CN114764100A - Novel detection kit for coronavirus and preparation method thereof - Google Patents

Novel detection kit for coronavirus and preparation method thereof Download PDF

Info

Publication number
CN114764100A
CN114764100A CN202210532626.1A CN202210532626A CN114764100A CN 114764100 A CN114764100 A CN 114764100A CN 202210532626 A CN202210532626 A CN 202210532626A CN 114764100 A CN114764100 A CN 114764100A
Authority
CN
China
Prior art keywords
solution
dtnb
pad
nitrocellulose membrane
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210532626.1A
Other languages
Chinese (zh)
Inventor
金涌
陈艳
邹明强
薛强
殷宏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chinese Academy of Inspection and Quarantine CAIQ
Original Assignee
Chinese Academy of Inspection and Quarantine CAIQ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chinese Academy of Inspection and Quarantine CAIQ filed Critical Chinese Academy of Inspection and Quarantine CAIQ
Priority to CN202210532626.1A priority Critical patent/CN114764100A/en
Publication of CN114764100A publication Critical patent/CN114764100A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F9/00—Making metallic powder or suspensions thereof
    • B22F9/16—Making metallic powder or suspensions thereof using chemical processes
    • B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
    • B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Composite Materials (AREA)
  • General Chemical & Material Sciences (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Manufacturing & Machinery (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The invention discloses a novel detection kit for coronavirus and a preparation method thereof. Belongs to the technical field of medical examination. The immunochromatography surface-enhanced Raman spectroscopy test strip comprises an immunochromatography surface-enhanced Raman spectroscopy test strip for detecting the new coronavirus and a sample extracting solution, wherein the test strip specifically comprises a Wu-DTNB @ Ag-Ab1 solution uniformly coated on a binding pad, a new crown N protein antibody 2 coated on a nitrocellulose membrane to form a detection line, and rabbit anti-mouse IgG coated on the nitrocellulose membrane to form a quality control line. The test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC backing, wherein the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are continuously adhered to the PVC backing in sequence. The method has the advantages of strong specificity, high sensitivity, short detection time, field operation, low detection cost, simple and convenient operation and the like.

