CN112782401A

CN112782401A - Method for rapidly detecting novel coronavirus in vitro and application

Info

Publication number: CN112782401A
Application number: CN202110158674.4A
Authority: CN
Inventors: 曹雪松
Original assignee: Liaocheng University
Current assignee: Liaocheng University
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-05-11

Abstract

The present invention relates to a method for quickly detecting novel coronavirus (SARS-CoV-2) in vitro and its application. The method can specifically detect Spike protein (S protein for short) of the novel coronavirus, namely the virus living body. Its advantages are: 1. has high specificity, high sensitivity and higher stability; 2. the required equipment and operation are simple, and the operation can be performed by non-professional personnel, so that the popularization is facilitated; 3. the visualization of point-of-care testing (POCT) and observation results can be realized, and particularly, the single-person sampling detection can be realized, and the novel coronavirus infectors or carriers can be screened on equipment in a high-throughput and large-scale manner; 4. particularly, the required reagent can be produced in a large scale according to the conventional technology, and the development of the kit is convenient for large-scale popularization and application.

Description

Method for rapidly detecting novel coronavirus in vitro and application

Technical Field

The invention relates to the field of molecular biology and organic chemistry, in particular to a method for rapidly detecting novel coronavirus (SARS-CoV-2) in vitro by using a fluorescence labeled polypeptide probe and application thereof.

Background

Since the outbreak of new coronavirus and the initiation of global circulation and spread, the development of specific, high-sensitivity and simple-operation detection technology is always the first guarantee for effectively preventing and controlling new coronavirus. The two types of technologies for detecting the novel coronavirus are nucleic acid detection methods, including reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and integrated Dual CRISPR-Cas12a (All-in-One Dual CRISPR-Cas12a AIOD-CRISPR). Although this type of technology has high sensitivity, specificity and relatively low false positive and false negative rates, and is currently the most widely used gold standard for detecting viruses, it requires high requirements for detection equipment or platforms, high-sensitivity RT-PCR instruments are expensive, high requirements for laboratory cleanliness and operator, and must be handled by specialized personnel (or trained). In addition, the nucleic acid detection takes longer time, and the report result can be usually obtained within 24 hours at the fastest speed in consideration of the conditions of sample transportation and sample overstock, and the point-of-care testing (POCT) is difficult to carry out, so that the method has the limitations of complex operation, long time consumption, need of centralized inspection and the like, and cannot meet the detection requirements of examination and diagnosis of a large number of rapidly-growing suspected patients, asymptomatic infectors and the like; another new type of coronavirus detection technology is antibody detection (also known as serological detection), and there are 3 types of serological antibody detection methods currently used in clinical practice: enzyme-linked immunosorbent assay, chemiluminescence immunoassay, and colloidal gold immunochromatography. The antibody detection reagent is used for detecting IgM or IgG antibodies generated by stimulating immunocytes after viruses enter a human body in serum, wherein the IgM antibodies appear earlier, and the IgG antibodies appear later. Virus antigen detection is primarily the detection of some proteins on the surface of the virus. The method has the advantages of realizing rapid and large-scale batch operation and reducing the exposure risk of medical staff in the respiratory tract sample collection process. The speed is fast, the operation is easy, and the method does not depend on professional laboratories and professionals. However, the method has the disadvantages that the detection sensitivity and specificity of the method are remarkably reduced for patients with mild and asymptomatic infection, and the window period exists from virus infection to antibody production, so that the detection is always delayed, the method cannot be used for screening infected patients at an early stage, and in addition, the preparation period of the antibody is long, and the large-scale production of the antibody is limited.

In view of the rapid increase of the number of new coronavirus infection cases, suspected cases and asymptomatic infectors, the development of a detection technology which is rapid, accurate, high in sensitivity, strong in specificity and simple in operation is urgently needed, and particularly, the detection technology can meet the requirement of point-of-care testing (POCT), namely, the detection technology can detect the existence of live viruses, and has very important significance for screening, early discovery, early isolation, early treatment and the like of asymptomatic and early infectors. At present, no report and application of a new coronavirus detection technology and a kit thereof which integrate the characteristics are found.

Therefore, the invention utilizes the recognition and combination characteristics between a Receptor Binding Domain (RBD) of a novel coronavirus Spike protein (S protein for short) and a human Receptor-angiotensin converting enzyme 2 (ACE 2) to simulate the amino acid composition of the ACE2 and S protein combination region, designs and synthesizes specific polypeptide, and utilizes fluorescent molecules with special properties to modify (fluorescence labeling polypeptide probes). TPE (thermoplastic elastomer) in fluorescent molecules 1- (4-carboxybenzene) -1,2, 2-triphenylethylene [1- (4-Carboxyphenyl) -1,2, 2-triphenylethylene ], (TPE-COOH for short) has Aggregation-induced emission (AIE), and when the TPE molecules are in a discrete state, the TPE molecules exist in an isolated molecular state, so that excited molecules can be attenuated to a ground state in a non-radiative transition mode through intense intramolecular rotation and vibration, namely, fluorescence is not generated. After the fluorescence labeling polypeptide probe is specifically combined with the new coronavirus spike protein, a so-called "restriction of intramolecular rotation" (RIR) effect is generated due to the aggregation of fluorescent molecules, so that a non-radiative transition mode effect is blocked, namely, the fluorescent molecules generate radiative transition, and then, fluorescent groups emit light, namely, an AIE effect is generated, namely, the fluorescent groups emit light due to the combination of specific polypeptides and the new coronavirus spike protein, and the fluorescent labeling polypeptide probe has strong binding force and specificity. The presence of new coronaviruses can be specifically detected. The invention has the characteristics of high specificity (designed according to the binding property of the virus S protein and the receptor ACE2 thereof), high sensitivity, simple required equipment, capability of realizing on-site instant test (POCT), simple operation (the operation can be carried out by non-professional personnel), and the like, and particularly can be used for single-person sampling detection and large-scale and high-flux detection on equipment (the specific operation and equipment are described in the following sections). The technology and the subsequently developed kit are established based on the current latest research results, and the method integrates the advantages of two methods of novel coronavirus nucleic acid detection and antibody detection and has the characteristic of unique live virus detection. So far, no relevant report is found.

Disclosure of Invention

The invention aims to provide a method for realizing high sensitivity, high specificity, good stability and quick point-of-care testing (POCT) of a new coronavirus living body aiming at the defects of the prior art.

The second purpose of the invention is to provide a method which is simple to operate (can be operated by non-professional personnel), has low requirement on equipment site, can visualize the detection result, and simultaneously satisfies the requirements of individuals and detects the new coronavirus at high flux.

The third purpose of the invention is to provide a technical method which has short production period and low cost of the detection reagent and can realize the production of large-scale kits to detect the new coronavirus.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for rapidly detecting the existence of a novel coronavirus living body in vitro and application thereof. The method is characterized in that specific recognition and combination characteristics between a Receptor Binding Domain (RBD) of a new coronavirus Spike protein (Spike protein) and a human Receptor-angiotensin converting enzyme 2 (ACE 2) are utilized to simulate the amino acid composition of the RBD binding domain in ACE2 and SARS-CoV-2, specific polypeptides are designed and synthesized, and fluorescent molecules with AIE properties are utilized to modify (fluorescence labeling polypeptide probes). When the fluorescent molecule (comprising the polypeptide) is in a discrete state, no fluorescence is produced. However, when the fluorescently labeled polypeptide probe specifically binds to the new coronavirus spike protein, the fluorophore is caused to emit light, that is, the fluorescence of the fluorophore is generated due to the specific binding of the polypeptide to the new coronavirus spike protein. The presence of new coronaviruses can be specifically detected.

In order to realize the second purpose, the invention adopts the technical scheme that: the fluorescent molecules in the fluorescence labeling polypeptide probe are as follows: 1- (4-Carboxyphenyl) -1,2, 2-triphenylethylene [1- (4-Carboxyphenyl) -1,2, 2-triphenylethylene](TPE-COOH for short). Molecular weight is 376.45, and structural formula is:

(color development is green fluorescence). The polypeptide amino acid sequence is as follows: h₂N-Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH. The sequence of the synthesized fluorescent labeled polypeptide probe is as follows: TPE-Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH [ note that TPE is 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene]。

During individual detection, a total black 96-hole plate can be used as a reaction container of a sample and a detection reagent, a portable ultraviolet lamp with the wavelength of 254nm is used for observation in a dark room, the portable ultraviolet lamp directly irradiates the sample hole, a positive reaction shows green fluorescence, and the positive reaction can be observed by naked eyes. When large-scale samples need to be detected, a black-edge bottom-penetrating 96-well plate is adopted, a full-function enzyme-labeling instrument is used for scanning, the fluorescence intensity is detected under the excitation wavelength of 320nm and the emission wavelength of 350-650nm, and the positive reaction shows a peak at the position of 455 nm.

In order to realize the third purpose, the invention adopts the technical scheme that: the fluorescence labeling polypeptide probe is modified and synthesized by professional polypeptide synthesis companies, the TPE-COOH molecules have the advantages of simple synthesis method, stable chemical property, easy modification, low use concentration and the like, the synthesis process flow and the technical route are conventional technologies, special technologies and raw materials are not needed, technical barriers do not exist, and large-scale synthesis production can be realized. In addition, due to higher sensitivity, the quantity of the fluorescence labeling polypeptide probes required by each reaction is extremely low, so that the production period is short and the cost is controllable in the subsequent development of the kit production process. And because the fluorescence labeling polypeptide probe has high purity and good stability and has no special high requirement on product transportation, the transportation and the kit production cost can be reduced.

The invention has the beneficial effects that:

1. the invention aims to provide a novel coronavirus detection technology which is rapid, high in sensitivity, strong in specificity and simple to operate, particularly can meet the requirement of point-of-care testing (POCT), namely can detect the existence of live viruses, and has important significance for screening, early finding, early isolating, early treating and the like of asymptomatic and early infected persons.

2. The polypeptide has obvious specificity, and aiming at the high specificity recognition and combination properties of the new coronavirus spike protein and the human receptor ACE2, the designed and synthesized polypeptide is key amino acid simulating the combination of the human receptor ACE2 and the new coronavirus spike protein, and the interaction between proteins has high specificity, so that the polypeptide also has obvious specificity.

3. The invention has high sensitivity, firstly, because the used fluorescent probe is an organic synthetic molecule, four benzene rings can generate fluorescence after being excited, and thus, the visual fluorescence can be generated only by a very small amount of molecules; secondly, even if no fluorescence can be observed by visual inspection due to low virus concentration, the fluorescence can be detected by a sensitive full-wavelength enzyme-labeling instrument or similar equipment; thirdly, under the condition of extremely low concentration of virus (S protein), strong fluorescence which can be seen by naked eyes can still be detected, the current test result shows that the S protein with the concentration as low as 1pMol can be detected (see figure 1), and when the fluorescent polypeptide probe is applied to a sample of an infected person, because the surface of each new coronavirus particle at least carries more than 10S proteins on average, namely, the fluorescent polypeptide probe at least can be combined with more than 10 fluorescent polypeptide probes, the fluorescent polypeptide probe can be completely detected by the technology. The method has incomparable advantages for quickly diagnosing new coronavirus infected persons (particularly mild, asymptomatic and early infected persons), and is the only method capable of specifically detecting live new coronavirus in the world at present. And the positive reaction result (generation of fluorescence) is stable, and can be stably developed for more than 30 hours at room temperature (different according to specific conditions such as temperature, environmental cleanliness during sampling, no pollution during operation and the like). The color can be stably developed after being stored in a refrigerator at 4 ℃ for 48 to 60 hours. This provides a guarantee for the reliability of the result of the retest (time difference) to the laboratory after the sample is collected on site.

4. The invention has the advantages of rapidness and simple operation, and the detection result can be completed within 5 minutes. The operation process is very simple, only field sampling is needed, only one reagent (fluorescence labeled polypeptide probe) is added, and the operation can be finished through one-step reaction at normal temperature (or room temperature), and the observation is carried out in a dark room by using a common portable ultraviolet lamp with the wavelength of 254nm through visual inspection. If no darkroom condition exists on the spot, the waste paper box can be used for simple manufacture, and the whole operation does not need professional personnel, professional training, other expensive and precise equipment and the like.

5. The invention has the advantage of realizing large-scale virus detection, and as long as the conditions permit (enough number of full-function microplate readers or similar equipment, enough space and the like), the sample amount which can be detected in unit time theoretically is as follows: and (3) calculating according to the detection of a 96-well plate and the operation of a single sample injection groove device, wherein the detectable number of one reaction is 96 multiplied by the number of devices. If an automatic sample injector is used for sample injection, and a multi-sample injection groove full-wavelength microplate reader or similar equipment is used for detection, the detection efficiency can be further improved, and the method has great technical advantages for large-scale screening of novel coronavirus infectors (especially early asymptomatic infectors).

6. The invention has the advantage of detecting the virus concentration (titer) in vitro (droplet, aerosol, excrement and urine and the like) discharged by virus infectors, and has the advantages of high sensitivity and result stability, so that the concentration of the virus discharged in vitro by patients can be preliminarily determined by adopting a parallel detection method aiming at the difference of the virus concentration carried by different infectors such as severe illness, mild illness, asymptomatic disease and the like according to the difference of the intensity of generated fluorescence, and the invention has special significance for quickly diagnosing and investigating the different infectors in early stage, particularly asymptomatic infectors, and the virus amount in the environment of places such as crowd gathering or hospital infectious department and the like.

7. The invention also provides an auxiliary visual detection means for developing novel coronavirus vaccines and infection blockers thereof, the development of antibody vaccines, polypeptides and other blockers aiming at the binding region of the novel coronavirus S protein and human ACE2 receptor is a hotspot of the current research, the invention can provide visual and sensitive detection means for the research, the antibodies, polypeptides and other blockers are firstly bound with the S protein by combining the technologies of biomembrane layer reflected light interference (Biolayer interference) and biomolecule crystal structure analysis, and the like, and then the fluorescence-labeled polypeptide probe is added into the reaction system by applying the technology of the invention, because the antibodies and the like are pre-bound and occupied with the S protein, the AIE (fluorescence) is weakened or disappeared, so that the strength of the binding capacity of the antibodies, polypeptides and blockers and the S protein can be presumed, namely, the high-flux detection means can be carried out aiming at the effectiveness of the novel coronavirus medicines, And (4) rapidly screening. At present, the technical application in the aspect is not reported.

Drawings

FIG. 1 shows the fluorescence detection results generated 5 minutes after the S protein is combined with the fluorescence-labeled polypeptide probe.

FIG. 2 shows the results of the full-function microplate reader (Synergy H1, Biotek).

FIG. 3 is a bar graph of fluorescence intensity detection.

FIG. 4 shows the fluorescence detection results 24 hours after the S protein is combined with the fluorescence-labeled polypeptide probe.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The invention designs and synthesizes specific polypeptide by utilizing the recognition and combination characteristics between Receptor Binding Domains (RBD) of the novel coronavirus spike protein and human Receptor-angiotensin converting enzyme 2 (ACE 2) according to the amino acid composition of the binding domains of ACE2 and RBD in SARS-CoV-2, and modifies the specific polypeptide by fluorescent molecules with AIE property (fluorescence labeling polypeptide probe). When the fluorescent molecule (comprising the polypeptide) is in a discrete state, no fluorescence is produced. However, when the fluorescence labeling polypeptide probe is specifically combined with the new coronavirus spike protein, the fluorescent group emits light, and orange fluorescence is generated. That is, the emission of the fluorophore is due to the binding of the specific polypeptide to the new coronavirus spike protein. The presence of new coronaviruses can be specifically detected. According to the generation of fluorescence, whether the infected new coronavirus is infected or not can be detected, and according to the intensity of the generated fluorescence, the titer of the virus carried by an infected person can be preliminarily determined.

The detection of the new coronavirus is to replace the virus particle with the full-length spike protein, and the principle is that the S protein of the new coronavirus is positioned on the surface of the virus particle, and the RBD (receptor binding domain) is positioned at the uppermost (outer) end of the whole S protein, so that the structural and molecular biological basis is established for simulating the virus particle by using the S protein, and the concentration of the detectable S protein (namely the corresponding virus concentration) can be accurately determined.

The method for detecting the new coronavirus S protein (including the new coronavirus) has the characteristics of intuition, accuracy, high efficiency, strong sensitivity and specificity, and has unique value for detecting the new coronavirus clinically and on site.

The fluorescent molecule in the fluorescence labeling polypeptide probe is as follows: 1- (4-Carboxyphenyl) -1,2, 2-triphenylethylene [1- (4-Carboxyphenyl) -1,2, 2-triphenylethylene](TPE-COOH for short). Molecular weight is 376.45, and structural formula is:

the polypeptide amino acid sequence of the invention is as follows: H2N-Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH. (Note: H)₂N represents an amino terminus, COOH represents a carboxyl terminus)

The sequence of the fluorescence labeling polypeptide probe is as follows: (TPE) -Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH [ note: TPE stands for 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene, modified at the amino terminus of the polypeptide ]. The TPE molecules can emit green fluorescence under the irradiation of an ultraviolet lamp with the wavelength of 254nm, and can be observed by visual inspection in a dark room. The accurate fluorescence intensity can be generated by scanning 650nm in the excitation wavelength of 320nm and the emission wavelength of 350-650nm by using a full-wavelength microplate reader, wherein the maximum peak is generated at 455 nm.

The fluorescence labeling polypeptide probe is synthesized by professional polypeptide companies, and the purity is 95%. Each 1mg of the stock solution was dissolved in 104. mu.l of dimethyl sulfoxide to a final concentration of 1 mMol.

The S protein sequence of the invention is (NCBI Accession No.: QHD 43416.1):

H₂N-VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP-COOH (Note: H)₂N represents an amino terminus, COOH represents a carboxyl terminus)

Protein S was purchased from a proprietary reagent company and was 99% pure. Each 50ug was dissolved in 83ul of sterile water to give 4.41uMol stock.

The invention provides a method for detecting new coronavirus living bodies, which has the advantages of high sensitivity, high specificity, good stability, simple operation, low requirement on equipment site, visual detection result and realization of rapid point-of-care testing (POCT).

Example 1 Synthesis of a fluorescently labeled polypeptide Probe sequence: (TPE) -Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH [ note: TPE stands for 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene, modified at the amino terminus of the polypeptide ]. The above sequences are modified and synthesized by Qiaozhou Biotechnology Ltd.

Each 1mg of the fluorescently labeled polypeptide was dissolved in 104.25ul of dimethyl sulfoxide to a final concentration of 1mMol in the stock solution.

The sequence of the novel coronavirus spike protein (S) (NCBI Access No.: QHD43416.1) was purchased from Acro biosystems. 50ug was dissolved in 83ul of sterile water to a final concentration of 4.4uMol stock solution.

50ul of S protein solution with final concentration of 0.2, 0.02 and 0.002uMol and 50ul of fluorescently-labeled polypeptide probe with final concentration of 40uMol are respectively added into wells A1, A2 and A3 in a full-black bottom transparent 96-well plate, and the S protein solution and the fluorescently-labeled polypeptide probe are respectively diluted to the required working concentration by 1% PBS (pH7.4) buffer solution and DMSO (dimethyl sulfoxide). The final concentrations of the S protein in the A1, A2 and A3 wells are 0.1, 0.01 and 0.001uMol respectively, and the final concentrations of the fluorescence labeling polypeptide probes are 20uMol respectively. The A1, A2 and A3 holes are used for detecting whether the S protein is combined with the fluorescence labeling polypeptide probe to react and generate green fluorescence.

Well a4 is a negative control containing only S protein. The final concentration of S protein was 0.01 uMol.

25ul of S protein solution with final concentration of 0.2, 0.02 and 0.002uMol and 25ul of fluorescently-labeled polypeptide probe with final concentration of 20uMol are respectively added into the wells B1, B2 and B3, and the S protein solution and the fluorescently-labeled polypeptide probe are diluted to the required working concentration by 1% PBS (pH7.4). The final concentrations of the S protein in the wells B1, B2 and B3 were 0.1, 0.01 and 0.001uMol, respectively, and the concentrations of the fluorescently-labeled polypeptide probes were 10 uMol. The holes B1, B2 and B3 are used for detecting whether the S protein is combined with the low-concentration fluorescence labeling polypeptide probe to react and generate bright green fluorescence.

By analogy, the wells C1, C2, C3, D1, D2 and D3 are used for detecting whether the S protein is combined with the low-concentration fluorescence-labeled polypeptide probe (the concentrations of the fluorescence-labeled polypeptide probe in the wells C1-C3 are all 1uMol, and the concentrations of the fluorescence-labeled polypeptide probe in the wells D1-D3 are all 0.1uMol) to react and generate bright green fluorescence.

Only 25ul of a 40uMol concentration of fluorescently labeled polypeptide probe was added to well E1 as a negative control. 25ul of PBS buffer was added to a final concentration of 20uMol of fluorescently labeled polypeptide probe.

25ul of dimethyl sulfoxide (DMSO) and 25ul of sterile water were added to wells E2 and E3, respectively, as negative controls. The final concentration of the fluorescently labeled polypeptide probe was 20 uMol.

Each of the above reactions was carried out at room temperature (25 ℃ C.) for 5 minutes.

The 96-well plate is placed under a portable ultraviolet lamp under 254nm wavelength for irradiation, and a common smart phone (the Huacheng nova6 mobile phone is used in the invention) is used for photographing. The results are shown in FIG. 1.

For the detection of visual visible fluorescence, the minimum concentration of the detectable S protein is 0.000001 uMol.

Attached table 1. statistical table of corresponding concentrations of S protein and fluorescent labeled polypeptide in each well of the 96-well plate of FIG. 1.

Example 2 the 96-well plate of example 1 was examined with a full-wavelength microplate reader (Synergy H1, Biotek). The parameters used were, fluorescence spectrum: random; and (3) fixed excitation: 405 nm; and (3) starting transmission: 350 nm; stopping: 650 nm; the method comprises the following steps: 5 nm; an optical element: top, gain: 5 nm; xenon flash lamp as light source, energy of light source: high; speed of normal, delay of 100msec, measurement/data point of 10 were detected. The detection height is 1 mm. The positive reactions (well No. a1, a2, A3, B1, B2, B3) exhibited a peak at about 455 nm. The results are shown in FIG. 2.

As shown in FIG. 2, when the final concentration of the fluorescently labeled polypeptide probe was 20uMol, the fluorescence intensities produced by the reactions with the S protein at final concentrations of 0.001, 0.00001, and 0.000001uMol, respectively, were 7946, 7519, and 5863, respectively. By analogy, when the final concentration of the fluorescence labeling polypeptide probe is 10uMol, the fluorescence intensities generated by the reactions with the S proteins of 0.001, 0.00001 and 0.000001uMol are 2729, 2981 and 2935 respectively, and when the final concentration of the fluorescence labeling polypeptide probe is less than or equal to 10uMol, the fluorescence intensities generated by the reactions with the S proteins of 0.001, 0.00001 and 0.000001uMol respectively are the same as those of a control (A4: only containing S protein; E1: only containing fluorescence labeling polypeptide probe; E2: only containing fluorescence labeling polypeptide probe + PBS buffer solution; E3: only containing fluorescence labeling polypeptide probe + DMSO), namely, the fluorescence intensity is not increased significantly. The fluorescence intensity of each reaction group is compared with that of FIG. 2. The above results demonstrate that the optimal final concentration of fluorescently labeled polypeptide probes used in the visual inspection (with a hand-held ultraviolet lamp) of the present invention is 20uMol, and the optimal final concentration of fluorescently labeled polypeptide probes used in the mechanical inspection (with a full-wavelength microplate reader) is 10uMol (to save costs, i.e., to reduce the amount of fluorescently labeled polypeptide probes used). The lowest concentration of the S protein of the new coronavirus that could be detected was 0.000001uMol, i.e. in theory infected samples as low as one new coronavirus particle could be detected.

Example 3 the 96-well plate of example 2 was left at room temperature (25 ℃ C.) for 24 hours, and the 96-well plate was irradiated with a portable ultraviolet lamp having a wavelength of 254nm, and photographed with a general smart phone (the invention uses a Huachenva 6 mobile phone). The results are shown in FIG. 4. The results show that each reaction well in the A1-A3 and B1-B3 groups still shows obvious fluorescence. The effect of detecting the new coronavirus by using the fluorescence-labeled polypeptide probe is proved to have higher stability, and the new coronavirus can be detected on site in any occasions (except the case of lower temperature in winter, the detection needs to be carried out indoors) without being stored at low temperature, namely, without low-temperature equipment such as a refrigerator and the like.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Sequence listing

<110> chat university

<120> method for rapidly detecting novel coronavirus in vitro and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> PRT

<213> polypeptide sequence (2 Ambystoma latex x Ambystoma jeffersonianum)

<400> 1

Thr Ala Gln Met Tyr Gly Gln Ala Lys Tyr Phe Leu Asp Lys Phe Asn

1 5 10 15

His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Gly Lys Gly Asp

20 25 30

Phe Arg Gly Tyr

35

<210> 2

<211> 1198

<212> PRT

<213> spike protein sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

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Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser

1 5 10 15

Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val

20 25 30

Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr

35 40 45

Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe

50 55 60

Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr

65 70 75 80

Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp

85 90 95

Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val

100 105 110

Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val

115 120 125

Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val

130 135 140

Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe

145 150 155 160

Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu

165 170 175

Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His

180 185 190

Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu

195 200 205

Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln

210 215 220

Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser

225 230 235 240

Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln

245 250 255

Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp

260 265 270

Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu

275 280 285

Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg

290 295 300

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

305 310 315 320

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

325 330 335

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

340 345 350

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

355 360 365

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

370 375 380

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

385 390 395 400

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

405 410 415

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

420 425 430

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

435 440 445

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

450 455 460

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

465 470 475 480

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

485 490 495

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

500 505 510

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe

515 520 525

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

530 535 540

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

545 550 555 560

Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser

565 570 575

Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln

580 585 590

Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala

595 600 605

Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

610 615 620

Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His

625 630 635 640

Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys

645 650 655

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val

660 665 670

Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn

675 680 685

Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr

690 695 700

Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser

705 710 715 720

Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn

725 730 735

Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu

740 745 750

Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala

755 760 765

Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly

770 775 780

Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg

785 790 795 800

Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala

805 810 815

Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg

820 825 830

Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro

835 840 845

Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala

850 855 860

Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln

865 870 875 880

Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val

885 890 895

Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe

900 905 910

Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser

915 920 925

Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu

930 935 940

Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser

945 950 955 960

Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val

965 970 975

Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr

980 985 990

Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

995 1000 1005

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg

1010 1015 1020

Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser

1025 1030 1035 1040

Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln

1045 1050 1055

Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala

1060 1065 1070

His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe

1075 1080 1085

Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn

1090 1095 1100

Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn

1105 1110 1115 1120

Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu

1125 1130 1135

Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly

1140 1145 1150

Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile

1155 1160 1165

Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp

1170 1175 1180

Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro

1185 1190 1195

Claims

1. A method for rapidly detecting novel coronavirus spike protein in vitro is characterized by comprising the following steps: (1) marking the N end of the polypeptide for identifying the novel coronavirus spike protein by using a fluorescent molecule to obtain a fluorescence-marked polypeptide probe;

(2) adding a fluorescence labeling polypeptide probe and novel coronavirus spike protein into a PBS buffer solution, and adding a fluorescence labeling polypeptide probe into the PBS buffer solution as a negative control in the other reaction;

standing at room temperature for 5 minutes, and directly irradiating the reaction solution with an ultraviolet lamp of 254nm to obtain green fluorescence; or detecting the fluorescence intensity under the excitation wavelength of 320nm and the emission wavelength of 350-650nm, and drawing corresponding curves of the novel coronavirus spike protein or RBD thereof with different concentrations and the fluorescence intensity;

determining the presence or concentration of the novel coronavirus spike protein based on whether the former exhibits orange fluorescence or an increase in the fluorescence intensity of the latter, as compared to a negative control group.

2. The method for rapidly detecting the novel coronavirus spike protein in vitro as claimed in claim 1, wherein the polypeptide sequence is as follows: H2N-Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH.

3. The method for rapidly detecting the novel coronavirus spike protein in vitro as claimed in claim 1, wherein the fluorescent molecule is 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene, the molecular weight of which is 376.45, and the structural formula of which is:

4. the method for rapidly detecting the novel coronavirus spike protein in vitro as claimed in claim 1, wherein the preparation method of the fluorescence-labeled polypeptide probe comprises the following steps:

using methyl ester and ethyl ester as protecting groups of carboxylic acid, then removing one molecule of water from the carboxyl (-COOH) of the 1- (4-carboxybenzene) -1,2, 2-triphenylethylene and the amino (-NH 2) of the polypeptide simultaneously, and carrying out dehydration condensation reaction to connect the 1- (4-carboxybenzene) -1,2, 2-triphenylethylene to the amino terminal of the polypeptide; the sequence of the novel coronavirus, namely the spike protein is (NCBIAccess No.: QHD 43416.1).

5. The method for rapidly detecting the novel coronavirus spike protein in vitro as claimed in claim 4, wherein the synthesized fluorescent-labeled polypeptide probe sequence is as follows: TPE-Thr-Ala-Gln-Met-Tyr-Gly-Gln-Ala-Lys-Tyr-Phe-Leu-Asp-Lys-Phe-Asn-His-Glu-Ala-Glu-Asp-Leu-Phe-Tyr-Gln-Ser-Ser-Leu-Gly-Lys-Gly-Asp-Phe-Arg-Gly-Tyr-COOH, wherein the TPE is 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene and is modified at the amino terminal of the polypeptide.

6. The application of the in vitro rapid detection of the novel coronavirus spike protein as claimed in claim 1, wherein the sample to be detected is a novel coronavirus throat swab, a nose swab or a lung lavage fluid, a fluorescence-labeled polypeptide probe is added into a PBS buffer solution, the mixture is left to stand and react for 5 minutes at room temperature, and the reaction solution is directly irradiated by a 254nm ultraviolet lamp to show green fluorescence; or detecting the fluorescence intensity under the excitation wavelength of 320nm and the emission wavelength of 350-650nm, and drawing a corresponding curve of the novel coronavirus spike protein or RBD thereof with different concentrations and the fluorescence intensity.

7. The kit for rapidly detecting the novel coronavirus spike protein in vitro is characterized by comprising a fluorescence-labeled polypeptide probe, wherein the fluorescence-labeled polypeptide probe is as follows: the polypeptide N end which is marked by fluorescent molecules and can identify the novel coronavirus spike protein.