WO2021218250A1 - 同时检测pedv和tgev的速测卡及其制备、使用方法 - Google Patents

同时检测pedv和tgev的速测卡及其制备、使用方法 Download PDF

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WO2021218250A1
WO2021218250A1 PCT/CN2021/073807 CN2021073807W WO2021218250A1 WO 2021218250 A1 WO2021218250 A1 WO 2021218250A1 CN 2021073807 W CN2021073807 W CN 2021073807W WO 2021218250 A1 WO2021218250 A1 WO 2021218250A1
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tgev
pedv
specific monoclonal
monoclonal antibody
line
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PCT/CN2021/073807
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English (en)
French (fr)
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李彬
范宝超
郭荣利
周金柱
赵永祥
何孔旺
倪艳秀
常新见
时丹怡
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江苏省农业科学院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—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
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of animal pathogen detection, in particular to an immune colloidal gold test card for simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine gastroenteritis virus (TGEV), and preparation and use methods thereof.
  • PEDV porcine epidemic diarrhea virus
  • TGEV porcine gastroenteritis virus
  • Porcine epidemic diarrhea virus can cause diarrhea, vomiting, dehydration, and eventually death in infected pigs. Pigs of all ages can be infected, but suckling piglets are most susceptible. Since the outbreak of the new porcine viral diarrhea (PED) in 2010, it has caused serious economic losses to the global pig industry. Studies have confirmed that the PEDV variant is the culprit of this epidemic, and its pathogenicity is stronger, which can cause 100% death of newborn piglets. Based on the virus's characteristics of easy mutation and recombination, infections of different genotype strains are currently occurring in many countries in Asia, America and Europe. This also makes the use of vaccines unsatisfactory and cannot be completely controlled so far.
  • PED porcine viral diarrhea
  • TGEV Porcine transmissible gastroenteritis virus
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a rapid test card for simultaneous detection of PEDV and TGEV, which is used for rapid, on-site and sensitive detection of PEDV and TGEV in pig feces samples.
  • the rapid test card for detecting PEDV and TGEV at the same time includes a card housing and a test strip set in the card housing.
  • a pad, a nitrocellulose membrane and an absorbent pad, wherein a sample hole and a result observation window are arranged on the card shell.
  • An anti-PEDV specific monoclonal antibody and an anti-TGEV specific monoclonal antibody are immobilized on the nitrocellulose membrane to form a first detection line and a second detection line, respectively, and a colloidal gold-labeled anti-PEDV is fixed on the binding pad
  • Specific monoclonal antibodies and colloidal gold labeled anti-TGEV specific monoclonal antibodies are immobilized on the nitrocellulose membrane to form a first detection line and a second detection line, respectively.
  • the anti-PEDV-specific monoclonal antibody is a hybridoma cell line FL011-26 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019, with a deposit number of CCTCC NO: C2019170 And/or prepared by the hybridoma cell line FL011-33 with the deposit number CCTCC NO: C2019194 deposited in the Chinese Type Culture Collection at Wuhan University, Wuhan, China.
  • the anti-TGEV-specific monoclonal antibody is a hybridoma cell line FL111-01 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019, with a deposit number of CCTCC NO: C2019195 And/or prepared by the hybridoma cell line FL011-07 with the deposit number CCTCC NO: C2019196 deposited in the Chinese Type Culture Collection at Wuhan University, Wuhan, China.
  • the nitrocellulose membrane is immobilized with the hybridoma cell line FL011-26 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019, with a deposit number of CCTCC NO: C2019170.
  • the colloidal gold label immobilized on the binding pad is produced by the hybridoma cell line FL011-33 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019, with the deposit number of CCTCC NO: C2019194
  • the use of anti-PEDV-specific monoclonal antibodies and colloidal gold markers were deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019, with the deposit number of CCTCC NO: C2019196 hybridoma cell line FL011-07 Prepared anti-TGEV specific monoclonal antibody.
  • the amount of the anti-PEDV specific monoclonal antibody and the anti-TGEV specific monoclonal antibody immobilized on the nitrocellulose membrane is 0.4 ⁇ g-1.2 ⁇ g, respectively, and the colloid fixed on the binding pad
  • the gold-labeled anti-PEDV-specific monoclonal antibody and the colloidal gold-labeled anti-TGEV-specific monoclonal antibody are 5ng-50ng, respectively.
  • Another aspect of the present invention also provides a method for preparing a rapid test card for simultaneous detection of PEDV and TGEV.
  • One of the detection line and the second detection line is coated with an anti-TGEV specific monoclonal antibody on a nitrocellulose membrane to form the other of the first detection line and the second detection line, and the goat anti- The mouse IgG secondary antibody is coated on the nitrocellulose membrane to form a quality control line to obtain a nitrocellulose membrane containing the first detection line, the second detection line and the quality control line; (2) preparing a fixed Colloidal gold-labeled anti-PEDV-specific monoclonal antibody and colloidal gold-labeled anti-TGEV-specific monoclonal antibody binding pad; (3) Assemble the test strip: paste the sample pad, the binding pad, The nitrocellulose membrane and the absorbent pad are used to obtain the test strip, wherein the sample pad is located above the binding pad and the binding pad and the sample pad have an overlapping area of 1mm-1.5mm, and the nitric acid The cellulose membrane
  • the anti-PEDV monoclonal antibody is a hybridoma cell line FL011-26 and/ Or it was prepared from the hybridoma cell line FL011-33 deposited at the China Type Culture Collection on September 11, 2019 with the deposit number CCTCC NO: C2019194.
  • the anti-TGEV monoclonal antibody is the hybridoma cell line FL111-01 deposited on September 11, 2019 at the Chinese Type Culture Collection at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2019195 or September 2019
  • the hybridoma cell line FL011-07 was deposited at the China Type Culture Collection at Wuhan University, Wuhan, China on the 11th, with a deposit number of CCTCC NO: C2019196.
  • the nitrocellulose membrane is immobilized with the hybridoma cell line FL011-26 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019, with a deposit number of CCTCC NO: C2019170.
  • the colloidal gold label immobilized on the binding pad utilizes the anti-PEDV specific monoclonal produced by the hybridoma cell line FL011-33 with the deposit number CCTCC NO: C2019194 deposited in the Chinese Type Culture Collection on September 11, 2019 Anti-TGEV specificity produced by the hybridoma cell line FL011-07 with the deposit number CCTCC NO: C2019196 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11, 2019 Monoclonal antibodies.
  • the amount of the anti-PEDV specific monoclonal antibody and the anti-TGEV specific monoclonal antibody immobilized on the nitrocellulose membrane is 0.4 ⁇ g-1.2 ⁇ g, respectively, and the colloid fixed on the binding pad
  • the gold-labeled anti-PEDV-specific monoclonal antibody and the colloidal gold-labeled anti-TGEV-specific monoclonal antibody are 5ng-50ng, respectively.
  • the present invention also provides a method for simultaneously detecting PEDV and TGEV by using the above-mentioned rapid test card, which includes the following steps: (1) Sample processing: adding pH 7.4, 0.2mol/L PBS buffer to fresh pig feces samples as Extract the buffer solution, stand or centrifuge, and take the supernatant; (2) Add sample: Take 100 ⁇ L of the supernatant in step (1) and add it to the sample well of the quick test card, and incubate for 8-10 min; (3) ) Judgment of result: Observe the color change of the first test line, second test line and quality control line in the quick test card from the result observation window.
  • the first test line and the second test line When only the quality control line is colored, the first test line and the second test line When there is no color development, it is determined that PEDV and TGEV in the sample to be tested are all negative. When the first detection line, the second detection line and the quality control line are all colored, it is determined that both PEDV and TGEV in the sample to be tested are positive. When the quality control line and one of the first test line or the second test line develop color at the same time, it is determined that the virus corresponding to the test line in the first test line or the second test line in the sample to be tested is positive .
  • the rapid test card provided by the present invention has a good anti-interference effect on stool samples, and can be widely used for rapid detection of swine diarrhea diseases.
  • the rapid test card of the present invention has the following advantages in particular: (1)
  • the rapid test card of the present invention does not simply physically glue the test cards for two pathogens, but The anti-PEDV-specific monoclonal antibody and the anti-TGEV-specific monoclonal antibody are fixed and coated on the same nitrocellulose membrane at the same time. Only one sample is required. The detection reaction is performed under the same conditions to achieve simultaneous detection of PEDV and TGEV.
  • the sample pretreatment is completely unified, and the operation is simple, and the test can be completed with only 100 ⁇ L of sample loading; (3)
  • the test time is short (5-8min), and the result judgment standard is unified.
  • the immune colloidal gold rapid test card for simultaneous detection of PEDV and TGEV provided by the present invention can provide rapid, on-site and sensitive detection means for actual detection departments, and is especially suitable for detection of pig tissue stool samples.
  • Fig. 1 is a diagram schematically showing the structure of a rapid test card of the present invention
  • Fig. 2 is a schematic diagram of an exploded structure composed of test strips in the rapid test card.
  • Fig. 3 is a black and white image showing the result of detecting a blank sample PBS with a quick test card of an embodiment of the present invention.
  • FIG. 4 is a black and white image showing the result of detecting a solution sample containing PEDV virus particles by using the rapid test card of the embodiment of the present invention.
  • FIG. 5 is a black and white image showing the result of detecting a solution sample containing TGEV virus particles by using a rapid test card of an embodiment of the present invention.
  • Fig. 6 is a black and white image showing the result of detecting a solution containing both PEDV and TGEV virus particles by using the rapid test card of the embodiment of the present invention.
  • FIG. 7 is a black and white image showing the result of detecting PEDV and TGEV double-negative stool sample extracts with the quick test card of the embodiment of the present invention.
  • Fig. 8 is a black and white image showing the result of detecting the PEDV single-positive stool sample extract with the quick test card of the embodiment of the present invention.
  • Fig. 9 is a black and white image showing the result of detecting the TGEV single-positive stool sample extract with the quick test card of the embodiment of the present invention.
  • FIG. 10 is a black and white image showing the result of detecting PEDV and TGEV double-positive stool sample extracts using the quick test card of the embodiment of the present invention.
  • the present invention provides an immune colloidal gold rapid test card capable of simultaneously detecting PEDV and TGEV.
  • Figure 1 schematically shows the general structure of the rapid test card.
  • the rapid test card mainly includes a card housing 1 and a test card arranged in the card housing 1.
  • the cartridge 1 is provided with a sample loading hole 3 and a result observation window 4, the size of the sample loading hole 3 is preferably 3 mm ⁇ 7 mm, and the size of the result observation window 4 is preferably 4 mm ⁇ 18 mm.
  • the test strip 2 is composed of a PVC bottom plate, a sample pad, a bonding pad, a nitrocellulose membrane (hereinafter referred to as NC membrane) and an absorbent pad which are successively pasted on the PVC bottom plate.
  • the sample pad is located above the bonding pad and the bonding pad and the sample pad have an overlapping area of 1mm-1.5mm
  • the NC film is located below the bonding pad and the NC film and the bonding pad have an overlapping area of 1mm-2mm
  • the absorbent pad is located above the NC film and The absorbent pad and the NC film have an overlapping area of 1mm-2mm.
  • the sample pad is preferably a glass fiber membrane with a size of 4 mm ⁇ 15 mm.
  • the bonding pad may be made of any suitable material known to those skilled in the art, and the size is preferably 3 mm ⁇ 4 mm.
  • the size of the NC film is preferably 4 mm ⁇ 28 mm.
  • the absorbent pad may be made of any suitable material known to those skilled in the art, and the size is preferably 4 mm ⁇ 19 mm.
  • Anti-PEDV specific monoclonal antibody, anti-TGEV specific monoclonal antibody and goat anti-mouse IgG secondary antibody are immobilized on the NC membrane, which are used to form two detection lines and quality control line C for the two viruses, respectively.
  • the amount of each monoclonal antibody on the NC membrane is preferably 0.4 ⁇ g-1.2 ⁇ g.
  • the detection line formed by the anti-PEDV-specific monoclonal antibody is referred to as the first detection line T1
  • the detection line formed by the anti-TGEV-specific monoclonal antibody is referred to as the second detection line T2.
  • first detection line T1 and the second detection line T2 are not limited to this, and the detection line formed by the anti-TGEV specific monoclonal antibody can also be referred to as the first detection line.
  • the detection line formed by the anti-PEDV-specific monoclonal antibody is called the second detection line.
  • a colloidal gold-labeled anti-PEDV-specific monoclonal antibody and a colloidal gold-labeled anti-TGEV-specific monoclonal antibody are immobilized on the binding pad, and the amount of each monoclonal antibody is preferably 5ng-50ng.
  • the anti-PEDV-specific monoclonal antibody is made using the hybridoma cell line FL011-26 and/or 2019 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China, with the deposit number of CCTCC NO: C2019170 It was prepared by the hybridoma cell line FL011-33 with the deposit number CCTCC NO: C2019194 deposited in the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11th.
  • the anti-TGEV-specific monoclonal antibody is made using the hybridoma cell line FL111-01 and/or 2019 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China, with the deposit number of CCTCC NO: C2019195 It was prepared by the hybridoma cell line FL011-07 with the deposit number CCTCC NO: C2019196 deposited at the Chinese Type Culture Collection at Wuhan University, Wuhan, China on September 11th.
  • the quality control line C develops color
  • the first detection line T1 develops color
  • the second detection line T2 does not develop color
  • the quality control line C develops color
  • the second detection line T2 develops color
  • the first detection line T1 does not develop color
  • it is determined that the PEDV in the tested sample is negative and the TGEV is positive.
  • the quality control line C develops color, but the first detection line T1 and the second detection line T2 are not colored, it is determined that both PEDV and TGEV in the tested sample are negative.
  • Example 1 Preparation of anti-PEDV-specific monoclonal antibodies and anti-TGEV-specific monoclonal antibodies
  • the hybridoma cell line FL011-26 with the deposit number of CCTCC NO: C2019170 secreting anti-PEDV specific monoclonal antibody for ease of description, the corresponding antibody obtained is referred to as PEDV antibody 1 below
  • CCTCC NO: C2019194 Tumor cell line FL011-33 for ease of description, the corresponding antibody obtained is referred to as PEDV antibody 2 below
  • the hybridoma cell line FL111-01 for ease of description, the corresponding antibody obtained is referred to as TGEV antibody 1) and CCTCC NO: C2019196 hybridoma cell line FL011-07 (for ease of description, the obtained corresponding antibody is referred to as TGEV antibody 2), respectively Expand the culture in the cell culture flask.
  • mice BALB/c mice aged 6-8 weeks were taken, and each mouse was injected intraperitoneally with 0.2 mL of liquid paraffin. After 10 days, the four hybridoma cells obtained by the above-mentioned culture were respectively inoculated into mice intraperitoneally to prepare corresponding monoclonal antibodies. When the abdomen of the mouse was significantly enlarged, the ascites was collected with an elbow dropper, and the titer of the ascites antibody was determined by indirect ELISA.
  • the protein G cartridge is used to further purify the crude antibody protein obtained above.
  • the new column is first passed through the column with 5ml ultrapure water, and then the purification cartridge is equilibrated with 5mL 0.4M PB buffer (pH 7.0); the antibody is passed through the column, in the process It is required to pass through the column slowly in order to better bind the antibody protein to the binding site; continue to equilibrate the purification cartridge with 10mL 0.4M PB buffer (pH 7.0); use 5mL eluent (0.1M glycine-hydrochloric acid buffer, pH 7.0) The antibody on the binding site is eluted, and 1M Tris-HCl (pH 2.7) is added to neutralize glycine and keep the pH at a neutral condition suitable for antibody storage.
  • the PEDV antibody 1 obtained in Example 1 was diluted to 1.0 mg/mL with a PBS buffer solution with a concentration of 10 mM and a pH of 7.4 to obtain a PEDV antibody 1 solution, which was used as a coating antibody for forming the first detection line T1.
  • the TGEV antibody 1 prepared in Example 1 was diluted to 1.0 mg/mL with a PBS buffer solution with a concentration of 10 mM and a pH of 7.4 to obtain a TGEV antibody 1 solution, which was used as a coating antibody for forming the second detection line T2.
  • the goat anti-mouse IgG secondary antibody (purchased from Hangzhou Longji Biotechnology Co., Ltd.) was diluted to 1 mg/mL with a PBS buffer solution with a concentration of 10 mM and a pH of 7.4, which was used as the coating antibody for quality control line C.
  • an antibody is coated on the NC film to form a first detection line T1, a second detection line T2, and a quality control line C.
  • the concentration of 1.0mg/mL PEDV antibody 1 solution, the concentration of 1.0mg/mL TGEV antibody 1 solution and the concentration of 1mg/mL goat anti-mouse IgG secondary antibody solution were respectively 1.0 ⁇ L/cm Scribe lines on the NC membrane (Pall vivid 170 nitrocellulose membrane) as the first detection line T1, the second detection line T2, and the quality control line C.
  • the NC film after scribing is dried at 37° C. for 24 hours to obtain an NC film containing the first detection line T1, the second detection line T2 and the quality control line C.
  • Antibody labeling Take 1.5 mL of the colloidal gold solution prepared in the above 1), adjust the pH value with 0.1M K 2 CO 3 , add 20ug PEDV antibody 2 and 20ug TGEV antibody 2 respectively and mix well, and react at room temperature for 40 minutes After that, 10% BSA was added to terminate the reaction, and it was allowed to stand for 30 minutes.
  • the 0.4mg/ml colloidal gold-labeled PEDV antibody 2 antibody solution and 0.4mg/ml colloidal gold-labeled TGEV antibody 2 solution prepared above were mixed 1:1 to obtain a mixed solution, and then the mixed solution was added to 2.0 ⁇ L /cm spray on the pretreated binding pad and dry to obtain a binding pad with a specific monoclonal antibody labeled with colloidal gold.
  • the sample pad, the bonding pad, the NC film and the absorbent pad in sequence on the PVC bottom plate to form a test strip.
  • one end of the sample pad is covered on the bonding pad, so that the sample pad and the bonding pad have an overlapping area of 1mm-1.5mm
  • the NC film is located under the bonding pad and the NC film and the bonding pad have an overlapping area of 1mm-2mm
  • the absorbent pad is located in the NC Above the film, the absorbent pad and the NC film have an overlapping area of 1mm-2mm.
  • the card cover and install the card cover on the test strip so that the sample hole is located above the sample pad to expose a part of the sample pad.
  • the observation window is located above the NC film to expose the first test line T1, the second test line T2 and the quality control line C Location. This completes the assembly of the quick test card.
  • Example 3 Use the rapid test card of Example 2 to detect PEDV and TGEV virus particles in the solution
  • the test card prepared according to Example 2 was used to test the following three solutions containing PEDV and/or TGEV virus particles prepared with PBS buffer to evaluate the effect of the test card: solution containing 1 ⁇ g/ml PEDV virus particles , A solution containing 1 ⁇ g/ml TGEV virus particles, and a solution containing both 1 ⁇ g/ml PEDV virus particles and 1 ⁇ g/ml TGEV virus particles.
  • solution containing 1 ⁇ g/ml PEDV virus particles A solution containing 1 ⁇ g/ml TGEV virus particles
  • a solution containing both 1 ⁇ g/ml PEDV virus particles and 1 ⁇ g/ml TGEV virus particles 0.2mol/L PBS buffer (pH 7.4) (specific formula: 8g sodium chloride, 3.35g disodium hydrogen phosphate dodecahydrate, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, dissolved in double distilled water Dilute to 1L) as a blank control.
  • Example 4 Using the rapid test card of Example 2 to detect PEDV and TGEV in stool samples
  • the rapid test card prepared according to Example 2 was used to detect the presence of PEDV and TGEV in the following stool samples: PEDV and TGEV were both negative, PEDV positive, TGEV positive, and both PEDV and TGEV were positive stool.
  • PBS 0.2mol/L
  • the specific preparation method of PBS is 8g sodium chloride, 3.35g disodium hydrogen phosphate dodecahydrate, 0.2g diphosphate Potassium hydride, 0.2g potassium chloride, dissolved in double distilled water to make the volume 1L;
  • the above results show that the rapid test card prepared according to Example 2 can accurately detect and identify PEDV and TGEV in fecal samples.
  • the rapid test card can be used for rapid, on-site and sensitive detection of PEDV and TGEV in pig tissue fecal samples. Detection.

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Abstract

同时检测猪流行性腹泻病毒（PEDV）和猪肠胃炎病毒（TGEV）的速测卡及其制备、使用方法。速测卡包括卡壳（1）和其内的试纸条（2），试纸条（2）包括PVC底板、依次相连地粘贴在PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸收垫，卡壳（1）上设置有加样孔（3）和结果观察窗（4），硝酸纤维素膜上固定有抗PEDV特异性单克隆抗体和抗TGEV特异性单克隆抗体，分别形成第一检测线（T1）和第二检测线（T2），结合垫上固定有胶体金标记的抗PEDV特异性单克隆抗体和胶体金标记的抗TGEV特异性单克隆抗体。该速测卡能够同时检测猪粪便样本中的PEDV、TGEV，加样只需1次，反应均在统一条件下进行，具有快速、准确性好、灵敏度高、操作简便等优点。

Description

同时检测PEDV和TGEV的速测卡及其制备、使用方法 技术领域
本发明涉及动物病原检测的技术领域，具体涉及一种同时检测猪流行性腹泻病毒(PEDV)和猪肠胃炎病毒(TGEV)的免疫胶体金速测卡及其制备、使用方法。
背景技术
猪流行性腹泻病毒(porcine epidemic diarrhea virus，以下简称PEDV)可造成感染猪体腹泻、呕吐、脱水，并最终导致死亡，各年龄段猪均可感染，但哺乳仔猪最为易感。自2010年新的猪病毒性腹泻(PED)疫情爆发以来，给全球养猪业造成了严重的经济损失。研究已证实PEDV变异株为此次疫情的元凶，且其致病力更强，可造成新生仔猪100％死亡。基于该病毒易突变和重组的特性，目前位于亚洲、美洲和欧洲的多个国家均发生不同基因型毒株的感染疫情，这也使得疫苗的使用效果并不理想，到目前为止无法彻底控制。
猪肠胃炎病毒(porcine transmissible gastroenteritis virus，以下简称TGEV)与PEDV相同，同属于α-冠状病毒属，也同样造成仔猪以腹泻、脱水，甚至严重至死亡为特征的疾病。Doyle和Hutchings首次报道了美国由TGEV引起的疾病，随后传播至各个大陆：北美(1989年的加拿大)、欧洲(1957年的英国)和亚洲(1956年的日本、2000年的韩国和2000年的泰国)。在中国，20世纪60年代报道了该病，自2010年以来发生了广泛的疫情。
作为猪病毒性腹泻疾病最为重要的两种病原体，两者之间也常常存在混合感染的情况。对于发生腹泻疫情的企业及养殖用户而言，能够及时有效地明确感染病原微生物，对预防和控制疫情的发展起到极其重要的作用。目前常规检测PEDV和TGEV的方法为PCR，但该方法需要专门的实验室场地，需要借助专门的仪器设备，并且对操作人员要求较高，需要有相当的专业知识、技能和操作经验，检测过程前处理复杂，所需时间很长。
因此，开发一种能够同时快速检测并鉴别PEDV和TGEV的技术手段对猪 流行性腹泻疾病的防控具有重要的意义。
发明内容
本发明的目的在于克服现有技术的不足，提供一种同时检测PEDV和TGEV的速测卡，用于对猪的粪便样本中的PEDV和TGEV进行快速、现场和灵敏的检测。
本发明所提供的同时检测PEDV和TGEV的速测卡包括卡壳和设置在卡壳内的试纸条，所述试纸条包括PVC底板、依次相连地粘贴在所述PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸收垫，所述卡壳上设置有加样孔和结果观察窗。所述硝酸纤维素膜上固定有抗PEDV特异性单克隆抗体和抗TGEV特异性单克隆抗体用于分别形成第一检测线和第二检测线，所述结合垫上固定有胶体金标记的抗PEDV特异性单克隆抗体和胶体金标记的抗TGEV特异性单克隆抗体。
优选地，所述抗PEDV特异性单克隆抗体是利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26和/或保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33制备得到的。
优选地，所述抗TGEV特异性单克隆抗体是利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01和/或保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07制备得到的。
优选地，所述硝酸纤维素膜上固定有利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26所产生的抗PEDV特异性单克隆抗体和利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01所制备得到的抗TGEV特异性单克隆抗体。所述结合垫上固定有胶体金标记的利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO： C2019194的杂交瘤细胞株FL011-33所产生的抗PEDV特异性单克隆抗体和胶体金标记的利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07所制备得到的抗TGEV特异性单克隆抗体。
优选地，所述硝酸纤维素膜上固定的所述抗PEDV特异性单克隆抗体和所述抗TGEV特异性单克隆抗体的量分别为0.4μg-1.2μg，所述结合垫上固定的所述胶体金标记的抗PEDV特异性单克隆抗体和所述胶体金标记的抗TGEV特异性单克隆抗体分别为5ng-50ng。
本发明另一方面还提供一种制备同时检测PEDV和TGEV的速测卡的方法，包括以下步骤：(1)用抗PEDV特异性单克隆抗体在硝酸纤维素膜上进行包被固定形成第一检测线和第二检测线中的一个，用抗TGEV特异性单克隆抗体在硝酸纤维素膜上进行包被形成所述第一检测线和所述第二检测线中的另一个，用羊抗鼠IgG二抗在硝酸纤维素膜进行包被形成质控线，得到含有所述第一检测线、所述第二检测线和所述质控线的硝酸纤维素膜；(2)制备固定有胶体金标记的抗PEDV特异性单克隆抗体和胶体金标记的抗TGEV特异性单克隆抗体的结合垫；(3)组装试纸条：在PVC底板上依次粘贴设置样品垫、所述结合垫、所述硝酸纤维素膜和吸收垫来得到所述试纸条，其中所述样品垫位于所述结合垫上方且所述结合垫与所述样品垫具有1mm-1.5mm的重叠区，所述硝酸纤维素膜位于所述结合垫下方且所述硝酸纤维素膜与所述结合垫具有1mm-2mm的重叠区，所述吸收垫位于所述硝酸纤维素膜的上方且所述吸收垫与所述硝酸纤维素膜具有1mm-2mm的重叠区，所述样品垫是大小为4mm×15mm的玻璃纤维膜；(4)安装卡壳使得其覆盖住所述试纸条，其中所述卡壳上的加样孔位于所述样品垫上方，所述结果观察窗位于所述硝酸纤维素膜上方使得能露出所述第一检测线、所述第二检测线和所述质控线所在的位置。
优选地，所述抗PEDV单克隆抗体是利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26和/或2019年09月11日保藏在中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33制备得到的。 所述抗TGEV单克隆抗体是利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01或2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07制备得到的。
优选地，所述硝酸纤维素膜上固定有利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26所产生的抗PEDV特异性单克隆抗体和利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01所产生的抗TGEV特异性单克隆抗体。所述结合垫上固定有胶体金标记的利用2019年09月11日保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33所产生的抗PEDV特异性单克隆抗体和胶体金标记的利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07所产生的抗TGEV特异性单克隆抗体。
优选地，所述硝酸纤维素膜上固定的所述抗PEDV特异性单克隆抗体和所述抗TGEV特异性单克隆抗体的量分别为0.4μg-1.2μg，所述结合垫上固定的所述胶体金标记的抗PEDV特异性单克隆抗体和所述胶体金标记的抗TGEV特异性单克隆抗体分别为5ng-50ng。
本发明还提供一种应用上述速测卡同时检测PEDV和TGEV的方法，其包括以下步骤：(1)样本处理：向猪的新鲜粪便样品中加入pH 7.4、0.2mol/L的PBS缓冲液作为提取缓冲液，静置或离心，取上清；(2)加样：取100μL步骤(1)中的上清液加入到所述速测卡的加样孔中，孵育8-10min；(3)结果判定：从所述结果观察窗观察速测卡中第一检测线、第二检测线和质控线的颜色变化，当仅有质控线显色而第一检测线和第二检测线均不显色时，判定待检样品中PEDV和TGEV均是阴性，当第一检测线、第二检测线和质控线均显色时，判定待检样品中PEDV和TGEV均是阳性，当质控线以及第一检测线或第二检测线中的一个同时显色时，判定待检样品中与第一检测线或第二检测线中显色的那道检测线所对应的病毒为阳性。
本发明所提供的速测卡对粪便样本具有较好的抗干扰效果，可广泛应用于猪腹泻疾病的快速检测。与现有的速测卡操作相比较，本发明的速测卡尤其具有以下优势：(1)本发明的速测卡不是将分别针对两种病原的检测卡进行简单的物理性胶连，而是在同一张硝酸纤维素膜上同时固定包被抗PEDV特异性单克隆抗体和抗TGEV特异性单克隆抗体，仅需1次加样，在同一条件下进行检测反应，实现同时检测PEDV和TGEV；(2)样品前处理完全统一，操作简单，仅需100μL的样品加样量即可完成检测；(3)检测时间短(5-8min)，结果判定标准统一。
本发明提供的同时检测PEDV和TGEV的免疫胶体金速测卡可为实际检测部门提供快速、现场和灵敏的检测手段，尤其适用于猪组织粪便样本的检测。
附图说明
图1是示意性示出本发明的速测卡结构的图，图2是速测卡中试纸条组成的分解结构示意图。
图3是示出用本发明实施例的速测卡对空白样品PBS检测时结果的黑白图像。
图4是示出用本发明实施例的速测卡对含PEDV病毒粒子的溶液样本检测时结果的黑白图像。
图5是示出用本发明实施例的速测卡对含TGEV病毒粒子的溶液样本检测时结果的黑白图像。
图6是示出用本发明实施例的速测卡对同时含PEDV和TGEV病毒粒子的溶液检测时结果的黑白图像。
图7是示出用本发明实施例的速测卡对PEDV和TGEV双阴性粪便样本提取液检测时结果的黑白图像。
图8是示出用本发明实施例的速测卡对PEDV单阳性粪便样本提取液检测时结果的黑白图像。
图9是示出用本发明实施例的速测卡对TGEV单阳性粪便样本提取液检测时结果的黑白图像。
图10是示出用本发明实施例的速测卡对PEDV和TGEV双阳性粪便样本提取液检测时结果的黑白图像。
具体实施方式
为了帮助本领域技术人员进一步理解本申请的技术方案，将结合附图对本申请的具体实施方式进行详细的描述。
本发明提供一种能同时检测PEDV和TGEV的免疫胶体金速测卡，图1示意性地示出了速测卡的大体结构，该速测卡主要包括卡壳1和设置在卡壳1内的试纸条2。卡壳1上设置有加样孔3和结果观察窗4，加样孔3的尺寸优选为3mm×7mm，结果观察窗4的尺寸优选为4mm×18mm。
如图2所示，试纸条2由PVC底板以及依次相连地粘贴在PVC底板上的样品垫、结合垫、硝酸纤维素膜(下文简称为NC膜)和吸收垫组成。其中样品垫位于结合垫上方且结合垫与样品垫具有1mm-1.5mm的重叠区，NC膜位于结合垫下方且NC膜与结合垫具有1mm-2mm的重叠区，吸收垫位于NC膜的上方且吸收垫与NC膜具有1mm-2mm的重叠区。样品垫优选是尺寸为4mm×15mm的玻璃纤维膜。结合垫可以是由本领域技术人员已知的任何合适的材料制成的，尺寸优选为3mm×4mm。NC膜的尺寸优选为4mm×28mm。吸收垫可以是由本领域技术人员已知的任何合适的材料制成的，尺寸优选为4mm×19mm。
NC膜上固定有抗PEDV特异性单克隆抗体、抗TGEV特异性单克隆抗体和羊抗鼠IgG二抗，它们分别用于形成针对两种病毒的两道检测线和质控线C。NC膜上每种单克隆抗体的量优选为0.4μg-1.2μg。为了方便描述，下文将利用抗PEDV特异性单克隆抗体所形成的检测线称为第一检测线T1，利用抗TGEV特异性单克隆抗体而形成的检测线称为第二检测线T2。然而，本领域技术人员将会理解，第一检测线T1和第二检测线T2的限定方式并不仅限于此，也可以将利用抗TGEV特异性单克隆抗体而形成的检测线称为第一检测线，将利用抗PEDV特异性单克隆抗体所形成的检测线称为第二检测线。
结合垫上固定有胶体金标记的抗PEDV特异性单克隆抗体和胶体金标记的抗TGEV特异性单克隆抗体，每种单克隆抗体的量优选为5ng-50ng。
抗PEDV特异性单克隆抗体是利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26和/或2019年09月11日保藏在位于中国武汉武汉大学的中 国典型培养物保藏中心、保藏号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33制备得到的。抗TGEV特异性单克隆抗体是利用2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01和/或2019年09月11日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07制备得到的。
用上述速测卡检测猪组织粪便样品中的PEDV和/或TGEV时，首先向加样孔3中加入100uL预处理过的样品溶液并水平静置，样品溶液会沿着样品垫向结合垫的方向渗透，静置8-10min后，从结果观察窗4观察质控线C、第一检测线T1和第二检测线T2的颜色变化。当质控线C、第一检测线T1和第二检测线T2均显色时，判定被检样品中PEDV和TGEV都为阳性。当质控线C显色，第一检测线T1显色，而第二检测线T2不显色时，判定被检样品中PEDV阳性，TGEV阴性。当质控线C显色，第二检测线T2显色，而第一检测线T1不显色时，判定被检样品中PEDV阴性，TGEV阳性。当质控线C显色，而第一检测线T1和第二检测线T2均不显色时，判定被检样品中PEDV和TGEV都为阴性。
实施例1：抗PEDV特异性单克隆抗体和抗TGEV特异性单克隆抗体的制备
取分泌抗PEDV特异性单克隆抗体的保藏编号为CCTCC NO:C2019170的杂交瘤细胞株FL011-26(为了便于描述，下文中将获得的对应抗体称为PEDV抗体1)和CCTCC NO:C2019194的杂交瘤细胞株FL011-33(为了便于描述，下文中将获得的对应抗体称为PEDV抗体2)以及分泌抗TGEV特异性单克隆抗体的保藏编号为CCTCC NO:C2019195的杂交瘤细胞株FL111-01(为了便于描述，下文中将获得的对应抗体称为TGEV抗体1)和CCTCC NO:C2019196的杂交瘤细胞株FL011-07(为了便于描述，下文中将获得的对应抗体称为TGEV抗体2)，分别在细胞培养瓶中进行扩大培养。
取6-8周龄的BALB/c小鼠，每只小鼠腹腔注射0.2mL液体石蜡。10天后，将上述培养获得的四种杂交瘤细胞分别腹腔接种小鼠来制备相应的单克隆抗体。待小鼠腹部明显膨大时，用弯头滴管采集腹水，通过间接ELISA测定腹水抗体的效价。
将小鼠腹水在室温、2000rpm下离心15min，挑去上层油脂，在4℃搅拌下 逐滴缓慢加入饱和硫酸铵至半饱和，继续搅拌30min，4℃、13000rpm下离心30min，弃上清；将沉淀溶于适量0.01M的PBS(pH7.4)；在4℃搅拌下逐滴缓慢加入饱和硫酸铵至33％，继续搅拌30min，4℃、13000rpm下离心30min，弃上清，将沉淀溶于适量0.01M的PBS(pH7.4)，4℃下透析过夜，然后测定蛋白质含量。
采用Protein G小柱对上述得到的抗体粗蛋白质进行进一步纯化，新柱子先用5ml超纯水过柱，再用5mL 0.4M PB缓冲液(pH 7.0)平衡纯化小柱；抗体过柱，过程中要求缓慢过柱，以求抗体蛋白更好的结合在结合位点上；继续用10mL 0.4M PB缓冲液(pH 7.0)平衡纯化小柱；用5mL洗脱液(0.1M甘氨酸-盐酸缓冲液，pH 7.0)洗脱结合位点上的抗体，并加入1M Tris-HCl(pH 2.7)用于中和甘氨酸，使pH保持为适合抗体保存的中性条件。
实施例2：速测卡的制造
1、制备含有第一检测线T1、第二检测线T2和质控线C的NC膜
首先，配制进行包被所需的抗体溶液。将实施例1得到的PEDV抗体1用浓度为10mM、pH值为7.4的PBS缓冲溶液稀释到1.0mg/mL得到PEDV抗体1溶液，用作形成第一检测线T1的包被抗体。将实施例1制备得到的TGEV抗体1用浓度为10mM、pH值为7.4的PBS缓冲溶液稀释到1.0mg/mL得到TGEV抗体1溶液，用作形成第二检测线T2的包被抗体。将羊抗鼠IgG二抗(购自杭州隆基生物技术有限公司)用浓度为10mM、pH值为7.4的PBS缓冲溶液稀释到1mg/mL，用作质控线C的包被抗体。
接下来，在NC膜上包被抗体以形成第一检测线T1、第二检测线T2和质控线C。
用划膜喷金机将浓度为1.0mg/mL的PEDV抗体1溶液、浓度为1.0mg/mL的TGEV抗体1溶液和浓度为1mg/mL的羊抗鼠IgG二抗溶液分别以1.0μL/cm在NC膜(Pall vivid 170硝酸纤维素膜)上分别划线，作为第一检测线T1、第二检测线T2和质控线C。
将划线之后的NC膜在37℃烘干24小时，得到含有第一检测线T1、第二检测线T2和质控线C的NC膜。
2、制备固定有胶体金标记的特异性单克隆抗体的结合垫
(1)胶体金制备
I)准备：将500mL烧杯、20mL小烧杯、转子、棕色瓶、玻璃棒等洗净后放入酸缸(重铬酸钾：浓硫酸：超纯水＝120g:200ml:1000ml)中浸泡24小时。取出先用自来水冲洗3-4次，再用超纯水冲洗3-4次，置于37℃烘箱中烘干备用。
II)烧金溶液A的配制：用塑料称量匙称取1g氯金酸粉末(购自sigma)于棕色瓶中，加入99ml超纯水充分溶解，4℃避光保存。
III)烧金溶液B的配制：称取1g柠檬酸三钠(购自sigma)溶解于99ml超纯水中，混匀。
IV)胶体金的制备：量取99ml超纯水于烧杯中，加入1ml烧金溶液A，置于恒温磁力搅拌器上搅拌混匀，开启加热至溶液沸腾，迅速加入2ml新制备的烧金溶液B，继续搅拌加热，溶液逐渐变为蓝黑色，然后紫黑，再加热出现红色，继续沸煮出现透明的橙红色，继续沸煮10min，自然冷却至室温，加超纯水定容至100ml，倒入棕色瓶，4℃避光保存，得到胶体金溶液(其中胶体金的粒径为40nm，浓度为0.01％)。
(2)抗体标记
I)抗体的标记：取1.5mL上述1)制取好的胶体金溶液，用0.1M的K 2CO 3调节pH值，分别加入20ug PEDV抗体2和20ug TGEV抗体2并混合均匀，室温反应40min后加入10％的BSA终止反应，静置30min。
II)标记抗体纯化：将上述静置产物先低速(1500r/min)离心并弃去由凝聚的金胶粒形成的沉淀，收集上清液；再将上清液高速(8500r/min)离心30分钟，仔细吸去上清液，收集沉淀，用含1％(质量百分含量)BSA的0.1M PBS(PH7.4)复溶沉淀，得到胶体金标记的PEDV抗体2溶液(浓度为0.4mg/ml)和胶体金标记的TGEV抗体2溶液(浓度为0.4mg/ml)，4℃保存。
(3)喷金与划膜
将上述制备的0.4mg/ml的胶体金标记的PEDV抗体2抗体溶液和0.4mg/ml的胶体金标记的TGEV抗体2溶液按1:1混匀，得到混合溶液，再将混合溶液以2.0μL/cm喷于预处理过的结合垫，烘干，得到固定有胶体金标记的特异性单克隆抗体的结合垫。
3、组装形成速测卡
在PVC底板上依次相连地粘贴样品垫、结合垫、NC膜和吸收垫，形成试纸条。其中，样品垫的一端覆盖在结合垫上，使得样品垫与结合垫具有1mm-1.5mm的重叠区，NC膜位于结合垫下方且NC膜与结合垫具有1mm-2mm的重叠区，吸收垫位于NC膜的上方且吸收垫与NC膜具有1mm-2mm的重叠区。
将卡壳覆盖安装在试纸条上，使得加样孔位于样品垫上方以便露出样品垫的一部分，结果观察窗位于NC膜上方以便露出第一检测线T1、第二检测线T2和质控线C所在的位置。至此完成速测卡的组装。
实施例3：用实施例2的速测卡检测溶液中的PEDV和TGEV病毒粒子
用按实施例2制备的速测卡对用PBS缓冲液配制的以下三种含有PEDV和/或TGEV病毒粒子的溶液进行检测以评价该速测卡的效果：含1μg/ml PEDV病毒粒子的溶液，含1μg/ml TGEV病毒粒子的溶液，以及同时含有1μg/ml PEDV病毒粒子和1μg/ml TGEV病毒粒子的溶液。同时用0.2mol/L PBS缓冲液(pH 7.4)(具体配方为：8g氯化钠、3.35g十二水合磷酸氢二钠，0.2g磷酸二氢钾，0.2g氯化钾，双蒸水溶解定容至1L)作为空白对照。
向各速测卡的加样孔中分别加入100uL待测溶液，在室温条件下反应8分钟后，从结果观察窗观察显色结果并以拍照的方式记录检测结果。
对于空白对照，结果如图3所示，从结果观察窗可观察到质控线C处呈现明显显色，第一检测线T1和第二检测线T2处不显色。
对于含1μg/ml PEDV病毒粒子的溶液，结果如图4所示，从结果观察窗可观察到质控线C和第一检测线T1处呈现明显显色，第二检测线T2处不显色。
对于含1μg/ml TGEV病毒粒子的溶液，结果如图5所示，从结果观察窗可观察到质控线C和第二检测线T2处呈现明显显色，第二检测线T1处不无色。
对于同时含有1μg/ml PEDV病毒粒子和1μg/ml TGEV病毒粒子的溶液，结果如图6所示，从结果观察窗可观察到质控线C、第一检测线T1和第二检测线T2处均呈现明显显色。
上述结果表明，按实施例2所制备的速测卡能够准确地检测并鉴别出样品中的PEDV病毒粒子和TGEV病毒粒子。
实施例4：用实施例2的速测卡检测粪便样本中的PEDV和TGEV
用按实施例2制备的速测卡对以下粪便样本中的PEDV和TGEV存在情况进行检测：PEDV和TGEV均为阴性的粪便，PEDV阳性的粪便，TGEV阳性的粪便，PEDV和TGEV均为阳性的粪便。
首先，按照以下步骤制备各粪便样本的提取液。
(1)用大吸管吸取1mL 0.2mol/L PBS(pH 7.4)作为提取缓冲液加入离心管，PBS的具体配制方法为8g氯化钠、3.35g十二水合磷酸氢二钠，0.2g磷酸二氢钾，0.2g氯化钾，双蒸水溶解定容至1L；
(2)棉签直接蘸取新鲜粪便后立即插入到装有提取缓冲液的离心管，将棉签在管壁上反复用力旋转至少10次，并与管中的溶液混匀，然后在液面上方的管壁上挤压棉签，使液体尽可能被挤出，丢弃棉签；
(3)静置1-2分钟，优选4000rpm下离心5分钟；
(4)取上清作为提取液用于后续的检测。
接下来，将上述制备的提取液各取100uL分别加入到速测卡的加样孔中，在室温条件下反应8分钟后，从结果观察窗观察显色结果并以拍照的方式记录检测结果。
对于PEDV和TGEV均为阴性的粪便样本，结果如图7所示，从结果观察窗中可观察到质控线C处呈现明显显色，第一检测线T1和第二检测线T2处不显色。
对于PEDV阳性的粪便样本，结果如图8所示，从结果观察窗可观察到质控线C和第一检测线T1处呈现明显显色，第二检测线T2处不显色。
对于TGEV阳性的粪便样本，结果如图9所示，从结果观察窗可观察到质控线C和第二检测线T2处呈现明显显色，第二检测线T1处不显色。
对于PEDV和TGEV均为阳性的粪便样本，结果如图10所示，从结果观察窗可观察到质控线C、第一检测线T1和第二检测线T2处均呈现明显显色。
上述结果表明，按实施例2所制备的速测卡能够准确地检测并鉴别出粪便样品中的PEDV和TGEV，该速测卡可用于针对猪组织粪便样本中PEDV、TGEV的快速、现场和灵敏的检测。
申请人声明，本发明专利通过上述实施例来说明本发明专利的技术方案，所属技术领域的技术人员应该明了，上述的说明不应被认为是对本发明的限制。在 本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的，对本发明专利申请进行的这些修改和替代，均落在本发明的专利保护范围内。

Claims (10)

  1. 一种同时检测PEDV和TGEV的速测卡，其包括卡壳和设置在卡壳内的试纸条，所述试纸条包括PVC底板、依次相连地粘贴在所述PVC底板上的样品垫、结合垫、硝酸纤维素膜和吸收垫，所述卡壳上设置有加样孔和结果观察窗，其特征在于：
    所述硝酸纤维素膜上固定有抗PEDV特异性单克隆抗体和抗TGEV特异性单克隆抗体用于分别形成第一检测线和第二检测线，所述结合垫上固定有胶体金标记的抗PEDV特异性单克隆抗体和胶体金标记的抗TGEV特异性单克隆抗体。
  2. 根据权利要求1所述的速测卡，其特征在于：
    所述抗PEDV特异性单克隆抗体是利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26和/或保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33制备得到的。
  3. 根据权利要求1所述的速测卡，其特征在于：
    所述抗TGEV特异性单克隆抗体是利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01和/或保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07制备得到的。
  4. 根据权利要求1所述的速测卡，其特征在于：
    所述硝酸纤维素膜上固定有利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26所产生的抗PEDV特异性单克隆抗体和利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01所制备得到的抗TGEV特异性单克隆抗体，
    所述结合垫上固定有胶体金标记的利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33所产生的抗PEDV特异性单克隆抗体和胶体金标记的利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07所制备得到的抗TGEV特异性单克隆抗体。
  5. 根据权利要求1所述的速测卡，其特征在于：
    所述硝酸纤维素膜上固定的所述抗PEDV特异性单克隆抗体和所述抗 TGEV特异性单克隆抗体的量分别为0.4μg-1.2μg，
    所述结合垫上固定的所述胶体金标记的抗PEDV特异性单克隆抗体和所述胶体金标记的抗TGEV特异性单克隆抗体分别为5ng-50ng。
  6. 一种制备同时检测PEDV和TGEV的速测卡的方法，其特征在于：包括以下步骤：
    (1)用抗PEDV特异性单克隆抗体在硝酸纤维素膜上进行包被固定形成第一检测线和第二检测线中的一个，用抗TGEV特异性单克隆抗体在硝酸纤维素膜上进行包被形成所述第一检测线和所述第二检测线中的另一个，用羊抗鼠IgG二抗在硝酸纤维素膜进行包被形成质控线(C)，得到含有所述第一检测线、所述第二检测线和所述质控线的硝酸纤维素膜；
    (2)制备固定有胶体金标记的抗PEDV特异性单克隆抗体和胶体金标记的抗TGEV特异性单克隆抗体的结合垫；
    (3)组装试纸条：在PVC底板上依次粘贴设置样品垫、所述结合垫、所述硝酸纤维素膜和吸收垫来得到所述试纸条，其中所述样品垫位于所述结合垫上方且所述结合垫与所述样品垫具有1mm-1.5mm的重叠区，所述硝酸纤维素膜位于所述结合垫下方且所述硝酸纤维素膜与所述结合垫具有1mm-2mm的重叠区，所述吸收垫位于所述硝酸纤维素膜的上方且所述吸收垫与所述硝酸纤维素膜具有1mm-2mm的重叠区，所述样品垫是大小为4mm×15mm的玻璃纤维膜；
    (4)安装卡壳使得其覆盖住所述试纸条，其中所述卡壳上的加样孔位于所述样品垫上方，所述结果观察窗位于所述硝酸纤维素膜上方使得能露出所述第一检测线、所述第二检测线和所述质控线所在的位置。
  7. 根据权利要求6所述的制备同时检测PEDV和TGEV的速测卡的方法，其特征在于：
    所述抗PEDV单克隆抗体是利用保藏在中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26和/或保藏在中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33制备得到的，
    所述抗TGEV单克隆抗体是利用保藏在中国典型培养物保藏中心、保藏编号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01或保藏在中国典型培养物 保藏中心、保藏编号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07制备得到的。
  8. 根据权利要求6所述的制备同时检测PEDV和TGEV的速测卡的方法，其特征在于：
    所述硝酸纤维素膜上固定有利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019170的杂交瘤细胞株FL011-26所产生的抗PEDV特异性单克隆抗体和利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019195的杂交瘤细胞株FL111-01所产生的抗TGEV特异性单克隆抗体，
    所述结合垫上固定有胶体金标记的利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019194的杂交瘤细胞株FL011-33所产生的抗PEDV特异性单克隆抗体和胶体金标记的利用保藏在中国典型培养物保藏中心、保藏号为CCTCC NO：C2019196的杂交瘤细胞株FL011-07所产生的抗TGEV特异性单克隆抗体。
  9. 根据权利要求6所述的制备同时检测PEDV和TGEV的速测卡的方法，其特征在于：
    所述硝酸纤维素膜上固定的所述抗PEDV特异性单克隆抗体和所述抗TGEV特异性单克隆抗体的量分别为0.4μg-1.2μg，
    所述结合垫上固定的所述胶体金标记的抗PEDV特异性单克隆抗体和所述胶体金标记的抗TGEV特异性单克隆抗体分别为5ng-50ng。
  10. 一种应用权利要求1-5中任一项所述的速测卡同时检测PEDV和TGEV的方法，其特征在于，包括以下步骤：
    (1)样本处理：向猪的新鲜粪便样品中加入pH 7.4、0.2mol/L的PBS缓冲液作为提取缓冲液，静置或离心，取上清；
    (2)加样：取100μL步骤(1)中的上清液加入到所述速测卡的加样孔中，孵育8-10min；
    (3)结果判定：通过所述结果结果观察窗观察速测卡中第一检测线、第二检测线和质控线的颜色变化，当仅有质控线显色而第一检测线和第二检测线均不显色时，判定待检样品中PEDV和TGEV均是阴性，当第一检测线、第二检测线和质控线均显色时，判定待检样品中PEDV和TGEV均是阳性，当质控线以 及第一检测线或第二检测线中的一个同时显色时，判定待检样品中与第一检测线或第二检测线中显色的那道检测线所对应的病毒为阳性。
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