Description

Novel coronavirus detection kit and preparation method thereof
Technical Field
The invention relates to the technical field of medical examination, in particular to a novel detection kit for coronavirus and a preparation method thereof.
Background
Nucleic acid detection as a diagnostic method plays an important role in clinical diagnosis of patients and investigation of suspected patients. However, the suspected patient cannot be diagnosed and treated by shunting in time due to the limitations of complicated operation, long time consumption, need of centralized inspection, and the like, and the detection also has the defect of false negative.
The immune detection detects infection by detecting in vivo virus protein (antigen) or in vivo specific antibody against virus protein by the principle of specific reaction between antibody and antigen, and is widely applied to detection of major infectious diseases such as AIDS virus, hepatitis B, hepatitis C and the like.
The novel coronavirus detection realizes the detection function by the specific combination of the novel coronavirus and a novel coronavirus antibody. The method has the characteristics of rapid result generation and simple and convenient operation, and is an important detection means for diagnosis, especially primary screening and investigation of the new coronary pneumonia. The product is rapidly detected on the use site, suspected patients and close-contact people are rapidly screened, and the method has great significance for rapidly controlling the transmission.
However, although immunoassay becomes an auxiliary detection means for the confirmed screening of new coronavirus, the low sensitivity is the biggest short board.
Therefore, how to provide a novel coronavirus detection kit to overcome the above technical defects is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the invention provides a novel detection kit for coronavirus and a preparation method thereof. Compared with the conventional colloidal gold chromatography reagent, the detection sensitivity is improved by more than one order of magnitude, the bottleneck technical problem of low immunodetection sensitivity is solved, the original congenital defects of immunodiagnosis products are overcome, and the kit is suitable for being used for rapid screening and diagnosis in primary medical institutions and public health emergency monitoring places.
Specifically, a Surface Enhanced Raman Spectroscopy (SERS) and an immunoassay technology are combined to establish an SERS immunochromatography detection technology, so that the SERS immunochromatography detection technology has high sensitivity and spectrum distinguishability of SERS, and also has high specificity and convenience of immunoassay. The tungsten-coated silver core-shell structure is used as the SERS substrate, so that the stability of the tungsten nanoparticles is realized, and the Raman enhancement effect of the silver nanoparticles is realized. Modifying a Raman signal molecule DTNB and a COVID-19 antibody on the tungsten silver core-shell surface, combining a surface enhanced Raman spectrum technology with an immunochromatography technology, and developing a novel ultrasensitive COVID-19 surface enhanced Raman spectrum immunochromatography detection method.
The test strip adopts a double-antibody sandwich method to detect whether a sample to be detected contains the novel coronavirus. When a positive sample is detected, the novel coronavirus in the sample is combined with a novel coronavirus antibody 1 connected to a Raman immune probe (Wu-DTNB @ Ag-Ab1), the compound moves forwards in the nitrocellulose membrane through chromatography, and is combined with a coated novel coronavirus antibody 2 when passing through a detection area to form a Raman immune probe-novel coronavirus antibody 1-novel coronavirus antibody 2 compound, so that a T line is banded, and the result is positive; when a negative sample is detected, the sample does not contain the neocorona antigen, and the immune complex cannot be formed, so that a T line does not have a strip, and the result is negative. And measuring a probe signal on the T line by using a Raman spectrometer, and measuring the content of the new coronavirus.
In order to achieve the purpose, the invention adopts the following technical scheme:
a detection kit for a novel coronavirus comprises an immunochromatography surface-enhanced Raman spectroscopy test strip for detecting the novel coronavirus and a sample extracting solution, wherein the test strip comprises a Wu-DTNB @ Ag-Ab1 solution uniformly coated on a binding pad, a novel corona N protein antibody 2 coated on a nitrocellulose membrane to form a detection line, and rabbit anti-mouse IgG coated on the nitrocellulose membrane to form a quality control line; the preparation method of the Wu-DTNB @ Ag-Ab1 solution comprises the following steps:
