CN114019160A - Release agent for releasing N protein from coronavirus, and preparation method and application thereof - Google Patents
Release agent for releasing N protein from coronavirus, and preparation method and application thereof Download PDFInfo
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- CN114019160A CN114019160A CN202210003860.5A CN202210003860A CN114019160A CN 114019160 A CN114019160 A CN 114019160A CN 202210003860 A CN202210003860 A CN 202210003860A CN 114019160 A CN114019160 A CN 114019160A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
A releasing agent for releasing N protein from coronavirus and its preparation method and application; belongs to the technical field of virus detection; the composite material comprises the following components in percentage by mass: 0.01-0.1% of metal chelate, 0.8-1.2% of nonylphenol polyoxyethylene ether, 0.8-1.2% of hexadecyl trimethyl ammonium bromide, 0.1-0.5% of surfactant, 0.8-1.2% of glycerol, 60000.8-1.2% of polyethylene glycol and the balance of NaCl solution. The release agent of the invention overcomes the problem of N protein release in the detection of N proteins of coronavirus series; the N protein can be effectively released from the coronavirus, the detection accuracy is improved, and screen leakage caused by false negative is avoided, so that the method has important significance on the whole coronavirus project research.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a releasing agent for releasing N protein of coronavirus, and a preparation method and application thereof.
Background
Coronaviruses are a large family of viruses, and mature coronavirus particles are diverse in shape, about 60-220nm in diameter, mainly spherical, and few are elliptical or polygonal. Coronaviruses are composed of proteins and RNA, and their membrane surface is composed mainly of 3 structural proteins: spike protein (S protein), envelope protein (E protein), and membrane protein (M protein); the RNA, which is the nucleic acid material responsible for virus propagation, inside the virus is surrounded and protected by the nucleocapsid protein (N protein).
There are 7 members of coronaviruses that have been found to be associated with human diseases, including human coronavirus 229E (HCoV 229E), human coronavirus NL63 (HCoV NL 63), human coronavirus OC43 (HCoV OC 43), human coronavirus HKU1 (HCoV HKU 1), severe acute respiratory syndrome coronavirus (SARS-CoV), and middle east respiratory syndrome virus (MERS-CoV). Among them, HCoV 229E, HCoV NL63, HCoV OC43 and HCoV HKU1 are pathogens of global epidemics that cause common cold symptoms, while SARS-CoV and MERS-CoV are highly pathogenic HCoV that cause severe acute respiratory syndrome and have a high mortality rate. Another pathogenic HCoV appeared in 2019, a new coronavirus in 2019 (2019-nCoV), whose infection could cause mild, moderate or severe disease (including severe pneumonia, sepsis and ARDS, etc.). The detection of coronavirus is an important link to deal with the spread of disease.
The main detection modes of the coronavirus at present comprise: nucleic acid detection, antibody detection and antigen detection. The antigen detection mainly comprises detection aiming at S protein and detection aiming at N protein, wherein the N protein is virus structural protein with most abundant content in the infection process of the new coronavirus, and can be expressed in a large amount after the virus infects human cells to cause strong immune response. The gene sequence of the N protein is relatively conserved and less mutations occur over time on the N protein than on the S protein. Because of good immunogenicity and stability, the N protein can be used as an antigen detection target of the coronavirus, but the N protein is positioned in the capsular sac in the structure of the coronavirus, so that the difficulty in detecting the N protein is high; taking the new coronavirus (2019-nCoV) as an example, the new coronavirus is composed of five components: one strand of RNA genes and four proteins. The outermost layer is spike glycoprotein (S protein), the viral envelope composed of small envelope glycoprotein (E protein) and membrane glycoprotein (M protein) is arranged under the spike, and the core hidden in the envelope is a spiral folding structure composed of RNA gene chain and nucleocapsid protein (N protein). Structurally, the N protein is innermost, and thus if it is desired to detect the N protein, the N protein needs to be released first. Therefore, how to release the N protein in the sample becomes a difficult problem in coronavirus detection. The existing antigen detection means can not effectively release N protein in coronavirus, so that the detection accuracy is low, and false negative phenomenon is easy to occur.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a releasing agent for releasing N protein from coronavirus, which can effectively release the N protein from coronavirus, improve the detection accuracy and reduce the false negative rate.
The second object of the present invention is to provide a method for preparing a releasing agent for N protein release of coronavirus.
The invention also aims to provide an application of the releasing agent for releasing the N protein of the coronavirus.
One of the purposes of the invention is realized by adopting the following technical scheme:
a releasing agent for releasing N protein from coronavirus comprises the following components in percentage by mass:
0.01-0.1% of metal chelate, 0.8-1.2% of nonylphenol polyoxyethylene ether, 0.8-1.2% of hexadecyl trimethyl ammonium bromide, 0.1-0.5% of surfactant, 0.8-1.2% of glycerol, 60000.8-1.2% of polyethylene glycol and the balance of NaCl solution.
Further, the metal chelate is DTPA and/or EDTA. EDTA and DTPA can be combined with metal ions to generate a water-soluble complex; and Mg is required for the action of most of the nucleases and some of the proteases2+Therefore, EDTA and DTPA can be used as nuclease and protease inhibitors.
Further, the surfactant is one or more than two of Tween-20, Tween-40 and sodium dodecyl sulfate.
Further, the mass concentration of NaCl in the NaCl solution is 0.008-0.012 mol/L.
Further, the paint comprises the following components in percentage by mass:
0.04-0.06% of DTPA, 0.8-1.2% of nonylphenol polyoxyethylene ether, 0.8-1.2% of hexadecyl trimethyl ammonium bromide, 0.2-400.2% of Tween-0.3%, 0.8-1.2% of glycerol, 60000.8-1.2% of polyethylene glycol, and the balance of NaCl solution.
The second purpose of the invention is realized by adopting the following technical scheme:
a method for preparing a releasing agent for N protein release from coronaviruses, comprising the steps of:
1) preparing a NaCl solution; 2) Adding the metal chelate, nonylphenol polyoxyethylene ether, cetyl trimethyl ammonium bromide, a surfactant, glycerol and polyethylene glycol 6000 into the NaCl solution, and uniformly mixing to obtain the N protein releasing agent for coronavirus.
Further, the preservation temperature of the releasing agent is 2-8 ℃.
The third purpose of the invention is realized by adopting the following technical scheme:
use of a releasing agent for N protein release from a coronavirus, which is at least one of a HCoV 229E virus, a HCoV NL63 virus, a HCoV OC43 virus, a HCoV HKU1 virus, a SARS-CoV virus, a MERS-CoV virus and a 2019-nCoV virus, in a reagent for detecting a coronavirus.
Compared with the prior art, the invention has the beneficial effects that:
according to the releasing agent for releasing the N protein from the coronavirus, the NaCl solution is used as a buffering agent, the metal chelate is matched with the detergent, the outer envelope of the virus can be cracked, the N protein is effectively released from the coronavirus, the released N protein is kept in a free state under the action of the surfactant, the aggregation is reduced, good detection conditions are provided for subsequent detection treatment, and the detection accuracy is improved. Glycerol as a co-surfactant enables the insertion of a non-ionic surfactant into the structure of the reverse micelles to increase the size of the reverse micelles, thereby enabling the solubilization of proteins of greater relative molecular mass.
The preparation method of the releasing agent for releasing the N protein by the coronavirus is simple in preparation process, and the obtained releasing agent is stable in performance and strong in cracking property.
The application of a releasing agent for releasing N protein from coronavirus in coronavirus detection can improve the accuracy of coronavirus detection and reduce the false negative rate.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Example 1
A releasing agent for releasing N protein from coronavirus comprises the following components in percentage by mass: 0.05% of metal chelate, 1% of detergent, 0.25% of surfactant, 1.5% of auxiliary agent and the balance of NaCl solution with the concentration of 0.008 mol/L.
Wherein the metal chelate is DTPA; the detergent is nonylphenol polyoxyethylene ether and hexadecyl trimethyl ammonium bromide; the surfactant is Tween-40; the auxiliary agent is glycerol and 0.5 percent of polyethylene glycol 6000.
The preparation method of the releasing agent for releasing the N protein by the coronavirus comprises the following steps:
preparing 1L of NaCl solution with the concentration of 0.008mol/L, adding 0.05wt% of DTPA, 0.5 wt% of nonylphenol polyoxyethylene ether, 0.5 wt% of hexadecyl trimethyl ammonium bromide, 0.25 wt% of Tween-40, 1.0 wt% of glycerol and 0.5 wt% of polyethylene glycol 6000, uniformly mixing, and storing at the temperature of 2-8 ℃.
Example 2
A releasing agent for releasing N protein from coronavirus comprises the following components in percentage by mass: 0.05% of metal chelate, 2% of detergent, 0.25% of surfactant, 2% of auxiliary agent and the balance of NaCl solution with the concentration of 0.01 mol/L.
Wherein the metal chelate is DTPA; the detergent is nonylphenol polyoxyethylene ether and hexadecyl trimethyl ammonium bromide; the surfactant is Tween-40; the auxiliary agent is glycerol and 0.5 percent of polyethylene glycol 6000.
The preparation method of the releasing agent for releasing the N protein by the coronavirus comprises the following steps:
preparing 1L of NaCl solution with the concentration of 0.01mol/L, adding 0.05wt% of DTPA, 1.0 wt% of nonylphenol polyoxyethylene ether, 1.0 wt% of hexadecyl trimethyl ammonium bromide, 0.25 wt% of Tween-40, 1.0 wt% of glycerol and 1.0 wt% of polyethylene glycol 6000, uniformly mixing, and storing at the temperature of 2-8 ℃.
Example 3
A releasing agent for releasing N protein from coronavirus comprises the following components in percentage by mass: 0.05% of metal chelate, 3% of detergent, 0.25% of surfactant, 2.5% of auxiliary agent and the balance of NaCl solution with the concentration of 0.01 mol/L.
Wherein the metal chelate is DTPA; the detergent is nonylphenol polyoxyethylene ether and hexadecyl trimethyl ammonium bromide; the surfactant is Tween-40; the auxiliary agent is glycerol and 0.5 percent of polyethylene glycol 6000.
The preparation method of the releasing agent for releasing the N protein by the coronavirus comprises the following steps:
preparing 1L of NaCl solution with the concentration of 0.01mol/L, adding 0.05wt% of DTPA, 1.5 wt% of nonylphenol polyoxyethylene ether, 1.5 wt% of hexadecyl trimethyl ammonium bromide, 0.25 wt% of Tween-40, 1.0 wt% of glycerol and 1.5 wt% of polyethylene glycol 6000, uniformly mixing, and storing at the temperature of 2-8 ℃.
Example 4
A releasing agent for releasing N protein from coronavirus comprises the following components in percentage by mass: 0.08 percent of metal chelate, 2.4 percent of detergent, 0.25 percent of surfactant, 2 percent of auxiliary agent and the balance of NaCl solution with the concentration of 0.012 mol/L.
Wherein the metal chelate is EDTA; the detergent is nonylphenol polyoxyethylene ether and hexadecyl trimethyl ammonium bromide; the surfactant is Tween-40; the auxiliary agent is glycerol and 0.5 percent of polyethylene glycol 6000.
The preparation method of the releasing agent for releasing the N protein by the coronavirus comprises the following steps:
preparing 1L of NaCl solution with the concentration of 0.012mol/L, adding 0.08wt% of EDTA, 1.2 wt% of nonylphenol polyoxyethylene ether, 1.2 wt% of hexadecyl trimethyl ammonium bromide, 0.25 wt% of Tween-40, 1.0 wt% of glycerol and 1.0 wt% of polyethylene glycol 6000, uniformly mixing, and storing at the temperature of 2-8 ℃.
Performance detection
The number of 20 positive specimens is 1-20, 10ul of the positive specimens are respectively added into a reaction cup, each specimen is divided into 3 tubes, and 80 tubes in total, the numbers of the samples are respectively A1-A20, B1-B20, C1-C20 and D1-D20, the numbers of the untreated specimens are E1-E20, and the untreated specimens are respectively treated by the release agent prepared in the embodiment.
Wherein 50. mu.L of the releasing agent of example 1 was added to each of the A1-A20 reaction cups, 50. mu.L of the releasing agent of example 2 was added to each of the B1-B20 reaction cups, 50. mu.L of the releasing agent of example 3 was added to each of the C1-C20 reaction cups, 50. mu.L of the releasing agent of example 4 was added to each of the D1-D20 reaction cups, the mixture was allowed to stand at room temperature for 10 minutes after mixing, and then an on-machine experiment was carried out by a chemiluminescence immunoassay (CLIA), and the results of the measurements were compared with the results of the measurements carried out without pretreatment with the releasing agent, and the results are shown in Table 1.
TABLE 1 test and comparison results
As shown in Table 1, the samples without the release agent pretreatment were all negative, so that it could be concluded that the N protein in the samples was not released and could not be detected, while the samples pretreated with the release agent prepared in examples 1-4 were all positive, wherein the positive coincidence rate of example 1 was 80%, the positive coincidence rate of example 2 was 100%, and the positive coincidence rate of examples 3 and 4 was 95%. According to the exploration of the mass ratio of the components in the release agent, the release effect of the release agent in example 2 is most obvious, and the condition that false negative causes screen leakage does not occur.
The releasing agent can release N protein in various coronaviruses from the interior of the coronaviruses, the releasing agent can be used for pretreating clinical specimens, the N protein in the specimens can be released from the coronaviruses to facilitate the operation of the next experiment, the measured experiment result is more accurate and more in line with the clinical requirement, screen leakage caused by false negative is avoided, the releasing agent has important significance for the experiment research and development of the whole coronaviruses project, and the subsequent research and development and optimization of the experiment project are facilitated.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (8)
1. A releasing agent for releasing N protein from coronavirus is characterized by comprising the following components in percentage by mass:
0.01-0.1% of metal chelate, 0.8-1.2% of nonylphenol polyoxyethylene ether, 0.8-1.2% of hexadecyl trimethyl ammonium bromide, 0.1-0.5% of surfactant, 0.8-1.2% of glycerol, 60000.8-1.2% of polyethylene glycol and the balance of NaCl solution.
2. A releasing agent for N protein released from coronavirus according to claim 1, wherein: the metal chelate is DTPA and/or EDTA.
3. A releasing agent for N protein released from coronavirus according to claim 1, wherein: the surfactant is one or more than two of Tween-20, Tween-40 and sodium dodecyl sulfate.
4. A releasing agent for N protein released from coronavirus according to claim 1, wherein: the mass concentration of NaCl in the NaCl solution is 0.008-0.012 mol/L.
5. A releasing agent for N protein released by coronavirus according to any one of claims 1 to 4, characterized by comprising the following components in percentage by mass:
0.04-0.06% of DTPA, 0.8-1.2% of nonylphenol polyoxyethylene ether, 0.8-1.2% of hexadecyl trimethyl ammonium bromide, 0.2-400.2% of Tween-0.3%, 0.8-1.2% of glycerol, 60000.8-1.2% of polyethylene glycol, and the balance of NaCl solution.
6. A process for preparing a releasing agent for N protein release from coronavirus according to any one of claims 1 to 5, comprising the steps of:
1) preparing a NaCl solution; 2) Adding the metal chelate, nonylphenol polyoxyethylene ether, cetyl trimethyl ammonium bromide, a surfactant, glycerol and polyethylene glycol 6000 into the NaCl solution, and uniformly mixing to obtain the N protein releasing agent for coronavirus.
7. The method of claim 6, wherein the release agent has a storage temperature of 2-8 ℃.
8. Use of a releasing agent for N protein release from coronavirus according to any one of claims 1 to 5, in the preparation of a reagent for coronavirus detection, wherein: the coronavirus is at least one of HCoV 229E virus, HCoV NL63 virus, HCoV OC43 virus, HCoV HKU1 virus, SARS-CoV virus, MERS-CoV virus and 2019-nCoV virus.
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