CN111763765A

CN111763765A - Novel method for preventing RNA degradation of novel coronavirus sample

Info

Publication number: CN111763765A
Application number: CN202010096543.3A
Authority: CN
Inventors: 张缨
Original assignee: Individual
Current assignee: Qinhuai Medical Area General Hospital of Eastern Theater of Pla
Priority date: 2020-02-18
Filing date: 2020-02-18
Publication date: 2020-10-13
Anticipated expiration: 2040-02-18
Also published as: CN111763765B

Abstract

The invention relates to a novel method for preventing RNA degradation of a novel coronavirus sample, belonging to the field of medical treatment. According to the invention, cardiac glycoside is innovatively used as a main component for preventing RNA degradation, various implementation modes are provided, an innovative idea is provided for detecting 2019 novel coronavirus, and the method can also be used for preventing other RNA degradation.

Description

Novel method for preventing RNA degradation of novel coronavirus sample

Technical Field

The invention relates to a novel method for preventing RNA degradation of a novel coronavirus sample, belonging to the field of medical treatment.

Background

Novel Coronavirus Pneumonia (Novel Coronavirus pneumoconia, NCP) is an acute infectious disease caused by 2019 infection with Novel Coronavirus (2019 Novel Coronavirus, 2019-nCoV). The diagnostic gold criteria for determining the presence of 2019-nCoV infection is to find the presence of 2019-nCoV in the patient. To find 2019-nCoV and to make a clear determination, the 2019-nCoV nucleic acid is determined. Therefore, the nucleic acid detection positive rate of 2019-nCoV is ensured, and the control quality of the epidemic situation is directly related.

2019A Novel Coronavirus (2019 Novel Coronavir, 2019-nCoV) is a RNA type virus. RNA virus is a kind of virus, belonging to the first class. Their genetic material is ribonucleic acid (RNA ribonucleic acid). 2019 the novel coronavirus mainly infects respiratory tract. Detecting and determining whether the 2019 novel coronavirus infection exists, firstly collecting sputum, a throat swab sample, alveolar lavage fluid and the like, namely, firstly completing specimen collection, transportation and storage, then carrying out fluorescence-PCR operation, and finally determining the result.

RNA is known by the English name Ribonucleic Acid and by the Chinese name Ribonucleic Acid. Genetic information carriers present in biological cells and in parts of viruses, viroids. RNA is a long chain molecule formed by the condensation of ribonucleotides via phosphodiester bonds. Thus, RNA is protein in nature. RNA is easily degraded after being separated from the body, and the detection rate is low or even false negative. There are many causes for degradation of RNA, but the main cause is that, on the one hand, RNA is extremely unstable and, on the other hand, there are many enzymes that can break down RNA in the sample. Therefore, it is easy to see that the RNA is ensured not to be degraded or slightly degraded in the storage stage of the sample, which is a very key basic link and directly relates to the subsequent operation quality and the detection rate. Therefore, after the sample is collected and before the sample enters the formal detection process, technical means are needed to ensure that the viral RNA in the sample is not degraded so as to ensure the detection rate of subsequent detection.

At present, the method for preventing the degradation of viral RNA in a sample mainly uses protein denaturants such as guanidine isothiocyanate, guanidine hydrochloride, urea, phenols and the like in a preservation solution. These conventional methods have their disadvantages. Such as guanidines, urea, etc., must be present in high concentrations to be effective, which can result in crystallization and precipitation at low temperatures during storage and transportation. Phenols and other substances are original toxic substances, water solution of the phenols and other substances easily causes systemic poisoning through skin, and vapor of the phenols and other substances is inhaled from respiratory tracts and has larger damage to nervous systems. In addition, the method is not ideal in terms of methodology because the heating is needed.

The inventor is familiar with the sample preservation technology because of the long-term pathological technology and diagnosis work and the variety of the used sample preservation solution is more. After the outbreak of the new coronavirus epidemic situation, inspired by the long-term use of the plant extract-containing cell preservation solution technology, a new technical route capable of preventing the RNA degradation of the virus sample is explored.

Disclosure of Invention

The solution studied by the inventors is to achieve the need to prevent RNA degradation during the preservation of viral samples, for the detection of 2019-nCoV nucleic acids. Further, RNA degradation can be prevented during storage for a variety of viral samples.

The inventors have found through research that three technical goals are to be achieved by a technical route for preventing RNA degradation, which aims to support nucleic acid detection. Firstly, after a virus sample enters a preservation solution system, RNA needs to be quickly denatured to prevent degradation. Secondly, a plurality of enzymes contained in the sample that can degrade RNA are inactivated to prevent the enzymes from degrading RNA. Finally, the denatured RNA should be revived to facilitate subsequent amplification and other detection procedures.

Based on years of experience and exploration and practice, the inventor finds that certain glycoside compounds have the functions of rapidly solidifying proteins and inactivating various enzymes. Glycosides, also known as glycosides or aglycones, are a class of organic compounds whose molecules consist of an alcohol or alcohol-like group (ligand, aglycone or aglycone) bound to a varying number of sugar molecules. Then, the inventors have found that cardiac glycosides are suitable for the use of the present invention, among glycoside compounds, through optimization. Cardiac glycoside (cardiac glycoside), also called cardiac glycoside, is a glycoside molecular structure, in which the ligand contains steroid nucleus (steroid nucleus), the 17-position carbon atom of which is connected with an unsaturated lactone ring, and the 3-position carbon atom of which is connected with a sugar molecule. Cardiac glycoside is a kind of medicine with selective cardiac function originally used in clinic, and is mainly used in treating chronic cardiac insufficiency and some arrhythmia, especially supraventricular arrhythmia. However, the inventors have found that cardiac glycosides do not have a specific affinity for cardiac muscle, but act on the human body by inhibiting Na and K-ATPases. That is, cardiac glycosides have a function of inactivating various enzymes, but this function has not attracted much attention in the fields of pathology and laboratory science. Meanwhile, the inventor finds that the cardiac glycoside solution with a certain concentration can denature RNA, and can revive the RNA and precipitate and extract the RNA under the action of alcohol.

Therefore, the inventor summarizes and practices to innovatively use cardiac glycoside as the main component for preventing RNA degradation, and alcohol (such as ethanol, isopropanol, etc.) as the auxiliary agent for recovering and precipitating RNA for collection.

The cardiac glycoside is a general term of glycoside compounds, can be theoretically used in the invention, has rich sources, is mainly extracted from leaves of angiosperm plants through natural plants, and has easy solution of the source of finished products. However, each cardiac glycoside is used in the present invention at a different concentration and in a different solvent. The inventor considers that the variety with better water solubility, such as digitoxin C, is generally selected. Other species with poor water solubility but better solubility in organic solvents, such as digitoxin, can be used as preservative solutions for special applications. In the present invention, the species having good water solubility is mainly used, and the preservation solution also uses water as a solvent.

In the research process of the invention, the inventor mainly selects digitoxin C. Digitonin C, abbreviated as digitonin C, is a rapid acting cardiac glycoside extracted from Digitalis lanata, which is the precursor of deacetyl-digoxin. The inventor uses PBS buffer solution with pH value of 7.4 as solvent, uses analytically pure hairy flower glycoside to prepare preservation solution with concentration of 0.1% -0.5%, and uses analytically pure isopropanol as auxiliary reagent. The concentration of the hairy flower glycoside C solution is preferably 0.3-0.5%, more preferably 0.35%. When in use, fresh samples are directly soaked in the preservation solution of the invention and are refrigerated at 4 ℃ for storage, thus realizing the sample preservation function of preventing RNA degradation. In the preservation solution of the invention, RNA can be kept undegraded or degraded only a little for at least 72 hours, and the whole process of sampling, transporting and reaching the destination for short-time preservation can meet the requirement in terms of time limit. Before detection, isopropanol with the same amount as the preservation solution is added, mixed evenly and kept stand for 10-15 minutes at normal temperature, and then RNA can be precipitated and extracted.

The invention innovatively uses the plant extract cardiac glycoside substances as raw materials, has safe use, wide sources, simple preparation process, convenient use and good performance, and can completely replace the traditional method. The invention provides an innovative idea for detecting 2019 novel coronavirus.

It is clear that the embodiments are not limited to the examples, as long as the cardiac glycosides are used as the starting material, and are within the scope of the present invention. The present invention can also be used to prevent degradation of other kinds of RNA.

Examples

In the implementation process of the invention, all the reagent raw materials are analytically pure, and the water is double distilled water. The specific implementation mode is as follows:

1) 8.0g of sodium chloride, 0.2g of potassium chloride, 2.9g of disodium hydrogen phosphate (dodecahydrate), 0.2g of potassium dihydrogen phosphate and 0.35g of eriocitrin were weighed by a 100g tray balance for later use.

2) In a 100ml glass beaker, about 80ml of double distilled water was added, and 2.9g of disodium hydrogenphosphate (dodecahydrate) was added, followed by stirring with a glass rod until completely dissolved. Then adding potassium chloride and sodium chloride, stirring and completely dissolving. And finally adding potassium dihydrogen phosphate, and stirring until the potassium dihydrogen phosphate is completely dissolved. Adding double distilled water to 100ml of constant volume to prepare a PBS solution.

3) The pH value of the PBS solution is measured to be about 7.4 by using a precise pH test paper with the measuring range of 6.8-8.0. Adding the eriocitrin C, and stirring by using a glass rod until the eriocitrin C is completely dissolved to obtain the preservation solution.

4, subpackaging the preserving fluid into small bottles according to the packaging amount of 5ml, and labeling to obtain the finished product.

The preservation solution of the invention is preserved at normal temperature, and the effective period is 12 months. After the sample is added into the preservation solution, the sample needs to be refrigerated at 4 ℃ for preservation or stored and transported by using an ice bag. The RNA in the sample is not degraded, or is degraded to a very slight extent, within at least 72 hours, and does not affect the subsequent detection.

Claims

1. A novel method for preventing RNA degradation in a sample of a novel coronavirus, characterized in that a cardiac glycoside is used as a starting material.

2. A novel method for preventing RNA degradation of a sample of a novel coronavirus is characterized in that a water-soluble cardiac glycoside is selected as a first choice.