(1) reacting trisodium citrate with silver nitrate to prepare a nano silver particle solution;
(2) adding Raman signal molecules DTNB into the nano silver particle solution prepared in the step (1) to obtain AgNPs-DTNB solution;
(3) adding tungsten atoms into the AgNPs-DTNB solution prepared in the step (2) to obtain Wu-DTNB @ Ag solution;
(4) and (4) adding the new crown N protein antibody 1 into the Wu-DTNB @ Ag solution prepared in the step (3) to obtain the Wu-DTNB @ Ag-Ab1 solution.
Preferably: the coating concentration of the new crown N protein antibody 2 is 0.5 mg/mL; the coating concentration of rabbit anti-mouse IgG was 1 mg/mL.
Preferably: the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC back lining; the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are sequentially adhered to the PVC backing in sequence.
The invention also provides a preparation method of the detection kit, which comprises the following steps:
(1) preparing a nano silver particle solution: weighing silver nitrate, dissolving the silver nitrate in ultrapure water to obtain a silver nitrate solution, heating, adding a trisodium citrate solution when the silver nitrate solution is boiled, continuing to heat, and cooling to obtain a nano silver particle solution;
(2) preparing a nano silver particle surface modification DTNB solution: adding the nano silver particle solution prepared in the step (1) into a DTNB solution, stirring for reaction, centrifuging, removing a supernatant, and suspending a precipitate with ultrapure water to obtain an AgNPs-DTNB solution;
(3) preparation of Wu-DTNB @ Ag: mixing the AgNPs-DTNB solution prepared in the step (2) and the tungsten atom solution in equal volume, reacting, centrifuging, suspending the precipitate with ultrapure water, and performing ultrasonic dispersion to obtain a Wu-DTNB @ Ag solution;
(4) preparation of Wu-DTNB @ Ag-Ab1 solution: adding potassium carbonate solution into the Wu-DTNB @ Ag solution prepared in the step (3), adding a new crown N protein antibody 1 into the solution, adding BSA solution after reaction, reacting, centrifuging, and suspending the precipitate with preservation solution to obtain Wu-DTNB @ Ag-Ab1 solution;
(5) coating of the bonding pad: after the bonding pad is soaked in a phosphate buffer solution, uniformly coating the Wu-DTNB @ Ag-Ab1 solution prepared in the step (4) on the bonding pad, and packaging for later use;
(6) Treatment of the sample pad: soaking the sample pad in phosphate buffer solution, and packaging for later use;
(7) coating of nitrocellulose membrane: diluting the new crown N protein antibody 2 by using a phosphate buffer solution, and coating the diluted new crown N protein antibody on a nitrocellulose membrane to be used as a detection line; diluting rabbit anti-mouse IgG antibody with phosphate buffer solution, and coating on nitrocellulose membrane as quality control line; packaging for later use;
(8) and (6) assembling the test strip.
Preferably, the following components: the mass-to-volume ratio of the silver nitrate, the ultrapure water and the trisodium citrate solution in the step (1) is 0.018 g: 100mL of: 4 mL; the heating conditions were: heating the mixture in an oil bath kettle at 130 ℃ while stirring; the mass concentration of trisodium citrate is 1 percent; the time for continuing heating is 1 h; cooling to 20-25 ℃;
the volume ratio of the nano silver particle solution, the DTNB solution and the ultrapure water in the step (2) is 10 mL: 20 μ L of: 2 mL; the concentration of the DTNB solution is 0.01 mol/L; the temperature of the stirring reaction is 130 ℃, and the time is 2 h; centrifuging at 12000r/min for 10 min;
the volume ratio of the AgNPs-DTNB solution to the tungsten atom solution in the step (3) is 2 mL: 2 mL; the reaction conditions are as follows: oil-bath pan at 50 deg.C for 1 hr; centrifuging: 8000r/min for 10 min; the using amount of the ultrapure water is 2 mL; ultrasonic dispersion: 500 w;
and (4) the volume-to-mass ratio of the Wu-DTNB @ Ag solution to the potassium carbonate solution to the new crown N protein antibody 1 is 1 mL: 6 mu L of the solution: 10 mu g of the solution; the concentration of the potassium carbonate solution is 0.05 mol/L; the mass concentration of the BSA solution is 10%, and the final concentration is adjusted to 1%; the reactions are as follows: stirring and reacting for 30 min; centrifuging: 4 ℃, 10000r/min and 10 min; preservation solution: 0.02mol/L sodium borate buffer solution, containing 2% BSA by mass, 0.25% Proclin-300 by mass, and pH 7.4.
Preferably: step (5) phosphate buffer: 0.0lM, pH7.4, soaking for 30min, and oven drying at 37 deg.C; the Wu-DTNB @ Ag-Ab1 solution was diluted 20 times, coated on a conjugate pad, and then oven-dried at 37 ℃.
Preferably: step (6) phosphate buffer: 0.0lM, containing BSA with the mass concentration of 1%, 0.5% Tween-20 with the mass concentration of 7.4; soaking for 20min, and oven drying at 37 deg.C.
Preferably: the phosphate buffer solution in the step (7) is: 0.0lM, pH 7.4; diluting the new crown N protein antibody 2 to 0.5 mg/mL; the coating is as follows: using a Biodot membrane instrument; diluting rabbit anti-mouse IgG antibody to 1 mg/mL; and (3) the distance between the detection line and two lines of the quality control line is 5-8 mm, and drying is carried out at 37 ℃.
Preferably, the following components: the step (8) is specifically as follows: and sequentially adhering the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the PVC backing, cutting into small strips with the width of 3mm, carrying out vacuum packaging, and storing at 4-8 ℃.
According to the technical scheme, compared with the prior art, the invention discloses and provides the novel coronavirus detection kit and the preparation method thereof, and the technical effects are that the kit has the advantages of strong specificity, high sensitivity, short detection time, field operation, low detection cost, simplicity and convenience in operation and the like, wherein the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC backing lining, and the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially and continuously adhered to the PVC backing lining. The bonding pad is in lap joint with the nitrocellulose membrane, the nitrocellulose membrane is in lap joint with the water absorption pad, the bonding pad is coated with a tungsten silver core-shell compound-new crown N protein antibody 1 and a Raman signal molecule DTNB (Wu-DTNB @ Ag-Ab1) marker, and the nitrocellulose membrane is respectively coated with a detection line T formed by the new crown N protein antibody 2 and a quality control line C formed by rabbit anti-mouse IgG.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic side view of the structure provided by the present invention, wherein the structure comprises a 1-PVC backing, a 2-sample pad, a 3-conjugate pad, a 4-nitrocellulose membrane, a 5-absorbent pad, a T-detection line, and a C-quality control line.
Fig. 2 is a schematic top view of the structure provided by the present invention.
Figure 3 the accompanying drawing is a standard graph provided by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a novel coronavirus detection kit and a preparation method thereof.
Raw materials and reagents which are not mentioned in the examples are obtained from conventional commercial sources, and the required equipment is conventional test equipment, for example, the new crown N protein antibody 1 and the new crown N protein antibody 2 are purchased from Fipeng biological corporation; rabbit anti-mouse IgG was purchased from the Loyang Bai Outon center for experimental materials and is not described in detail herein.
Example 1
Novel detection kit for coronavirus
The immunochromatography surface-enhanced Raman spectroscopy test strip for detecting the new coronavirus is structurally shown in fig. 1 and 2 and comprises a sample pad 2, a combination pad 3, a nitrocellulose membrane 4, a water absorption pad 5 and a PVC backing 1.
A sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are continuously adhered to the PVC backing in sequence, a lap joint is arranged between the combination pad and the nitrocellulose membrane, and a lap joint is arranged between the nitrocellulose membrane and the water absorption pad; the binding pad is coated with a Wu-DTNB @ Ag-Ab1 marker, and the nitrocellulose membrane is respectively coated with a detection line T formed by a new crown N protein antibody 2 and a quality control line C formed by rabbit anti-mouse IgG.
The coating concentration of the new crown N protein antibody 2 is 0.5 mg/mL; the coating concentration of the rabbit anti-mouse IgG is 1mg/mL
When in use, a sample to be detected is treated by a sample extracting solution (0.01M phosphate buffer solution containing 1% Twen 20, 1% EDTA, pH7.4), and then the sample extracting solution is dripped on a sample pad. Special sample adding holes can be made on the sample pad.
The novel coronavirus detection principle is that a double-antibody sandwich method is adopted to detect whether a sample to be detected contains the novel coronavirus. When a positive sample is detected, the novel coronavirus in the sample is combined with a novel coronavirus antibody 1 connected to a Raman immune probe (Wu-DTNB @ Ag-Ab1), the compound moves forwards in the nitrocellulose membrane through chromatography, and is combined with a coated novel coronavirus antibody 2 when passing through a detection area to form a Raman immune probe-novel coronavirus antibody 1-novel coronavirus antibody 2 compound, so that a T line is banded, and the result is positive; when a negative sample is detected, the sample does not contain the neocorona antigen, and the immune complex cannot be formed, so that a T line does not have a strip, and the result is negative. And measuring a probe signal on the T line by using a Raman spectrometer, and measuring the content of the new coronavirus.
Example 2
Preparation method of novel coronavirus detection kit
(1) Preparing a nano silver particle solution: weighing 0.018g of silver nitrate, dissolving the silver nitrate in 100mL of ultrapure water, placing the prepared silver nitrate solution in an oil bath pot (130 ℃) and heating while stirring, adding 4mL of 1% trisodium citrate solution when the silver nitrate solution is boiled, continuing heating for 1 hour, and cooling to 20-25 ℃ to obtain a nano silver particle solution (AgNPs);
(2) surface modification of the nano silver particles with DTNB: taking 10mL of the nano silver particle solution prepared in the step (1), adding 20 mu L of 0.01mol/L DTNB solution, stirring and reacting for 2h at room temperature, 12000 r/min, centrifuging for 10min, removing supernatant, and suspending precipitate with 2mL of ultrapure water to obtain AgNPs-DTNB solution;
(3) preparing a tungsten-coated AgNPs-DTNB core-shell structure: taking 2mL of AgNPs-DTNB solution prepared in the step (2) and a tungsten atom solution, mixing the AgNPs-DTNB solution and the tungsten atom solution in equal volume, carrying out oil bath reaction at 50 ℃ for 1h at 8000r/min, centrifuging for 10min, suspending the precipitate with 2mL of ultrapure water, and carrying out ultrasonic dispersion at 500w to obtain a tungsten-coated AgNPs-DTNB core-shell compound (Wu-DTNB @ Ag);
(4) preparing a Raman immune probe: and (3) taking 1mL of the Wu-DTNB @ Ag solution prepared in the step (3), adding 6 mu L of 0.05mol/L potassium carbonate solution, adding 10 mu g of new crown N protein antibody 1 into the solution, stirring for reaction for 30min, adding 10% BSA solution until the final concentration is 1%, and stirring for reaction for 30 min. Centrifuging at 4 ℃ at 10000r/min for 10min, suspending the precipitate with a sodium borate buffer solution (containing 2% BSA, 0.25% Proclin-300 and pH7.4) of 0.02mol/L of a preservation solution to obtain a tungsten-silver core-shell complex labeled new crown N protein antibody 1 and a Raman signal molecule DTNB solution (Wu-DTNB @ Ag-Ab 1);
(5) Coating of the bonding pad: soaking the bonding pad in 0.0l M phosphate buffer solution with pH of 7.4 for 30min, and oven drying at 37 deg.C; diluting the Wu-DTNB @ Ag-Ab1 marker prepared in the step (4) by 20 times, uniformly coating the marker on a bonding pad, drying the marker at 37 ℃, and packaging the marker for later use;
(6) treatment of the sample pad: soaking the sample pad in 0.0l M phosphate buffer (containing 1% BSA, 0.5% Tween-20, pH7.4) for 20min, oven drying at 37 deg.C, and packaging;
(7) coating of nitrocellulose membrane: the new crown N protein antibody 2 is diluted to 0.5mg/mL by 0.0l M phosphate buffer solution (pH7.4), and is coated on a nitrocellulose membrane by a Biodot membrane analyzer to be used as a detection line; diluting the rabbit anti-mouse IgG antibody to 1mg/mL by using 0.0l M phosphate buffer solution (pH7.4), coating the diluted rabbit anti-mouse IgG antibody on a nitrocellulose membrane by using a Biodot membrane analyzer to serve as a quality control line, and keeping the distance between a detection line and two lines of the quality control line to be 5-8 mm. Drying at 37 ℃, and packaging for later use;
(8) assembling the test strip: and sequentially adhering the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the PVC backing, cutting into small strips with the width of 3mm, carrying out vacuum packaging, and storing at 4-8 ℃.
Example 3
Establishment of a Standard Curve
Taking new crown recombinant N protein, diluting with a sample extracting solution to obtain the concentrations of 1pg/mL, 10 pg/mL, 100pg/mL and 500pg/mL, testing with the test strip, detecting the characteristic peak intensity of T-line Raman signal molecule DTNB by a laser confocal Raman spectrometer, and drawing a standard curve, which is shown in figure 3.
Technical effects
Specificity test
The pathogenic bacteria or recombinant proteins for specificity test include SARS-N protein, MERS-N protein, and A-type H1N1 virus isolate.
Diluting the concentration of the substance to be detected to be 100ng/mL, accurately sucking 80 mu L of sample to be detected by a liquid transfer device, vertically and slowly dripping the sample to be detected into a sample adding hole of a detection card, starting timing after sample adding, and reading the result after 10 minutes of sample adding. The test results are shown in table 1:
TABLE 1 test paper strip of the present invention for specificity test results
Figure BDA0003636932200000071
Figure BDA0003636932200000081
The results show that: SARS-N protein, MERS-N protein, A type H1N1 virus isolate and the like have no cross reaction with the kit, and the kit has good specificity.
Example 4
In practice use
1. Sample pretreatment
(1) And if the sample is a nasopharyngeal swab, immediately inserting the swab into a sample collection tube with a sample extracting solution from top to bottom after the sample is collected, rotating the nasopharyngeal swab 5-10 times, covering the nasopharyngeal swab with a cover, uniformly mixing, and waiting for 1-2 minutes for detection.
(2) If the dropper is used for sucking saliva, the dropper can be directly used for sucking the saliva, the saliva pipe is inserted into the sample collecting pipe after the saliva is collected, the sample collecting pipe is uniformly mixed, and the detection is carried out after the sample collecting pipe waits for 1-2 minutes.
2. Detection of
(1) The test card and sample collection tube were returned to room temperature.
(2) And (3) accurately sucking 80 mu L of sample to be detected by a pipettor, vertically and slowly dripping the sample to be detected into the sample adding hole of the detection card, timing after sample adding, and reading the result in 10 minutes.
(3) And substituting the reading result into a standard curve to calculate the content of the novel coronavirus in the sample.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. A novel detection kit for coronavirus is characterized by comprising an immunochromatography surface-enhanced Raman spectroscopy test strip for detecting the novel coronavirus and a sample extracting solution, wherein the test strip comprises a Wu-DTNB @ Ag-Ab1 solution uniformly coated on a binding pad, a novel corona N protein antibody 2 coated on a nitrocellulose membrane to form a detection line, and a rabbit anti-mouse IgG coated on the nitrocellulose membrane to form a quality control line; the preparation method of the Wu-DTNB @ Ag-Ab1 solution comprises the following steps:
(1) reacting trisodium citrate with silver nitrate to prepare a nano silver particle solution;
(2) adding Raman signal molecules DTNB into the nano silver particle solution prepared in the step (1) to obtain AgNPs-DTNB solution;
(3) adding tungsten atoms into the AgNPs-DTNB solution prepared in the step (2) to obtain a Wu-DTNB @ Ag solution;
(4) and (4) adding the new crown N protein antibody 1 into the Wu-DTNB @ Ag solution prepared in the step (3) to obtain the Wu-DTNB @ Ag-Ab1 solution.
2. The detection kit of claim 1, wherein the coating concentration of the neo-coronary N protein antibody 2 is 0.5 mg/mL; the coating concentration of the rabbit anti-mouse IgG is 1 mg/mL.
3. The test kit of claim 2, wherein the test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, a bibulous pad, and a PVC backing; and the PVC back lining is sequentially adhered with a sample pad, a combined pad, a nitrocellulose membrane and a water absorption pad.
4. The method for preparing the detection kit according to any one of claims 1 to 3, characterized by comprising the steps of:
(1) preparing a nano silver particle solution: weighing silver nitrate, dissolving the silver nitrate in ultrapure water to obtain a silver nitrate solution, heating, adding a trisodium citrate solution when the silver nitrate solution is boiled, continuing to heat, and cooling to obtain a nano silver particle solution;
(2) preparing a nano silver particle surface modification DTNB solution: adding the nano silver particle solution prepared in the step (1) into a DTNB solution, stirring for reaction, centrifuging, removing a supernatant, and suspending a precipitate with ultrapure water to obtain an AgNPs-DTNB solution;
(3) preparation of Wu-DTNB @ Ag: mixing the AgNPs-DTNB solution prepared in the step (2) and the tungsten atom solution in equal volume, reacting, centrifuging, suspending the precipitate with ultrapure water, and performing ultrasonic dispersion to obtain a Wu-DTNB @ Ag solution;
(4) preparation of Wu-DTNB @ Ag-Ab1 solution: adding potassium carbonate solution into the Wu-DTNB @ Ag solution prepared in the step (3), adding a new crown N protein antibody 1 into the solution, adding BSA solution after reaction, reacting, centrifuging, and suspending the precipitate with preservation solution to obtain Wu-DTNB @ Ag-Ab1 solution;
(5) coating of the bonding pad: after the bonding pad is soaked in a phosphate buffer, uniformly coating the Wu-DTNB @ Ag-Ab1 solution prepared in the step (4) on the bonding pad, and packaging for later use;
(6) Treatment of the sample pad: soaking the sample pad in phosphate buffer solution, and packaging for later use;
(7) coating of nitrocellulose membrane: diluting the new crown N protein antibody 2 by using a phosphate buffer solution, and coating the diluted new crown N protein antibody on a nitrocellulose membrane to be used as a detection line; diluting rabbit anti-mouse IgG antibody with phosphate buffer solution, and coating on nitrocellulose membrane as quality control line; packaging for later use;
(8) and (6) assembling the test strip.
5. The preparation method of claim 4, wherein the mass-to-volume ratio of the silver nitrate, the ultrapure water and the trisodium citrate solution in the step (1) is 0.018 g: 100mL of: 4 mL; the heating conditions are as follows: heating the mixture in an oil bath kettle at 130 ℃ while stirring; the mass concentration of the trisodium citrate is 1 percent; the time for continuing heating is 1 h; cooling to 20-25 ℃;
the volume ratio of the nano silver particle solution, the DTNB solution and the ultrapure water in the step (2) is 10 mL: 20 μ L of: 2 mL; the concentration of the DTNB solution is 0.01 mol/L; the temperature of the stirring reaction is 130 ℃, and the time is 2 h; the centrifugation is 12000r/min, and the time is 10 min;
the volume ratio of the AgNPs-DTNB solution to the tungsten atom solution in the step (3) is 2 mL: 2 mL; the reaction conditions are as follows: oil-bath pan at 50 deg.C for 1 hr; and (3) centrifuging: 8000r/min for 10 min; the using amount of the ultrapure water is 2 mL; and (3) ultrasonic dispersion: 500 w;
The volume-mass ratio of the Wu-DTNB @ Ag solution, the potassium carbonate solution and the neo-corona-N protein antibody 1 in the step (4) is 1 mL: 6 mu L of the solution: 10 mu g of the solution; the concentration of the potassium carbonate solution is 0.05 mol/L; the mass concentration of the BSA solution is 10%, and the final concentration is adjusted to 1%; the reactions are all as follows: stirring and reacting for 30 min; and (3) centrifuging: 4 ℃, 10000r/min and 10 min; the preservation solution comprises: 0.02mol/L sodium borate buffer solution, containing 2% BSA by mass, 0.25% Proclin-300 by mass, pH 7.4.
6. The method according to claim 5, wherein the phosphate buffer solution of step (5): 0.01M, pH7.4, soaking for 30min, and drying at 37 deg.C; the Wu-DTNB @ Ag-Ab1 solution is diluted by 20 times, coated on a bonding pad and dried at 37 ℃.
7. The method according to claim 6, wherein the phosphate buffer of step (6): 0.0lM, containing BSA with the mass concentration of 1%, 0.5% Tween-20 with the mass concentration of 7.4; the soaking time is 20min, and the soaked product is dried at 37 ℃.
8. The method according to claim 7, wherein the phosphate buffer of step (7) is: 0.01M, pH 7.4; the new crown N protein antibody 2 is diluted to 0.5 mg/mL; the coatings are all as follows: using a Biodot membrane instrument; diluting the rabbit anti-mouse IgG antibody to 1 mg/mL; and the distance between the detection line and two lines of the quality control line is 5-8 mm, and the drying is carried out at 37 ℃.
9. The preparation method according to claim 8, wherein the step (8) is specifically: and sequentially adhering the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on the PVC backing, cutting into small strips with the width of 3mm, carrying out vacuum packaging, and storing at 4-8 ℃.
CN202210532626.1A 2022-05-10 2022-05-10 Novel detection kit for coronavirus and preparation method thereof Pending CN114764100A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210532626.1A CN114764100A (en) 2022-05-10 2022-05-10 Novel detection kit for coronavirus and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210532626.1A CN114764100A (en) 2022-05-10 2022-05-10 Novel detection kit for coronavirus and preparation method thereof

Publications (1)

Publication Number Publication Date
CN114764100A true CN114764100A (en) 2022-07-19

Family

ID=82364875

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210532626.1A Pending CN114764100A (en) 2022-05-10 2022-05-10 Novel detection kit for coronavirus and preparation method thereof

Country Status (1)

Country Link
CN (1) CN114764100A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111474351A (en) * 2020-04-28 2020-07-31 上海泰辉生物科技有限公司 Immunochromatographic test strip and kit for detecting coronavirus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111474351A (en) * 2020-04-28 2020-07-31 上海泰辉生物科技有限公司 Immunochromatographic test strip and kit for detecting coronavirus
CN111474351B (en) * 2020-04-28 2024-01-16 上海泰辉生物科技有限公司 Immunochromatography test strip and kit for detecting coronaviruses

Similar Documents

Publication Publication Date Title
Wang et al. Novel fabrication of immunochromatographic assay based on up conversion phosphors for sensitive detection of clenbuterol
CN108254563B (en) Detect time-resolved fluoroimmunoassay chromatograph test strip, the kit and preparation method thereof of cTnI
Fang et al. Barcode lateral flow immunochromatographic strip for prostate acid phosphatase determination
WO2018120854A1 (en) Time-resolved fluorescent immunochromatographic test strip and kit for detecting ck-mb, and preparation method therefor
CN108535485A (en) A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method quantitatively detecting CA153 in blood
CN107121548A (en) Quantitatively detect test paper, preparation method and the detection method of tumor markers
CN111781351A (en) Electrochemical immunochromatographic test strip for quantitative detection of novel coronavirus antigen and preparation method thereof
CN110470832A (en) The hollow bimetallic test strips and preparation method thereof of IL-6, IL-4 and TNF-α are detected simultaneously
CN107328942A (en) A kind of fluorogenic quantitative detection PAPP A immunochromatography reagent bar and preparation method thereof
CN114764100A (en) Novel detection kit for coronavirus and preparation method thereof
JP4223163B2 (en) Immunochromatographic test strip and chromatographic analysis method
CN110988365A (en) Quantitative immune colloidal gold detection card and kit for cystatin C, microalbuminuria and urinary creatinine
CN106596968A (en) Dot gold infiltration kit for detecting urine microalbumin, and application of dot gold infiltration kit
JP2001033454A (en) Method and apparatus for determining substance to be detected on porous solid-phase ligand measuring test piece
CN108535495A (en) A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method quantitatively detecting CYFRA21-1 in blood
CN106645043A (en) Kit and method for fast quantitatively detecting small molecule compound
JP6998626B2 (en) An immunological composition for diagnosing lung cancer using an autoantibody-antigen conjugate, a method for diagnosing lung cancer using the same, and a kit for diagnosing lung cancer containing the same.
WO2020094103A1 (en) Test strip for quantificationally detecting marker cyfra21-1 and preparation method therefor and detection method thereof
JPH0646893A (en) Method and means for measuring analyte
CN116773807A (en) Method for detecting African swine fever virus by using time-resolved fluorescence immunoassay probe
WO2004081568A1 (en) Method of examining specimen and specimen container to be used in the examinaton method
JPH03272466A (en) Multiple immunologocal evaluating analyzing system
CN113504377A (en) Test strip for bimodal detection of CRP (C-reactive protein), preparation method and detection method
CN112505319A (en) Immune quantitative detection device, detection method and application of to-be-detected marker
CN108535484A (en) A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method quantitatively detecting fPSA in blood

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination