formulation contents, and for the sake of simplicity, the following experimental formulations were used: 150mg/ml of glycine, 1mg/ml of aprotinin, 1mg/ml of wortmannin, 20mg/ml of glutathione and 100mg/ml of sodium thiocyanate. Group 3 blood virus RNA protective agents were formulated in the following table, with the various reagents required being dissolved in DEPC water.

TABLE 1 EDTA-3K based anticoagulant formulations

TABLE 2 EDDHA-Na anticoagulant-based formulations

TABLE 3 EDTA-3K and EDDHA-Na based anticoagulant formulations

And filling the protective agent into a glass blood collection tube according to the blood collection amount of 0.02ml per ml, and vacuumizing to prepare the negative pressure blood collection tube.

Example 2 comparison of hemolytic Performance of samples

3 male volunteers 22-25 years old (subjected to physical examination, normal blood routine index, no physical examination tumor indication and no inflammation indication) are collected, and 100ml of blood is respectively collected (formula 1-3 blood collection tubes, Congjie heparin lithium blood collection tubes, Streck non-invasive vacuum blood collection tubes and blank glass blood collection tubes). The mixture was stored at rest in a room temperature incubator at 23 ℃ until the start of the experiment.

Each sample was tested on day 0 (2 hours post-blood draw), day 2, day 5, day 10, day 14, and day 21 as follows:

hemolysis, and detecting 414nm OD value.

The results are as follows:

TABLE 2 haemolysis

The results show that except for the Congjie heparin lithium blood collection tube and the blank glass blood collection tube, the hemolysis conditions of other blood collection tubes are better within 21 days, particularly within 15 days, and the best effect is the Streck noninvasive vacuum blood collection tube and formulas 9-14 based on EDTA-3K and EDDHA-Na anticoagulants.

Example 3: experiment on preservation Effect of viral RNA

The source of the experimental sample is as follows: hepatitis C Virus (HCV) positive patients belonging to the first hospital affiliated to Zhejiang medical university in 2016;

collecting samples: blood samples are collected by using blood collection tubes of formulas 1-3, Streck non-invasive vacuum blood collection tubes and blank glass blood collection tubes, 5 blood collection tubes are used for each blood collection tube, 1ml of each blood collection tube is used for each blood collection tube, and the blood collection tubes are placed in a room temperature incubator at 23 ℃ for storage until an experiment is started.

Performing virus detection on each sample on 0, 2, 5, 10 and 14 days;

detection of HCV viral RNA:

extracting nucleic acid by using a Roche nucleic acid extraction column, and amplifying and detecting by using an HCV fluorescent quantitative PCR detection kit of Shenzhen P.K. company and an Opticon fluorescent quantitative PCR detector of American Research company; to avoid batch-to-batch variation in nucleic acid quantification, each sample was RUN in the same RUN.

The results of the experiment are shown in the following table:

TABLE 3 HCV viral RNA detection results

The results show that in blood samples of the blood virus RNA protective agent obtained by the formulas 1 to 9 in the embodiment, the RNA virus content is slightly reduced in 7 days, but the degradation is not obvious and is less than or equal to 0.27 Log, namely, the RNA virus content is not more than 53.8 percent of the original virus titer; the reduction of the RNA virus by the Streck tube for 7 days was 0.60, which corresponds to a reduction of 25.1% of the original virus titer.

Claims

1. The blood virus RNA protective agent is characterized by being prepared by dissolving the following components in DEPC water: 150mg/ml of glycine, 1mg/ml of aprotinin, 1mg/ml of wortmannin, 20mg/ml of glutathione, 100mg/ml of sodium thiocyanate, 1.0mg/ml of EDTA-3K, 1.0mg/ml of EDDHA-Na, 6.0 mg/ml of sorbitol and 6.0 mg/ml of trehalose.

2. The blood virus RNA protective agent is characterized by being prepared by dissolving the following components in DEPC water: 150mg/ml of glycine, 1mg/ml of aprotinin, 1mg/ml of wortmannin, 20mg/ml of glutathione, 100mg/ml of sodium thiocyanate, 1.0mg/ml of EDTA-3K, 1.0mg/ml of EDDHA-Na and 10mg/ml of rhamnose.

3. The blood virus RNA protective agent is characterized by being prepared by dissolving the following components in DEPC water: 150mg/ml of glycine, 1mg/ml of aprotinin, 1mg/ml of wortmannin, 20mg/ml of glutathione, 100mg/ml of sodium thiocyanate, 1.0mg/ml of EDTA-3K, 1.0mg/ml of EDDHA-Na, 6.0 mg/ml of rhamnose and 6.0 mg/ml of sorbitol.

4. A blood collection tube containing the agent for protecting a blood virus RNA according to any one of claims 1 to 3.