CN114137209B - Immunofluorescence detection test strip for rapidly detecting oyster herpesvirus antigen and application thereof - Google Patents

Immunofluorescence detection test strip for rapidly detecting oyster herpesvirus antigen and application thereof Download PDF

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CN114137209B
CN114137209B CN202110133142.5A CN202110133142A CN114137209B CN 114137209 B CN114137209 B CN 114137209B CN 202110133142 A CN202110133142 A CN 202110133142A CN 114137209 B CN114137209 B CN 114137209B
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CN114137209A (en
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辛鲁生
于江南
白昌明
王崇明
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • 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
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an immunofluorescence detection test strip for rapidly and qualitatively detecting oyster herpes virus (Ostreidheres virus1, osHV-1) antigen and application thereof. The immunofluorescence test strip provided by the invention takes ORF104 protein shown as SEQ ID NO.1 as an antigen molecule to be detected. The invention has simple operation steps, high detection speed, qualitative observation and quantitative measurement of the result, and low detection cost. The sample to be detected is simple to prepare, and only tissue is crushed by matching with tissue lysate, so that a complicated nucleic acid extraction process is not needed.

Description

Immunofluorescence detection test strip for rapidly detecting oyster herpesvirus antigen and application thereof
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an immunofluorescence detection test strip for rapidly and qualitatively detecting oyster herpes virus (Ostreidheres virus1, osHV-1) antigen and application thereof.
Background
Oyster herpes virus (OsHV-1), belonging to the order Herpesvirales (Herpesvirales), family molluscae herpesviridae (Malarohalidevidae), and the host range relates to a plurality of cultured bivalve shellfish in China, including oyster, scallop, clam and the like. After the oyster herpes virus infects the cultured shellfish population, the shellfish population can die in a large scale. Because the bivalve shellfish culture mode belongs to open water culture, the shellfish is difficult to apply after the shellfish is ill, so that the risk management and control of the pathogen in the culture process is more important. In order to realize the periodic monitoring task of the oyster herpesvirus pathogen, a rapid and efficient detection method needs to be developed.
At present, the detection of the OsHV-1 is based on the detection of the virus nucleic acid level, and the requirement on the business level of operators engaged in detection is higher; the detection place needs to be matched with related equipment and a reagent storage space, and high cleanliness is required to be kept so as to avoid pollution of positive samples. The current nucleic acid detection technology has a longer treatment period, and the purpose of on-site rapid detection cannot be realized.
Disclosure of Invention
Aiming at the problems existing in the prior art, the main purpose of the invention is to provide an antigen immunofluorescence test strip for rapidly detecting oyster herpesvirus (OsHV-1), which can realize qualitative and quantitative detection of oyster herpesvirus, is applied to regular monitoring of OsHV-1 in the breeding process, and realizes risk management and control of the pathogen; in addition, a method for quantifying viruses other than nucleic acid levels may be provided to researchers.
The invention also provides application of the antigen immunofluorescence test strip.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention provides an immunofluorescence test strip for rapidly detecting oyster herpes virus OsHV-1 antigen, which takes ORF104 protein shown as SEQ ID NO.1 as an antigen molecule to be detected; optimally, the immunofluorescence test strip takes a peptide segment shown as SEQ ID NO.2 as an immunogen to prepare a polyclonal antibody.
The immunofluorescence test strip prepared by the invention specifically comprises a PVC bottom plate, wherein a sample pad, a bonding pad, a detection pad and a water absorption pad are sequentially stacked on the upper layer of the bottom plate; the binding pad is provided with an EPI polyclonal antibody marked by fluorescent microspheres; the detection pad consists of a nitrocellulose membrane, and is sprayed with a goat anti-mouse IgG quality control line C and a detection line T coated with an EPI polyclonal antibody.
The preparation method of the EPI polyclonal antibody specifically comprises the following steps:
(1) Recombinant expression of EPI protein by pGEX-4-1 and pET28a-SUMO plasmids respectively, and purification by glutathione and Ni ion coupled agarose resin respectively;
(2) Taking the EPI protein expressed by pGEX-4-1 in a recombinant way to immunize mice;
(3) Purifying by octanoic acid-ammonium sulfate precipitation method to obtain mouse polyclonal antibody IgG corresponding to EPI protein, and recombining and expressing EPI protein by pET28a-SUMO to be used as antigen to be detected.
Further, in the step (2), the immunization dose of each mouse is 200 μg recombinant protein, and the number of immunization times is 7.
Further, in step (3), the ELISA titer of the purified polyclonal antibody is 1:10 3 ~1:10 6 。
The invention also provides an application of the=immunofluorescence test strip for rapidly, qualitatively and quantitatively detecting oyster herpes viruses, and the specific detection method comprises the following steps:
a. collecting a shellfish mantle sample to be detected, adding a tissue lysate, grinding or mashing the tissue to obtain a solution to be detected;
b. dripping the solution to be detected into a sample pad of the fluorescent immunity detection test paper strip, and horizontally placing for 3-5 min to observe a result;
c. carrying out rapid qualitative detection by adopting a handheld ultraviolet lamp with the wavelength of 365nm or so for irradiation;
d. the EPI standard is diluted by the negative sample tissue lysate in a gradient way, and a log linear regression standard curve between the sample concentration and the T/C ratio is drawn; and testing the T/C value of the sample to be detected, calculating the antigen content in the sample through a standard curve, and carrying out antigen quantitative detection.
Further, in step (a), the tissue lysate comprises 0.1M Tris-HCl,0.5% Tween20,1.5% Triton X-100,0.03% NaN 3 。
Further, in step (c), the qualitative detection is: when the quality control line C and the detection line T of the detection pad show two fluorescent strips, the sample to be detected contains the antigen to be detected; if only the quality control line C shows a fluorescent strip, the antigen to be detected is not detected in the sample to be detected.
According to the analysis of the structural characteristics and the molecular expression level of OsHV-1 molecules, the invention optimizes that an OsHV-1 nucleocapsid protein ORF104 (SEQ ID NO. 1) is used as an antigen molecule to be detected and is applied to an OsHV-1 immunofluorescence rapid detection test strip. ORF104 is used as nucleocapsid-like protein, is a potential structural molecule of virus, and has quite high expression level during virus infection. The polyclonal antibody is prepared by predicting antigen epitope, preferably a section of antigen epitope EPI (SEQ ID NO. 2).
Compared with the prior art, the invention has the beneficial technical effects that:
1. the invention has simple operation steps, high detection speed, qualitative observation and quantitative measurement of the result, and low detection cost.
2. The sample to be detected is simple to prepare, and only tissue is crushed by matching with tissue lysate, so that a complicated nucleic acid extraction process is not needed.
Drawings
FIG. 1 shows SDS-PAGE of EPI protein expressed recombinantly by pGEX-4-1;
in the figure, M is Marker;1 is protein after EPI purification;
FIG. 2 is an SDS-PAGE electrophoresis of the recombinant expressed EPI protein of pET28 a-SUMO;
in the figure, M is Marker;1 is EPI pre-purification protein; 2 is protein after EPI purification;
FIG. 3 is a schematic diagram of the structure of an immunofluorescence rapid detection test strip;
in the figure, 1 is a bottom plate, 2 is a sample pad, 3 is a bonding pad, 4 is a detection pad, 5 is a water absorption pad, 6 is a detection line T, and 7 is a quality control line C.
FIG. 4 is a standard graph of viral antigen concentration detection;
in the figure, the X-axis represents the logarithmic value of the antigen concentration, and the Y-axis represents the logarithmic value of T/C.
Detailed Description
The technical scheme of the invention is further explained and illustrated by specific examples.
Example 1: preparation of OsHV-1 immunofluorescence detection test strip
1. Prokaryotic expression and purification of EPI proteins
1) According to the France 2013 strain sequence published by GenBank (GenBank: NC_ 005881.2), a recombinant vector containing an OsHV-1 EPI gene (SEQ ID NO. 3) was synthesized from Shanghai Biotechnology Co., ltd.) using pUC57 plasmid as a frame, and designated pUC57-EPI, respectively;
2) The synthesized recombinant vector pUC57-EPI is used as a template, and cloning EPI fragments are respectively amplified to prokaryotic expression vectors pGEX-4t-1 and pET28a-SUMO, so that prokaryotic expression vectors pGEX-4t-1-EPI and pET28a-EPI are constructed;
4) The prokaryotic expression vectors pGEX-4t-1-EPI and pET28a-p25 are respectively transformed into competent cells of escherichia coli BL21 (DE 3) to obtain expression strains BL 21-pGEX-4 t-1-EPI and BL21-pET28a-EPI;
5) The method utilizes an escherichia coli prokaryotic expression system to express and purify the OsHV-1 EPI protein respectively, and comprises the following specific steps:
transferring the recombinant bacteria cultured overnight into fresh LB culture medium according to the volume ratio of 1:50, and continuously culturing until the OD of the bacterial growth log phase is reached 600 =0.4 to 0.6; adding isopropyl-beta-D thiogalactoside (IPTG) with the final concentration of 1.0 mmol/L as an inducer, and inducing and expressing more than 12 h at 37 ℃ and 220 r/min; harvesting the expressed bacteria by centrifugation at 5000g at 4 ℃; adding PBS buffer solution into the precipitated thalli according to 1/15 of the culture volume for resuspension, and centrifugally collecting supernatant and precipitate after ultrasonic crushing; the recombinant protein of BL 21-pGEX-4 t-1-EPI strain is purified and collected by glutathione coupled gel filler and is used as an immunogen antigen; recombinant proteins of BL21-pET28a-EPI strain are purified and collected by Ni ion coupling gel filler and used as coating detection antigen, SDS-PAGE (SDS-PAGE) electricThe swim map is shown in figure 1 and figure 2 respectively, and is stored at-20deg.C for use.
EPI polyclonal antibody preparation
1) Immunization of animals
Mice were immunized by subcutaneous multipoint injection on the dorsum abdomen. Firstly, mixing and emulsifying Freund's complete adjuvant and purified EPI protein in equal volume, wherein the immune dose of each mouse is 200 mug protein; mixing and emulsifying with Freund's incomplete adjuvant and purified two proteins respectively after the first immunization for three weeks, and enhancing immunization with the same dosage of protein for 6 times at intervals of two weeks; 7 days after the last immunization, tail-end blood collection and ELISA detection of serum titers show that the mouse antiserum titers are 1:10 3 To 1:10 7 Between them.
2) Antibody serum mass collection and polyclonal antibody purification
Mice were bled by eye-drop. Standing overnight at 4deg.C, and centrifuging at 3000 g to collect antibody serum; purifying the polyclonal antibody by adopting an octanoic acid-ammonium sulfate precipitation method, and detecting the titer of the purified polyclonal antibody by ELISA, wherein the result shows that the titer of the purified polyclonal antibody can reach 1:10 6 And (3) storing at-80 ℃ for standby.
3. Preparation of fluorescent microsphere labeled antibody
1) Activation of fluorescent microspheres: a fluorescent polystyrene microsphere of rare earth europium element with the diameter of 300 nm is selected, and the fluorescent polystyrene microsphere contains a functional group carboxyl; diluting the 1 mg fluorescent microspheres with 0.1M MES (pH 6.0), and mixing by vortex; subsequently, add (20-50) μL of 50mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and (20-50) μL 50mg/mL N-hydroxysuccinimide (NHS) and react on a rotator for about 30min; EDC and NHS were both 0.05M MES buffer configuration.
2) Quenching after activation of the fluorescent microspheres: centrifuging at high speed for about 20 min, and collecting activated fluorescent microspheres; 1mL of 0.25% methanol (0.1M MES buffer) was added and resuspended by vortexing; repeating once.
3) Labeling of polyclonal antibodies: adding 50 mug of EPI polyclonal antibody into the fluorescent microsphere after ultrasonic resuspension, mixing uniformly by vortex, and placing the mixture on a rotator for reaction for about 30min;
4) Closing: adding 500 μl of 3% bovine serum albumin (0.04M ethanol amine), performing ultrasonic treatment for 3s, intermittently for 3s, repeating for 3 times, and placing on a rotator for continuous sealing reaction for 30min after ultrasonic treatment is completed;
5) And (3) re-dissolving: the blocked fluorescent microspheres were collected by high-speed centrifugation, 500. Mu.L of a complex solution of labeled fluorescent microspheres (containing 0.05% casein, 0.3% polyvinylpyrrolidone PVP, 0.05% glycerol, 0.05% Tween-20 and, 0.03% Proclin 300, 5% trehalose, 0.05% sodium azide, 0.05M Tris-HCl, pH 7.8) was added, and the above was sonicated to aid dissolution and repeated 3 times.
4. Preparation of bond pads
The fluorescent microsphere marked polyclonal antibody is sprayed on a glass fiber membrane by a quantitative film spraying instrument according to the amount of 4 mu L/cm, and the glass fiber membrane is put into a baking oven and dried in a dark place at 50 ℃.
5. Preparation of detection pad
The concentration of the EPI protein polyclonal antibody was adjusted to 1.0 mg/mL with a coating solution (0.05M PBS (pH 7.0-7.4), 3% trehalose, 0.05% NaN 3), and goat anti-mouse IgG antibody was treated in parallel with the same conditions. Spraying an EPI polyclonal antibody and a goat anti-mouse IgG antibody on the center of a nitrocellulose membrane by using a quantitative membrane spraying instrument to form a detection line T and a quality control line C print, wherein the interval is 5 mm; and (5) drying the detection film and preserving at room temperature for later use.
6. Sample pad and preparation of water absorbing pad
Soaking glass fiber cotton with TBS (pH 7.2) solution containing 0.01mol/L Tris-HCl,0.2% Tween20 (v/v) and 0.1% (w/v) NaN 3; drying to obtain a sample pad, adding a drying agent, and hermetically preserving at room temperature; the water-absorbing filter paper is selected as the water-absorbing pad.
7. Assembly of test strips
The detection pad (lower) and the sample pad (upper) are sequentially adhered to the sample end of the bottom plate (supporting plate), the layers are overlapped by about 2 mm, the detection film is adhered to the center of the supporting plate, and finally the water absorption pad is adhered to the other end of the detection film and is overlapped with the detection film by about 2 mm. The test strip structure is shown in FIG. 3:
the test strip comprises a bottom plate (supporting plate) 1, a sample pad 2, a combination pad 3, a detection pad 4 and a water absorption pad 5 which are fixed on the bottom plate, wherein the detection pad is provided with a quality control line 7 for spraying and printing goat anti-mouse IgG antibody and a detection line 6 for spraying and printing EPI polyclonal antibody.
Example 2: rapid detection of OsHV-1 antigen using test strip
1. Quick qualitative detection of OsHV-1 antigen
1.1 preparation of test sample solutions
Collecting the mantle tissue of the shellfish to be detected, adding a proper amount of tissue lysate, and grinding and crushing.
1.2 Sample detection
And dripping the sample solution to be detected on a sample pad of the detection test paper strip, and horizontally placing for 3-5 min to observe a result.
1.3 determination of detection results
Two fluorescent strips appear on the detection pad of the detection test strip (namely, the detection line and the quality control line are both developed), which indicates that the sample to be detected contains the OsHV-1 antigen; only one fluorescence band (quality control line) is displayed as negative, which indicates that the sample to be detected does not detect the OsHV-1 antigen; failure of the test strip to reveal any bands indicates improper detection or failure of the test strip.
2. Rapid quantitative detection of OsHV-1 antigen
2.1 Drawing a standard curve
The EPI recombinant protein is diluted to nine concentrations (0 ng/mL, 2 ng/mL, 4 ng/mL, 8 ng/mL, 15 ng/mL, 31 ng/mL, 62 ng/mL and 123 ng/mL) by using a negative sample tissue lysate, 50 ul is taken and dripped into a test paper sample end, and the test paper sample end is placed into a fluorescence analyzer for detection after reacting for 10 min. Logarithm of concentration and T/C value, and then linear fitting is carried out, wherein a fitting curve is shown in figure 4, R 2 >0.99。
2.2 Precision determination
And repeatedly measuring the diluted EPI recombinant protein for 10 times, calculating the measured concentration and the variation coefficient according to the measured T/C value and the fitted curve, and measuring the concentration variation coefficient CV (%) value by a test strip to be less than or equal to 10%.
2.3 Determination of sample concentration to be detected
And (3) in the qualitative detection process of sample preparation, 50 uL samples to be detected are dripped into the sample ends of the test strips, the T/C ratio is measured, and the sample concentration is calculated.
Sequence listing
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Ala Tyr Asn Lys Leu Phe Val Val Ser Thr Leu Met Gly Glu Gly Ala
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Asp Val Ala Asn Thr Ser Asp Asn Val Arg Leu Ser Tyr Ser Ser Thr
85 90 95
Ser His Asn Thr Pro Leu Tyr Asn Glu Phe Val Gln Thr Ile Val Arg
100 105 110
Pro Ala Ala Pro Met Arg Gly Gly Val Ile Glu Asp Thr Thr Ser Met
115 120 125
Ile Thr Met Met Arg Glu Tyr Asp Lys Val Glu Pro Thr Cys Met Thr
130 135 140
Thr Leu Pro Glu Ile Ala Val Asp Leu Ile Asn Lys Ala Arg Leu Asn
145 150 155 160
Ile Leu Asn Glu Asn Asn Gly Tyr Val Ser Glu Ser Thr Leu Ser Asp
165 170 175
Pro Thr Leu Asp Ala Gln Arg Arg Glu Thr Ala Met Ala Ala Leu Gln
180 185 190
Gln Ile Asn Ala Gly Phe Ser Gly Asn Val Ser Lys Met Gln Leu Ala
195 200 205
Leu Met Ala Ser Ile Pro Glu Gln Ser Gln Ala Ala Ile Glu Ala Glu
210 215 220
Ser Ile Pro Tyr Glu Leu Ala Gln Val Thr Asn Glu Leu Ser Ala Ser
225 230 235 240
Ile Val Gln Asn Thr Ala Gln Gly Ser Ile Asp Pro Ile Gly Glu Leu
245 250 255
Arg Asp Ala Asn His Thr Arg Ser Tyr Tyr Glu Glu Val Ala Ser Arg
260 265 270
Ser Leu Glu Asn Leu Phe Gly Val Ser Lys Ser Val Thr Lys Gly Thr
275 280 285
Met Gly Ala Phe Phe Ser Glu Leu Met Arg Thr Asn Arg Asp Phe Met
290 295 300
Thr Asn Leu Thr Thr Ser Asp Asn Thr Ala Ser Gly Val Val Val Met
305 310 315 320
Pro Ala Asp Gly Gln Pro Gln Leu
325
<210> 3
<211> 984
<212> DNA
<213> Meacorn
<400> 3
atgtcttttg tagcaccgca gagaggtatt gttaccgata gggttggaag gaatgttggt 60
aacatgccta acaagactag catcaaacca ttgatgcgag agattggaag gatgttggtt 120
aagggtaatc tgaacggaaa aggaccaatt acatctgaaa cggattggga gacgttactg 180
aataagtctg gtgcatacaa caaattattt gttgtgtcga cactcatggg agagggtgct 240
gatgtggcca acacatctga taatgttaga ctaagttatt catcaaccag ccacaacaca 300
cccttgtaca atgagtttgt acaaactata gtgagacccg cagcaccgat gagaggaggt 360
gttattgagg acaccacatc catgataacc atgatgagag agtatgacaa agtcgagcca 420
acctgcatga caacattgcc ggaaattgct gtagacctca tcaataaggc ccgtcttaac 480
atattgaatg aaaataacgg atatgtctct gagagcacat tgtctgatcc tactctggat 540
gcacagagga gggaaacagc catggctgca ctacagcaaa tcaatgctgg attcagtgga 600
aatgtttcca agatgcaact ggcattgatg gcctctattc cagaacagtc acaggcagcc 660
attgaagcag agtcaattcc atatgagctg gcgcaagtaa cgaatgaatt gagcgcatca 720
atcgtgcaga acaccgccca gggaagcatt gatccaattg gagaattgag agatgccaat 780
cacaccagga gttattatga ggaggtagcc tcacggtctc ttgaaaacct atttggcgtg 840
tcaaagagcg tgacaaaggg aaccatgggc gccttctttt ctgagttaat gaggaccaac 900
agggacttca tgaccaatct taccacctct gacaacaccg ccagtggagt ggttgtgatg 960
ccagccgatg gtcaaccaca gctt 984

Claims (7)

1. An immunofluorescence test strip for rapidly detecting oyster herpes virus OsHV-1 antigen is characterized in that the immunofluorescence test strip takes ORF104 protein shown as SEQ ID NO.1 as an antigen molecule to be detected; the immunofluorescence test strip takes peptide EPI shown in SEQ ID NO.2 as an immunogen to prepare a polyclonal antibody;
the device specifically comprises a PVC bottom plate, wherein a sample pad, a bonding pad, a detection pad and a water absorption pad are sequentially stacked on the upper layer of the bottom plate; the binding pad is provided with an EPI polyclonal antibody marked by fluorescent microspheres; the detection pad consists of a nitrocellulose membrane, and is sprayed with a goat anti-mouse IgG quality control line C and a detection line T coated with an EPI polyclonal antibody;
the detection sample of the immunofluorescence test strip is as follows: and (3) adding the shellfish mantle sample into the tissue lysate, grinding or mashing the tissue to obtain a solution to be detected.
2. The immunofluorescence test strip of claim 1, wherein the EPI polyclonal antibody is specifically prepared by the following method:
(1) Recombinant expression of EPI protein by pGEX-4-1 and pET28a-SUMO plasmids respectively, and purification by glutathione and Ni ion coupled agarose resin respectively;
(2) Taking the EPI protein expressed by pGEX-4-1 in a recombinant way to immunize mice;
(3) Purifying by octanoic acid-ammonium sulfate precipitation method to obtain mouse polyclonal antibody IgG corresponding to EPI protein, and recombining and expressing EPI protein by pET28a-SUMO to be used as antigen to be detected.
3. The immunofluorescence test strip of claim 2, wherein in step (2), the immunization dose per mouse is 200 μg of recombinant protein and the number of immunizations is 7.
4. The immunofluorescent test strip of claim 1 or 3, wherein in step (3), the ELISA titer of the purified polyclonal antibody is 1:10 3 ~1:10 6 。
5. Use of an immunofluorescent test strip according to any one of claims 1-4 for rapid qualitative and quantitative detection of oyster herpes viruses, comprising the steps of:
a. collecting a shellfish mantle sample to be detected, adding a tissue lysate, grinding or mashing the tissue to obtain a solution to be detected;
b. dripping the solution to be detected into a sample pad of the fluorescent immunity detection test paper strip, and horizontally placing for 3-5 min to observe a result;
c. carrying out rapid qualitative detection by adopting a handheld ultraviolet lamp with the wavelength of 365nm or so for irradiation;
d. the EPI standard is diluted by the negative sample tissue lysate in a gradient way, and a log linear regression standard curve between the sample concentration and the T/C ratio is drawn; and testing the T/C value of the sample to be detected, calculating the antigen content in the sample through a standard curve, and carrying out antigen quantitative detection.
6. The use according to claim 5, wherein in step (a) the tissue lysate comprises 0.1M Tris-HCl,0.5% Tween20,1.5% Triton X-100,0.03% NaN 3 。
7. The use according to claim 5 or 6, wherein in step (c), the qualitative detection is: when the quality control line C and the detection line T of the detection pad show two fluorescent strips, the sample to be detected contains the antigen to be detected; if only the quality control line C shows a fluorescent strip, the antigen to be detected is not detected in the sample to be detected.
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