CN111624348A

CN111624348A - Method for detecting novel coronavirus 2019-nCoV through multi-protein joint inspection combination

Info

Publication number: CN111624348A
Application number: CN202010442399.4A
Authority: CN
Inventors: 吴玉章; 吴长龙
Original assignee: Wuxi Fuwei Biomedical Technology Co ltd
Current assignee: Wuxi Fuwei Biomedical Technology Co ltd
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-09-04

Abstract

The invention relates to a method for detecting a novel coronavirus 2019-nCoV by a multi-protein joint inspection combination, and belongs to the technical field of novel coronavirus 2019-nCoV detection. The invention provides a method for detecting a novel coronavirus 2019-nCoV by a multi-protein joint inspection combination, which is characterized in that a novel high-purity and specific coronavirus antigen is directly scribed on a nitrocellulose membrane to prepare a detection test strip for detecting the novel coronavirus 2019-nCoV antibody, and IgG, IgM and/or IgA antibodies in a sample can be simultaneously detected by utilizing multiple antigens. The invention adopts 3 or 4 specific antigens for joint inspection, simultaneously carries out optimized design on the protein sequence of the antigen, effectively increases the sensitivity and specificity, and effectively avoids the problems of false negative and false positive.

Description

Method for detecting novel coronavirus 2019-nCoV through multi-protein joint inspection combination

Technical Field

The invention relates to a method for detecting a novel coronavirus 2019-nCoV by a multi-protein joint inspection combination, and belongs to the technical field of novel coronavirus 2019-nCoV detection.

Background

Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus which has never been found in human body before, and the disease caused by the coronavirus is named as 2019 coronavirus disease (COVID-19) and lethal virus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) by the world health organization.

The coronavirus genome encodes, in order, a spinous process protein (spike protein), an envelope protein (E protein), a Membrane protein (Membrane protein) and a Nucleocapsid protein (N protein). The spinous process protein (spike protein) is the most important surface membrane protein of coronaviruses and contains two subunits (subbunit), S1 and S2. Wherein S1 mainly contains receptor binding domain (RBD protein) responsible for recognizing cell receptor. S2 contains the basic elements required for the membrane fusion process. The spinous process protein plays a role in the combination of virus and host cell membrane receptor and membrane fusion, and is an important action site of host neutralizing antibody and a key target of vaccine design. The spinous process protein of SARS-CoV-2(2019-nCoV) interacts with human ACE2 to infect human airway epithelial cells. The nucleocapsid protein is the most abundant protein in coronaviruses. During virion assembly, the N protein binds to viral RNA and leads to the formation of a helical nucleocapsid. The nucleocapsid protein is a highly immunogenic phosphoprotein involved in viral genome replication and regulation of cellular signaling pathways.

At present, the serum antibody detection is mainly an immunochromatography method, and the method has simple operation and low requirement on equipment, even does not need detection equipment; the detection time is short; the requirement on the detection environment is not high, and the result can be obtained in about 15 minutes generally. The method has the disadvantages that the lateral chromatography method uses 1 or at most 2 proteins for detection, has no cleaning process, easily causes cross reaction of tests due to the fact that the blood of individual patients contains rheumatoid factors, heterophilic antibodies, autoantibodies, medicines, tumor cells and the like, and produces false positive results.

Therefore, a novel coronavirus detection method which has low requirement on detection environment, is suitable for large-scale population screening, can basically avoid the problems of false negative and false positive, has high sensitivity and specificity and can be used for disease course monitoring and vaccine effect evaluation is urgently needed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for detecting a novel coronavirus 2019-nCoV by a multi-protein joint inspection combination. The invention detects 3 or 4 kinds of specific antigens together, optimizes the protein sequence of the antigen, verifies multiple proteins mutually, changes the antibody combination mode (increasing cleaning and changing antibody markers) by using the immunoblotting method, effectively increases the sensitivity and specificity, can detect multiple kinds of specific antigens in the virus simultaneously, and effectively avoids the problems of false negative and false positive.

