CN114137209A

CN114137209A - Immunofluorescence detection test strip for rapidly detecting oyster herpesvirus antigen and application

Info

Publication number: CN114137209A
Application number: CN202110133142.5A
Authority: CN
Inventors: 辛鲁生; 于江南; 白昌明; 王崇明
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2022-03-04
Anticipated expiration: 2041-02-01
Also published as: CN114137209B

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an immunofluorescence detection test strip for rapidly, qualitatively and quantitatively detecting oyster herpesvirus1 (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 method has the advantages of simple operation steps, high detection speed, qualitative observation and quantitative measurement of results, and low detection cost. The sample to be detected is simple to prepare, only needs to be matched with the tissue lysate to break the tissue, and does not need a complicated nucleic acid extraction process.

Description

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

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to an immunofluorescence detection test strip for rapidly, qualitatively and quantitatively detecting oyster herpesvirus1 (OsHV-1) antigen and application thereof.

Background

Oyster herpesvirus (OsHV-1) belongs to Herpesvirales, mollusca herpesviridae (Malacoheres), and the host range relates to a plurality of cultured bivalve shellfish in China, including oysters, scallops, blood clam and the like. After the oyster herpesvirus infects cultured shellfish groups, the shellfish groups can be killed on a large scale. Because the bivalve shellfish culture mode belongs to open water body culture, the treatment is difficult to be carried out by applying the medicine after the shellfish is ill, so the control of the risk of the pathogen in the culture process is more important. In order to realize the task of regularly monitoring the oyster herpesvirus as a pathogen, a rapid and efficient detection method is urgently needed to be developed.

At present, the detection of OsHV-1 is based on the detection of the virus nucleic acid level, and the requirement on the service level of operators engaged in the detection is high; the detection site needs to be matched with related equipment and reagent storage space, and needs to keep high cleanliness so as to avoid positive sample pollution. The nucleic acid detection technology is long in processing period, and the aim of on-site rapid detection cannot be fulfilled.

Disclosure of Invention

Aiming at the problems in the prior art, the invention mainly aims 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 control of the pathogen; in addition, a method for quantifying viruses other than the nucleic acid level can be provided for scientific researchers.

The invention also provides application of the antigen immunofluorescence test strip.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the invention provides an immunofluorescence test strip for rapidly detecting oyster herpesvirus OsHV-1 antigen, wherein the immunofluorescence test strip takes ORF104 protein shown as SEQ ID NO.1 as an antigen molecule to be detected; preferably, the immunofluorescence test strip takes the peptide segment shown in SEQ ID NO.2 as immunogen to prepare the polyclonal antibody.

The immunofluorescence test strip specifically comprises a PVC base plate, wherein a sample pad, a combination pad, a detection pad and a water absorption pad are sequentially stacked on the upper layer of the base plate; the binding pad is provided with an EPI polyclonal antibody marked by fluorescent microspheres; the detection pad is composed of a nitrocellulose membrane, and is sprayed and printed with a quality control line C coated with goat anti-mouse IgG and a detection line T coated with an EPI polyclonal antibody.

The specific preparation method of the EPI polyclonal antibody comprises the following steps:

(1) respectively utilizing pGEX-4-1 and pET28a-SUMO plasmids to express EPI protein in a recombinant mode, and then respectively utilizing glutathione and Ni ions to couple agarose resin for purification;

(2) immunizing a mouse by taking the EPI protein expressed by the pGEX-4-1 in a recombinant mode;

(3) mouse polyclonal antibody IgG corresponding to the EPI protein is obtained by purification through an octanoic acid-ammonium sulfate precipitation method, and the EPI protein is expressed through pET28a-SUMO recombination and is used as an antigen to be detected.

Further, in the step (2), the immunization dose of each mouse is 200 μ g of the recombinant protein during the immunization, and the immunization times are 7 times.

Further, in the step (3), the ELISA titer of the purified polyclonal antibody is 1:10³~1:10⁶。

The invention also provides an application of the = immunofluorescence test strip for rapid qualitative and quantitative detection of oyster herpesvirus, and the specific detection method comprises the following steps:

a. collecting a mantle sample of the shellfish to be detected, adding a tissue lysate to grind or triturate tissues to obtain a solution to be detected;

b. dripping the solution to be detected into a sample pad of a fluorescence immunoassay test strip, and horizontally placing for 3-5 min to observe the result;

c. irradiating by adopting a handheld ultraviolet lamp with a wavelength of about 365nm to carry out rapid qualitative detection;

d. diluting an EPI standard substance by a negative sample tissue lysate gradient, and drawing a log linear regression standard curve between the sample concentration and the T/C ratio; and testing the T/C value of the sample to be detected, calculating the content of the antigen in the sample through a standard curve, and carrying out quantitative detection on the antigen.

Further, in step (a), the tissue lysate comprises0.1M Tris-HCl，0.5% Tween20，1.5% TritonX-100，0.03% NaN₃。

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 fluorescence strips, the detection pad indicates that the sample to be detected contains the antigen to be detected; if only one fluorescence band appears on the quality control line C, the antigen to be detected is not detected in the sample to be detected.

According to the invention, based on the analysis of the OsHV-1 molecular structure characteristics and the molecular expression quantity one by one, an OsHV-1 nucleocapsid protein ORF104 (SEQ ID NO. 1) is preferably selected as an antigen molecule to be detected and applied to an OsHV-1 immunofluorescence rapid detection test strip. ORF104, as a nucleocapsid-like protein, is a potential structural molecule of viruses and is expressed in quite high amounts during viral 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 method has the advantages of simple operation steps, high detection speed, qualitative observation and quantitative measurement of results, and low detection cost.

2. The sample to be detected is simple to prepare, only needs to be matched with the tissue lysate to break the tissue, and does not need a complicated nucleic acid extraction process.

Drawings

FIG. 1 is SDS-PAGE of EPI protein recombinantly expressed by pGEX-4-1;

in the figure, M is Marker; 1 is EPI purified protein;

FIG. 2 is an SDS-PAGE electrophoresis of EPI protein recombinantly expressed from pET28 a-SUMO;

in the figure, M is Marker; 1 is a protein before EPI purification; 2 is EPI purified protein;

FIG. 3 is a schematic view of a test strip for immunofluorescence rapid assay;

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 the detection of viral antigen concentration;

in the figure, the X-axis represents the logarithm of the antigen concentration, and the Y-axis represents the logarithm of T/C.

Detailed Description

The technical solution of the present invention is further explained and illustrated by the following specific examples.

Example 1: preparation of OsHV-1 immunofluorescence detection test strip

1. Prokaryotic expression and purification of EPI protein

1) According to the France 2013 strain sequence published by GenBank (GenBank: NC-005881.2), using pUC57 plasmid as frame, synthesizing recombinant vector containing OsHV-1 EPI gene (SEQ ID NO. 3) by Shanghai biological engineering Co., Ltd, and respectively naming as pUC 57-EPI;

2) respectively amplifying and cloning EPI fragments to prokaryotic expression vectors pGEX-4t-1 and pET28a-SUMO by taking the synthetic recombinant vector pUC57-EPI as a template to construct prokaryotic expression vectors pGEX-4t-1-EPI and pET28 a-EPI;

4) respectively transforming prokaryotic expression vectors pGEX-4t-1-EPI and pET28a-p25 into escherichia coli BL21(DE3) competent cells to obtain expression strains BL 21-pGEX-4 t-1-EPI and BL21-pET28 a-EPI;

5) respectively expressing and purifying OsHV-1 EPI protein by using an escherichia coli prokaryotic expression system, and specifically comprising the following steps:

transferring the overnight cultured recombinant bacteria into a fresh LB culture medium according to the volume ratio of 1:50, and continuously culturing until the bacteria grow to logarithmic phase OD₆₀₀= 0.4-0.6; adding isopropyl-beta-D thiogalactoside (IPTG) with the final concentration of 1.0 mmol/L as an inducer, and carrying out induced expression at 37 ℃ and 220 r/min for more than 12 h; carrying out centrifugation at 5000g at 4 ℃ to obtain expression bacteria; adding 1/15 of the culture volume of the precipitated thallus into PBS buffer solution for resuspension, centrifuging after ultrasonic crushing, and collecting supernatant and precipitate; BL 21-pGEX-4 t-1-EPI strain recombinant protein is purified and collected by glutathione coupled gel packing and is used as immunogen antigen; BL21-pET28a-EPI strain recombinant protein is purified and collected by Ni ion coupling gel filler, and is used as coating detection antigen, and SDS-PAGE electrophoresis images are respectively shown in figure 1 and figure 2 and are preserved at-20 ℃ for later use.

Preparation of EPI polyclonal antibodies

1) Animal immunization

Mice were injected subcutaneously at multiple points through the dorsoventral regionThe method of (3) immunizing. Firstly, mixing and emulsifying the purified EPI protein with Freund's complete adjuvant in equal volume, wherein the immune dose of each mouse is 200 mu g of protein; mixing and emulsifying the two purified protein isotypes by Freund incomplete adjuvant and purified protein isotypes respectively after three weeks of primary immunization, and performing booster immunization 6 times by using the same dosage of protein for two weeks at intervals; 7 days after the last immunization, blood is collected after tail breaking, the serum titer is detected by ELISA, and the result shows that the mouse antiserum titer is 1:10³To 1:10⁷In the meantime.

2) Antibody serum mass collection and polyclonal antibody purification

Mice were bled by the eyeball method. Standing overnight at 4 ℃, centrifuging 3000 g and collecting antibody serum; the octanoic acid-ammonium sulfate precipitation method is adopted to purify the polyclonal antibody, ELISA is adopted to detect the titer of the purified polyclonal antibody, and the result shows that the titer of the purified polyclonal antibody can reach 1:10⁶Storing at-80 deg.C.

3. Preparation of fluorescent microsphere labeled antibody

1) Activation of fluorescent microspheres: selecting a fluorescent polystyrene microsphere of rare earth europium element, wherein the diameter of the fluorescent polystyrene microsphere is 300 nm, and the fluorescent polystyrene microsphere comprises functional group carboxyl; diluting 1 mg of fluorescent microspheres with 0.1M MES (pH 6.0), and mixing by vortex; followed by addition of (20-50) μ L of 50mg/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and (20-50) μ L of 50mg/mL N-hydroxysuccinimide (NHS) and reaction on a rotator for about 30 min; EDC and NHS were both in 0.05M MES buffer.

2) Quenching after the activation of the fluorescent microspheres: centrifuging at high speed for about 20 min, and collecting activated fluorescent microspheres; 1mL of 0.25% methanol (in 0.1M MES buffer) was added and resuspended by vortexing; and repeating the steps once.

3) Labeling of polyclonal antibodies: adding 50 mu g of EPI polyclonal antibody into the fluorescent microspheres subjected to ultrasonic resuspension, uniformly mixing by vortex, and placing on a rotator for reaction for about 30 min;

4) and (3) sealing: adding 500 μ L of 3% bovine serum albumin (prepared from 0.04M ethanolamine), performing ultrasonic treatment for 3s, repeating for 3s, and placing on a rotator to continuously perform closed reaction for 30min after the ultrasonic treatment is completed;

5) redissolving: the fluorescent microspheres after being blocked are collected by high-speed centrifugation, 500 microliter of marked fluorescent microsphere complex solution (containing 0.05 percent of casein, 0.3 percent of polyvinylpyrrolidone PVP, 0.05 percent of glycerol, 0.05 percent of Tween-20, 0.03 percent of Proclin 300, 5 percent of trehalose, 0.05 percent of sodium azide, 0.05M Tris-HCl and pH 7.8) is added, the ultrasonic assisted dissolution is carried out in the same way, and the steps are repeated for 3 times.

4. Preparation of the conjugate pad

The fluorescent microsphere marked polyclonal antibody is sprayed on a glass fiber membrane by a quantitative membrane spraying instrument in an amount of 4 mu L/cm, and the glass fiber membrane is put into an oven and dried at 50 ℃ in a dark place.

5. Preparation of detection pad

The concentration of the EPI protein polyclonal antibody was adjusted to 1.0 mg/mL using a coating solution (0.05M PBS (pH 7.0-7.4), 3% trehalose, 0.05% NaN 3), and the goat anti-mouse IgG antibody was treated under the same conditions in parallel. Spraying and printing an EPI polyclonal antibody and a goat anti-mouse IgG antibody in 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 blot with a spacing of 5 mm; and drying the detection membrane, and storing at room temperature for later use.

6. Preparation of sample pad and absorbent pad

Soaking glass fiber cotton in 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 storing at room temperature; absorbent filter paper is selected as the absorbent pad.

7. Assembly of test strips

Firstly, the detection pad (lower) and the sample pad (upper) are sequentially pasted at the sample end of the bottom plate (support plate), the overlapping of each layer is about 2 mm, the detection membrane is pasted at the center of the support plate, and finally, the water absorption pad is pasted at the other end of the detection membrane and is overlapped with the detection membrane by about 2 mm. The test strip structure is shown in figure 3:

the test strip comprises a bottom plate (support plate) 1, a sample pad 2, a combination pad 3, a detection pad 4 and a water absorption pad 5, wherein the sample pad, the combination pad 3, the detection pad 4 and the water absorption pad 5 are fixed on the bottom plate, and a quality control line 7 for spray printing of goat anti-mouse IgG antibodies and a detection line 6 for spray printing of EPI polyclonal antibodies are arranged on the detection pad.

Example 2: rapid detection of OsHV-1 antigen by using test strip

1. Rapid qualitative detection of OsHV-1 antigen

1.1 preparation of test sample solution

Collecting the mantle tissue of the shellfish to be detected, adding a proper amount of tissue lysate for grinding and crushing.

1.2 sample detection

And dripping the sample solution to be detected on the sample pad of the detection test strip, and horizontally placing for 3-5 min to observe the result.

1.3 determination of detection result

The detection pad of the detection test strip shows two fluorescence strips (namely, the detection line and the quality control line are colored), which indicates that the sample to be detected contains the OsHV-1 antigen; only one fluorescence band (quality control line) is shown to be negative, which indicates that the OsHV-1 antigen is not detected in the sample to be detected; the absence of any bands on the test strip indicates improper testing or failure of the test strip.

2. Rapid quantitative detection of OsHV-1 antigen

2.1 drawing Standard Curve

Diluting EPI recombinant protein 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 negative sample tissue lysate, taking 50 ul, dripping the solution into a test paper sample end, reacting for 10min, and then putting the test paper sample end into a fluorescence analyzer for detection. The concentration and T/C values were logarithmized and then subjected to linear fitting, the fitted curve being shown in FIG. 4, R²>0.99。

2.2 determination of precision

And repeatedly measuring the diluted EPI recombinant protein for 10 times, calculating the measured concentration and the coefficient of variation according to the measured T/C value and a fitted curve, and measuring the coefficient of variation CV (%) value of the concentration by a test strip to be less than or equal to 10%.

2.3 determination of the concentration of the sample to be examined

And (3) preparing a sample to be detected, namely, in the same qualitative detection process, taking 50 uL of the sample to be detected, dripping the sample to be detected into the sample end of the test strip, measuring the T/C ratio, and calculating the concentration of the sample.

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Claims

1. An immunofluorescence test strip for rapidly detecting oyster herpesvirus (OsHV-1) antigen is characterized in that ORF104 protein shown as SEQ ID No.1 is used as an antigen molecule to be detected.

2. The immunofluorescence test strip of claim 1, wherein the immunofluorescence test strip is used for preparing polyclonal antibodies with the peptide fragment as shown in SEQ ID No.2 as an immunogen.

3. The immunofluorescence test strip according to claim 1 or 2, which is characterized by comprising a PVC bottom plate, wherein a sample pad, a combination 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 is composed of a nitrocellulose membrane, and is sprayed and printed with a quality control line C coated with goat anti-mouse IgG and a detection line T coated with an EPI polyclonal antibody.

4. The immunofluorescence test strip according to claim 3, wherein the EPI polyclonal antibody is specifically prepared by the following method:

5. The immunofluorescence test strip according to claim 4, wherein, in the step (2), the immunization dose of each mouse is 200 μ g of recombinant protein, and the immunization times are 7 times.

6. The immunofluorescence test strip according to claim 4 or 5, wherein in step (3), the ELISA titer of the purified polyclonal antibody is 1:10³~1:10⁶。

7. The application of the immunofluorescence test strip of any one of claims 1-6, which is used for rapid qualitative and quantitative detection of oyster herpesvirus, and the specific detection method comprises the following steps:

8. The use of claim 7, wherein in step (a), the tissue lysate comprises 0.1M Tris-HCl, 0.5% Tween20, 1.5% Triton X-100, 0.03% NaN₃。

9. Use according to claim 7 or 8, 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 fluorescence strips, the detection pad indicates that the sample to be detected contains the antigen to be detected; if only one fluorescence band appears on the quality control line C, the antigen to be detected is not detected in the sample to be detected.