The technical scheme of the invention is as follows:

a method for detecting novel coronavirus 2019-nCoV by a multi-protein joint test combination comprises the steps of detecting a human plasma or serum sample by using a detection test strip, and immersing the detection test strip into the human plasma or serum sample for one-time incubation during detection; cleaning; adding a detection antibody for secondary incubation; then cleaning is carried out; carrying out color development reaction; the detection test strip jointly uses 3 or 4 novel coronavirus antigens to qualitatively detect IgG, IgM and/or IgA antibodies in a sample; the detection strip is prepared by using a nitrocellulose membrane as a solid phase carrier and scribing 3 or 4 detection lines (T lines) and 1 quality control line (C line) on the nitrocellulose membrane; a novel coronavirus antigen is fixed on the detection line; the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant N protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant E protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein, a recombinant N protein and a recombinant E protein; the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody.

Further, the amino acid sequence of the recombinant S1 protein is shown as SEQ ID NO. 1.

Furthermore, the amino acid sequence of the recombinant RBD protein is shown as SEQ ID NO. 2.

Furthermore, the amino acid sequence of the recombinant N protein is shown as SEQ ID NO. 3.

Furthermore, the amino acid sequence of the recombinant E protein is shown as SEQ ID NO. 4.

Furthermore, goat anti-mouse IgG is fixed on the quality control line.

Further, the method specifically comprises the following steps:

(1) wetting: placing the test strip in a reaction tank, adding the sample diluent, and uniformly mixing for 1-2min at 15-25 ℃; the sample diluent is a phosphate buffer solution with the pH value of 7-8;

(2) primary incubation: adding human serum or plasma sample, and incubating at 15-25 deg.C for 30-60 min;

(3) cleaning: pouring out the liquid in the reaction tank, adding the eluent into the reaction tank, and incubating for 4-6min at 15-25 ℃; cleaning is repeated for one time;

(4) and (3) secondary incubation: pouring out the liquid in the reaction tank, adding the detection antibody into the reaction tank, and incubating for 30-60min at 15-25 ℃; the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody;

(5) cleaning: pouring out the liquid in the reaction tank, adding the eluent into the reaction tank, and incubating for 4-6min at 15-25 ℃;

(6) cleaning: pouring out the liquid in the reaction tank, and repeatedly cleaning once;

(7) color development: pouring out the liquid in the reaction tank, draining the reaction tank, adding a substrate into the reaction tank, and reacting for 2-10min in a dark place; if more than two bands are developed, the detection result of the novel coronavirus antibody is positive; if no band develops color, the detection result of the novel coronavirus antibody is negative.

Further, the streaked concentrations of the recombinant antigen S1 protein, the recombinant antigen RBD protein, the recombinant antigen N protein and the recombinant antigen E protein are respectively 0.2-0.5mg/mL (1 μ L/cm), 0.1-0.2mg/mL (1 μ L/cm) and 0.1-0.2mg/mL (1 μ L/cm).

Further, the goat anti-mouse IgG was streaked at a concentration of 0.2-1mg/mL (1. mu.L/cm).

The beneficial technical effects of the invention are as follows:

the invention detects 3 or 4 kinds of specific antigens simultaneously, optimizes the protein sequence of the antigen, effectively increases the sensitivity and specificity, can detect a plurality of specific antigens in the virus simultaneously, and effectively avoids the problems of false negative and false positive.

Drawings

Fig. 1 is a schematic side view of a test strip prepared by joint detection of multiple proteins in the present invention, wherein T1, T2, T3 and T4 represent different detection lines, and different detection lines are marked with different novel coronavirus antigen bands.

Fig. 2 is a schematic diagram of a test strip prepared by the joint detection of multiple proteins in embodiment 1 of the present invention.

Fig. 3 is a schematic diagram of a test strip prepared by the joint detection of multiple proteins in embodiment 2 of the present invention.

Fig. 4 is a schematic diagram of a test strip prepared by the joint detection of multiple proteins in embodiment 3 of the present invention.

Detailed Description

Preparing a sample diluent: 0.1M, ph7.4 phosphate buffer: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; adding 999mL of pure water to dissolve, sucking 1mL of proClin300 into the solution, stirring and mixing uniformly, and fixing the volume to 1000 mL. Standing at normal temperature for later use.

Preparation of an eluent: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; adding 997.5mL of pure water to dissolve, sucking 1mL of proClin300 and 1.5mL of Tween-20 into the solution respectively, stirring uniformly, and diluting to 1000 mL. Standing at normal temperature for use.

Preparation of a substrate A: trimethylolaminomethane (1.21 g) was weighed, dissolved in pure water (900 mL), and pH was adjusted to pH7.5 with HCl. The volume is up to 1000 mL. Standing at normal temperature for later use.

Preparation of a substrate B: measuring 30% H₂O₂0.1mL, 0.01g of 3, 3-diaminobenzidine was weighed out to 100 mL. Standing at 2-8 deg.C for use.

Preparation of a substrate: substrate A and substrate B were mixed in equal volumes. It is used as it is.

Preparing an antibody diluent: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; 20g of bovine serum albumin is weighed, 900mL of pure water is added for dissolution, and NaOH solution is added for adjusting the pH value to 7.5. Sucking 1mL of proClin300 into the solution, stirring and mixing uniformly, and fixing the volume to 1000 mL. Standing at 2-8 deg.C for use.

The preparation method of the detection antibody comprises the following steps:

the Horse Radish Peroxidase (HRP) marked mouse anti-human IgG antibody is diluted by using an antibody diluent to ensure that the final concentration is 0.5 mu g/mL, and the antibody is uniformly mixed and placed for standby at the temperature of 2-8 ℃.

Preparation of recombinant S1 protein: synthesizing a full-length gene for coding a novel coronavirus 2019-nCOVSpike protein, and constructing the synthesized gene on an expression vector pCMV6-c.mFC of a mouse IgG FC by using a PCR (polymerase chain reaction) method to obtain a recombinant plasmid; the recombinant plasmid is transfected into Hek293 cells to secrete and express the fusion protein, and affinity purification is carried out through ProteinA to obtain the recombinant plasmid. Theoretical Mw 76.5 kD.

Preparing a recombinant RBD protein: synthesizing genes encoding 319-Arg to 541-Phe of a novel coronavirus 2019-nCOV Spike protein, and constructing the synthesized genes on an expression vector pCMV6-c.mFC of a mouse IgG FC by using a PCR (polymerase chain reaction) method to obtain a recombinant plasmid; the recombinant plasmid is transfected into Hek293 cells to secrete and express the fusion protein, and affinity purification is carried out through ProteinA to obtain the recombinant plasmid. Theoretical Mw 52 kD.

Preparation of recombinant N protein: synthesizing a complete gene for encoding a novel coronavirus 2019-nCOV nucleocapsid protein, and constructing the synthesized gene on an expression vector of pET28A-N-His by using a PCR (polymerase chain reaction) method to obtain a recombinant plasmid; transferring the recombinant plasmid into escherichia coli, expressing recombinant protein, and performing affinity purification through a nickel column to obtain the recombinant protein. Theoretical Mw 46.5 kD.

Preparing recombinant E protein: the complete gene for encoding the envelope protein of the novel coronavirus 2019-nCOV is synthesized, and the synthesized gene is constructed on an expression vector of HIS-C-pET28A-N-GST by using a PCR method. And E, converting the plasmid with correct sequencing into escherichia coli after preparation, expressing the recombinant protein, and performing affinity purification through a nickel column to obtain the recombinant protein. Theoretical Mw 36.8 kD.

And (3) selecting the marking concentration of the protein on the test strip: the color depth is indicated by "+"; "+ + + +" represents the strongest.

The detection method comprises the following steps: after the protein is scribed to prepare the detection test strip, a colloidal gold platform false positive sample and a positive sample, a detection antibody and a substrate are added, and the color development intensity of the strip is observed. As shown in tables 1-5.

Table 1 effect of different goat anti-mouse IgG antibody streaking concentrations on color; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	+++	+++	+++
0.5mg/mL	+++	+++	+++	+++
					0.2mg/mL	++	++	++	++
0.1mg/mL	+	+	+	+

Table 2 effect of streaking concentration of different recombinant S1 proteins on color; unit: mg/mL (1. mu.L/cm)

Table 3 effect of streaking concentration of different recombinant RBD proteins on color; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	-	-	+
0.5mg/mL	+++	-	-	-
					0.2mg/mL	++	-	-	-
0.1mg/mL	-	-	-	-

Table 4 effect of streaking concentration of different recombinant N proteins on colour; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	-	-	+
0.5mg/mL	+++	-	-	+
					0.2mg/mL	+++	-	-	-
0.1mg/mL	++	-	-	-

Table 5 effect of streaking concentration of different recombinant E proteins on colour; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	+	-	-
0.5mg/mL	+++	-	-	-
					0.2mg/mL	+++	-	-	-
0.1mg/mL	++	-	-	-

The scribe line concentrations were determined as follows:

0.2-1mg/mL (1. mu.L/cm) of goat anti-mouse IgG antibody;

0.2-0.5mg/mL (1. mu.L/cm) of recombinant S1 protein;

0.2-0.5mg/mL (1 μ L/cm) of recombinant RBD protein;

0.1-0.2mg/mL (1. mu.L/cm) of recombinant N protein;

0.1-0.2mg/mL (1. mu.L/cm) of recombinant E protein.

Selection of detection time and temperature:

at room temperature, setting the sample incubation time to be 30min, 40min and 1 h; the incubation time for detecting the antibody is set to be 30min, 40min and 1 h;

according to different incubation times, detecting the detection color development intensity of the test strip on the positive sample, and determining the appropriate time for detecting incubation as follows: sample preparation: 40min, detection of antibody: and (4) 40 min.

The temperature was set at 4 ℃, 22 ℃, 37 ℃ and the incubation time was set as sample: 40min, detection of antibody: 40 min;

according to the detection color development intensity of the detection test strip on the positive sample at different temperatures, the appropriate detection incubation temperature is determined as follows: at room temperature (15-25 ℃).

The present invention will be described in detail with reference to examples.

The test strip used in the following examples has a width of 3.5-4.0mm, a PVC offset plate length of 5-6cm, a label paper length of 1.1-1.2cm printed with serial numbers, a nitrocellulose membrane length of 4-5cm, an overlapping portion of 0.1-0.2cm, a label paper with serial numbers printed on the overlapping portion, a nitrocellulose membrane below, a quality control line spaced 0.9-1.1cm from the adjacent test line, and test lines spaced 0.4-0.6cm from each other.

Example 1

Preparing a detection test strip:

1. preparation of a reagent:

1.1 protein dilution: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; adding 999mL of pure water to dissolve, sucking 1mL of proClin300 into the solution, stirring and mixing uniformly, and fixing the volume to 1000 mL. Standing at normal temperature for later use.

1.2 preparation of sealing liquid: 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate, 9g of sodium chloride and 10g of bovine serum albumin are weighed, 900mL of pure water is added for dissolution, and NaOH solution is added for adjusting the pH value to 7.4. Sucking 1mL of proClin300 into the solution, stirring uniformly, and diluting to 1000 mL. Standing at 2-8 deg.C for use.

2. The preparation method comprises the following steps:

2.1 cutting the nitrocellulose membrane into a size of 31cm × 5cm in length and width, scribing 4 high-purity specific recombinant S1 proteins, recombinant RBD proteins, recombinant N proteins and recombinant E proteins on the nitrocellulose membrane respectively to obtain 4 detection lines (the scribing concentrations are 0.2mg/mL, 0.1mg/mL and 0.1mg/mL respectively, and the scribing amounts are all 1 μ L/cm), as shown in fig. 1 and fig. 2 (the distribution sequence of each detection line on the nitrocellulose membrane does not have a significant effect on the detection results);

2.2, scribing the goat anti-mouse IgG on the nitrocellulose membrane (quality control line, C line; scribing concentration is 0.5mg/mL, and scribing amount is 1 muL/cm);

drying for 2 hours at the temperature of 2.337 ℃;

2.4 completely immersing the dried nitrocellulose membrane in the sealing solution for 1 hour at room temperature (15-25 ℃);

drying for 2 hours at the temperature of 2.537 ℃;

and 2.6 sequentially sticking the nitrocellulose membrane dried for the second time and the synthetic paper printed with serial numbers on a PVC (polyvinyl chloride) rubber plate, and cutting into pieces with the width of 3.5-4.0mm to obtain the novel coronavirus 2019-nCOV antibody spectrum detection test paper strip.

The method for detecting the novel coronavirus 2019-nCoV antibody by using the novel coronavirus 2019-nCOV antibody spectrum detection test strip comprises the following steps:

(1) wetting: placing a detection test strip into a reaction tank, adding 1mL of sample diluent, and uniformly mixing for 1min at room temperature (15-25 ℃);

(2) primary incubation: adding 1mL of sample (human plasma or serum), and incubating for 40min at room temperature (15-25 ℃);

(3) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 5min at room temperature (15-25 ℃); cleaning is repeated for one time;

(4) and (3) secondary incubation: pouring out liquid in the reaction tank, adding 1mL of detection antibody into the reaction tank, and incubating for 40min at room temperature (15-25 ℃);

(5) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 5min at room temperature (15-25 ℃);

(7) color development: and (3) pouring out liquid in the reaction tank, draining the reaction tank, adding 200 mu L of substrate into the reaction tank, and reflecting for 5min in a dark place.

And observing whether the strip is colored or not and the depth of the strip. When two or more bands are developed, the detection result of the novel coronavirus antibody is positive, and the deeper the development is, the higher the antibody content is; when all the strips are not developed, the detection result of the novel coronavirus antibody is negative.

Example 2

Preparing a detection test strip:

1. preparation of a reagent:

1.2 preparation of sealing liquid: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; 10g of bovine serum albumin was weighed, dissolved in 900mL of pure water, and adjusted to pH7.4 by adding NaOH solution. Sucking 1mL of proClin300 into the solution, stirring uniformly, and diluting to 1000 mL. Standing at 2-8 deg.C for use.

2. The preparation method comprises the following steps:

2.1, cutting the nitrocellulose membrane into a size of 31cm × 5cm in length and width, and scribing 3 high-purity specific recombinant S1 proteins, recombinant RBD proteins and recombinant N proteins on the nitrocellulose membrane respectively to obtain 3 detection lines (the scribing concentrations are 0.3mg/mL, 0.3mg/mL and 0.2mg/mL respectively, and the scribing amounts are all 1 μ L/cm), as shown in FIG. 3 (the distribution sequence of each detection line on the nitrocellulose membrane does not have a significant influence on the detection results);

2.2, scribing the goat anti-mouse IgG on the nitrocellulose membrane (quality control line, C line; scribing concentration is 0.2mg/mL, and scribing amount is 1 muL/cm);

drying for 2 hours at the temperature of 2.337 ℃;

drying for 2 hours at the temperature of 2.537 ℃;

(1) wetting: placing a detection test strip into a reaction tank, adding 1mL of sample diluent, and uniformly mixing for 90s at room temperature (15-25 ℃);

(2) primary incubation: adding 1mL of sample, and incubating for 50min at room temperature (15-25 ℃);

(3) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 4min at room temperature (15-25 ℃); cleaning is repeated for one time;

(4) and (3) secondary incubation: pouring out liquid in the reaction tank, adding 1mL of detection antibody into the reaction tank, and incubating for 50min at room temperature (15-25 ℃);

(5) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 4min at room temperature (15-25 ℃);

(7) color development: and (3) pouring out liquid in the reaction tank, draining the reaction tank, adding 200 mu L of substrate into the reaction tank, and reacting for 4min in a dark place.

Example 3

Preparing a detection test strip:

1. preparation of a reagent:

2. The preparation method comprises the following steps:

2.1, cutting the nitrocellulose membrane into a size of 31cm × 5cm in length and width, and scribing 3 high-purity specific recombinant S1 proteins, recombinant RBD proteins and recombinant E proteins on the nitrocellulose membrane respectively to obtain 3 detection lines (the scribing concentrations are 0.5mg/mL, 0.5mg/mL and 0.5mg/mL respectively, and the scribing amounts are all 1 μ L/cm), as shown in FIG. 4 (the distribution sequence of each detection line on the nitrocellulose membrane does not have a significant influence on the detection results);

2.2, scribing the goat anti-mouse IgG on the nitrocellulose membrane (quality control line, C line; scribing concentration is 1mg/mL, and scribing amount is 1 muL/cm);

drying for 2 hours at the temperature of 2.337 ℃;

drying for 2 hours at the temperature of 2.537 ℃;

(1) wetting: placing a detection test strip into a reaction tank, adding 1mL of sample diluent, and uniformly mixing for 2min at room temperature (15-25 ℃);

(2) primary incubation: adding 1mL of human serum or plasma sample, and incubating for 60min at room temperature (15-25 ℃);

(3) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 6min at room temperature (15-25 ℃); cleaning is repeated for one time;

(4) and (3) secondary incubation: pouring out liquid in the reaction tank, adding 1mL of detection antibody into the reaction tank, and incubating for 60min at room temperature (15-25 ℃);

(5) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 6min at room temperature (15-25 ℃);

(7) color development: and (3) pouring out liquid in the reaction tank, draining the reaction tank, adding 200 mu L of substrate into the reaction tank, and reacting for 6min in a dark place.

Test example

200 negative serum samples (including false positive samples detected by 9 colloidal gold kits; including 5 hepatitis B, positive hepatitis A and hepatitis B antibody and 2 hepatitis B antigen positive samples and the like) and 20 positive serum samples are detected, and the prediction sensitivity and specificity of the detection test strip prepared in the embodiment 1 are 100% and 100% respectively; the detection results of 200 samples can be obtained within 2h by using a full-automatic immunoassay analyzer. The operation process is convenient and quick, and the detection result is accurate. The test strips prepared in examples 2 and 3 also achieve these test effects.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Fuweier biomedical science and technology Co., Ltd, Wuxi, City

<120> method for detecting novel coronavirus 2019-nCoV by multi-protein joint inspection combination

<160>4

<170>PatentIn version 3.3

<210>1

<211>681

<212>PRT

<213> Artificial sequence

<400>1

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

ThrLeu 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 IleArg 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 GlnPhe 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 Ala His

660 665 670

His His His His His His His His His

675 680

<210>2

<211>457

<212>PRT

<213> Artificial sequence

<400>2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Asp Asp Asp Asp Lys Ala Val Pro Arg Asp Ser Gly Cys Lys Pro Cys

225 230 235 240

Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys

245 250 255

Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val

260 265 270

Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe

275 280 285

Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu

290 295 300

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

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala

325 330 335

Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg

340 345 350

Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met

355 360 365

Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro

370 375 380

Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn

385 390 395 400

Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val

405 410 415

Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr

420 425 430

Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu

435 440 445

Lys Ser Leu Ser His Ser Pro Gly Lys

450 455

<210>3

<211>425

<212>PRT

<213> Artificial sequence

<400>3

Met His His His His His His Ser Asp Asn Gly Pro Gln Asn Gln Arg

1 5 10 15

Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser

20 25 30

Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro

35 40 45

Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln

50 55 60

His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile

65 70 75 80

Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala

85 90 95

Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro

100 105 110

Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro

115 120 125

Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala

130 135 140

Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn

145 150 155 160

Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly

165 170 175

Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser

180 185 190

Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser

195 200 205

Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala

210 215 220

Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met

225 230 235 240

Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser

245 250 255

Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys

260 265 270

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

275 280 285

Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr

290 295 300

Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe

305 310 315 320

Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp

325 330 335

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

340 345 350

Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr

355 360 365

Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu

370 375 380

Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu

385 390 395 400

Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser

405 410 415

Met Ser Ser Ala Asp Ser Thr Gln Ala

420 425

<210>4

<211>81

<212>PRT

<213> Artificial sequence

<400>4

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 2530

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

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

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val His His His His His

65 70 75 80

His

Claims

1. A method for detecting novel coronavirus 2019-nCoV by a multi-protein joint test combination is characterized in that a test strip is used for detecting a human plasma or serum sample, and the test strip is immersed into the human plasma or serum sample for primary incubation during detection; cleaning; adding a detection antibody for secondary incubation; then cleaning is carried out; carrying out color development reaction; the detection test strip combines 3 or 4 novel coronavirus antigens to qualitatively detect IgG, IgM and/or IgA antibodies in a sample; the detection test strip is prepared by using a nitrocellulose membrane as a solid phase carrier and scribing 3 or 4 detection lines and 1 quality control line on the nitrocellulose membrane; a novel coronavirus antigen is fixed on the detection line; the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant N protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant E protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein, a recombinant N protein and a recombinant E protein; the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody.

2. The method according to claim 1, wherein the amino acid sequence of the recombinant S1 protein is shown as SEQ ID No. 1.

3. The method of claim 1, wherein said recombinant RBD protein has the amino acid sequence set forth in SEQ id No. 2.

4. The method according to claim 1, wherein the amino acid sequence of the recombinant N protein is as shown in SEQ ID No. 3.

5. The method according to claim 1, wherein the amino acid sequence of the recombinant E protein is shown as SEQ ID No. 4.

6. The method of claim 1, wherein goat anti-mouse IgG is immobilized on the control line.

7. The method according to claim 1, characterized in that it comprises in particular the steps of:

(4) and (3) secondary incubation: pouring out liquid in the reaction tank, adding the detection antibody into the reaction tank, and incubating for 30-60min at 15-25 ℃; the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody;