CN117147832A - Colloidal gold immune test strip for detecting betel nut APV1 virus and preparation method thereof - Google Patents
Colloidal gold immune test strip for detecting betel nut APV1 virus and preparation method thereof Download PDFInfo
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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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
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Abstract
The invention relates to the technical field of colloidal gold immunoassay, in particular to a colloidal gold immunoassay test strip for detecting betel nut APV1 virus and a preparation method thereof. The test strip comprises a sample pad, a colloidal gold combination pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line; the nitrocellulose membrane is stuck to the middle section of the surface of the PVC bottom plate, a colloidal gold bonding pad and a water absorption pad are respectively overlapped at two ends, and the overlapped area is 2mm; the sample pad is adhered to one end of the colloidal gold bonding pad, which is far away from the nitrocellulose membrane, and is used for dropwise adding a sample to be detected; the colloidal gold binding pad is coated with an APV1 antibody solution marked by colloidal gold; the detection line is coated with a mouse anti-APV 1 monoclonal antibody; the quality control line is coated with goat anti-mouse IgG antibody. The advantages are that: the test strip has the characteristics of high specificity, rapid response, simple operation, low cost and the like, and can finish the rapid detection of the areca sample APV1 virus within 15 minutes without instruments and equipment.
Description
Technical Field
The invention relates to the technical field of colloidal gold immunoassay, in particular to a colloidal gold immunoassay test strip for detecting betel nut APV1 virus and a preparation method thereof.
Background
Betel nut is located at the beginning of four big south medicines in China, the planting area in Hainan is close to 300 mu, and the betel nut becomes the most important tropical economic crop in Hainan province and is the main economic source of 200 mu rural population in Hainan province (2022 years data of rural agricultural halls in Hainan province). The betel nut industry plays a significant role in implementing country trembling strategy in the south of the sea. The betel nut yellowing disease (Yellow leaf disease, YLD) is reported in India for the first time in the 60 th century, and the current disease area of Hainan full province can reach more than 100 ten thousand mu, and the betel nut yellowing disease is spreading rapidly, so that a large number of betel nut trees die, and more than 20 hundred million yuan are lost each year. In 2015, wu Qingfa, university of china science and technology, taught team discovered and named APV1 virus for the first time in betel nut yellow disease samples by small RNA sequencing technology (Areca palm velarivirus 1). The virus was found to belong to the genus Velarivirus by phylogenetic tree analysis (Yu et al 2015). In order to identify the etiology of betel nut yellow disease, in previous studies, we analyzed using transcriptome sequencing and digital expression profiling, APV1 virus was also found from the differentially expressed genes, and cloned to obtain the full length genomic sequence (MK 956940) of 17,546nt APV1-WNY virus strain. The morphology of the APV1 virus particles was observed by virus isolation and purification and electron microscopy to be in a curved line shape, and the length was about 1200-1500nm, which accords with the characteristics of viruses of the family of the longline virus (Wang et al, 2020). We sampled throughout Hainan and examined APV1 virus by reverse transcription polymerase chain reaction (Reverse Transcription-Polymerase Chain Reaction, RT-PCR for short), found that there was a high correlation between APV1 virus infection and betel nut etiolation (Wang et al 2020) from the results of the examination of 76 parts of diseased leaves APV1 from the etiolation disease area, of 100 parts of asymptomatic leaves APV1 from the etiolation disease area, of 12% from the etiolation disease area, and of 0% from 22 parts of asymptomatic leaves APV1 from the etiolation disease non-diseased area. In order to further understand how the APV1 virus spreads in betel nut gardens, we collect pests from a plurality of areas of Hainan where betel nut yellowing disease occurs and perform RT-PCR detection, and find that the APV1 virus is commonly carried by scale insects in betel nut gardens in a plurality of disaster areas. The scale insects carrying the APV1 virus are inoculated to healthy areca seedlings, so that the healthy areca seedlings have typical areca yellow disease symptoms, and the scale insects are insect transmission media (Zhang et al 2021) of the APV1 virus, wherein the APV1 virus is the etiology of the areca yellow disease.
At present, no effective chemical control technology exists for viral diseases of plants, and prevention and control measures mainly reduce spread of diseases through quarantine of diseases and popularization of healthy seedlings, so that an effective detection technology must be established, and the detection technology published and applied at present mainly is an RT-PCR detection technology, and the main problems of the detection technology include: (1) The cost is high, and expensive consumables such as an RNA extraction kit, an RNA reverse transcriptase kit and the like are needed; (2) Laboratory hardware requirements are high, and expensive instruments and equipment such as centrifuges and PCR instruments are needed; (3) time consuming, requiring 24 hours to test a batch of samples. The limiting factors greatly limit the detection technology of the betel nut APV1 virus, and seriously influence the popularization of healthy betel nuts and the prevention and control of betel nut yellowing diseases. Therefore, it is important to develop a rapid detection technology which is low in cost, simple and convenient to operate and independent of laboratory hardware conditions so as to meet social demands.
Disclosure of Invention
The invention provides a colloidal gold immune test strip for detecting betel nut APV1 virus and a preparation method thereof.
The first aim of the invention is to provide a colloidal gold immune test strip for detecting betel nut APV1 virus, which comprises a sample pad, a colloidal gold combination pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line;
the nitrocellulose membrane is adhered to the middle section of the surface of the PVC bottom plate, the two ends of the nitrocellulose membrane are respectively overlapped with the colloidal gold bonding pad and the water absorption pad, and the overlapped area is 2mm;
the sample pad is adhered to one end of the colloidal gold bonding pad, which is far away from the nitrocellulose membrane, and is used for dropwise adding a sample to be detected;
the colloidal gold binding pad is coated with 0.6-1 mu L of colloidal gold-labeled APV1 antibody solution, and the concentration of the colloidal gold-labeled APV1 antibody solution is 0.15-0.25 mg/mL;
the detection line is coated with a mouse anti-APV 1 monoclonal antibody, and 0.8-1.5 mu L of the mouse anti-APV 1 monoclonal antibody with the concentration of 0.8-1.2 mg/mL is coated on a test strip with the width of each centimeter;
the quality control line is coated with the goat anti-mouse IgG antibody, and the concentration of the goat anti-mouse IgG antibody is 0.8-1.2 mg/mL per centimeter of wide test strip, and the concentration of the goat anti-mouse IgG antibody is 0.8-1.5 mu L. Preferably, the distance between the detection line and the quality control line is 5-10 mm.
Preferably, the length of the colloidal gold bonding pad is 7.5-8.5 mm; the length of the nitrocellulose membrane is 13.5-14.5 mm; the length of the water absorbing pad is 31-33 mm.
Preferably, the colloidal gold-labeled APV1 antibody solution is a colloidal gold-labeled APV1 virus monoclonal antibody solution or a colloidal gold-labeled APV1 virus polyclonal antibody solution.
The second aim of the invention is to provide a preparation method of a colloidal gold immune test strip for detecting betel nut APV1 virus, which comprises the following steps:
s1, treating a colloidal gold bonding pad: firstly, treating a colloidal gold binding pad by using PBS buffer solution containing 0.5-1.5% of sucrose and 0.5-1.5% of BSA, and drying;
s2, preparing a gold mark pad: spraying the APV1 antibody solution marked by the colloidal gold on a dried colloidal gold bonding pad, and drying for 1-3 hours at the temperature of 35-38 ℃ to obtain a gold-marked pad;
s3, preparing a detection line and a quality control line: spraying a mouse anti-APV 1 monoclonal antibody onto a nitrocellulose membrane to serve as a detection line, spraying a sheep anti-mouse IgG antibody onto the nitrocellulose membrane to serve as a quality control line, and drying the nitrocellulose membrane at 35-38 ℃ for 6-10 h;
s4, assembling a test strip: the dried nitrocellulose membrane is stuck to the middle section of the surface of the PVC bottom plate, and then a water absorption pad and a gold mark pad are respectively stuck to the two ends of the nitrocellulose membrane, wherein the water absorption pad and the gold mark pad are overlapped above the nitrocellulose membrane by 2mm; the sample pad is adhered to one end of the gold mark pad far away from the nitrocellulose membrane; cutting into test strips with the width of 3.5-4.5 mm after assembly, mounting a clamping shell, sealing, drying and preserving.
Preferably, the colloidal gold-labeled APV1 antibody solution in step S2 is a colloidal gold-labeled APV1 virus monoclonal antibody solution, and the specific preparation method comprises the following steps:
s201, taking 8-12 ml of colloidal gold particle solution, and using K with the concentration of 0.02mol/l 2 CO 3 The pH of the solution is adjusted to 8;
s202, stirring the colloidal gold particle solution, dropwise adding 35-45 ul of purified APV1 virus monoclonal antibody solution with the concentration of 0.5-1.5 mg/ml while stirring, and continuing stirring for 15-25 min;
s203, adding 2-3 ml of polyethylene glycol 2000 with the concentration of 15-25 mg/ml dropwise, and continuously stirring for 15-25 min;
s204, centrifuging the solution obtained in the step S203 at 7000-8000 r/min for 30-50 min at 0-4 ℃; the supernatant was discarded, and the pellet was resuspended in PBS buffer containing 1.5-2.5% BSA and filtered at 0.22 μm to give a colloidal gold-labeled APV1 virus monoclonal antibody solution.
Preferably, the particle size of the colloidal gold particles in the colloidal gold particle solution is 38-42 nm; the colloidal gold particle solution is prepared by adding trisodium citrate into a boiling chloroauric acid solution.
Preferably, the concentration of the APV1 virus monoclonal antibody solution purified in step S202 is 1mg/ml.
Preferably, the concentration of polyethylene glycol 2000 in step S203 is 20mg/ml, and the stirring time is 20min.
The third object of the present invention is to provide a method for detecting betel nut APV1 virus, comprising: preparing a sample to be detected into a solution, dripping 150-200 mu l into a sample adding hole of the colloidal gold immune test strip for detecting the betel nut APV1 virus, and placing the sample at room temperature for 5-15min, wherein positive reaction shows two red lines of a detection line and a quality control line, and the sample contains betel nut APV1 virus; the negative reaction only shows a red line on the quality control line, which indicates that the betel nut APV1 virus is not present in the sample.
Compared with the prior art, the invention has the following beneficial effects:
(1) The complex operations of extracting RNA from the plant RNA, reverse transcription, polymerase chain reaction, gel electrophoresis and the like of the user are omitted, the betel nut sample APV1 virus can be rapidly detected within 15 minutes without any instrument and equipment in the field, the waiting time of the user is effectively shortened, and the user experience is improved;
(2) The test strip has the characteristics of high specificity, rapid reaction, simple operation, low cost and the like, utilizes colloidal gold to mark antigens or antibodies as tracers, uses a nitrocellulose membrane as a carrier of the reaction, combines liquid to be tested containing free colloidal gold particles at a detection line through the combination of specific antigen and antibodies according to the capillary chromatography principle of the microporous membrane, gathers on a nitrocellulose membrane capture area to form macroscopic color development, has short whole reaction process time, high detection accuracy and wide development prospect in the detection field.
Drawings
FIG. 1 shows the results of serum immunotiter assays of mice injected with prokaryotic expression of purified APV1CP protein according to embodiments of the present invention; 1APV1CP 3A-1-4 shows the results of serum immunotiter detection of four mice, respectively.
FIG. 2 is a chart showing the results of ELISA assay titers for APV1 polyclonal antibodies provided in accordance with an embodiment of the invention; wherein, the Anti-1APV1CP3A-a and the Anti-1APV1CP3A-B are different rabbit serum samples.
Fig. 3 is a schematic diagram of a colloidal gold immunoassay test strip for detecting betel nut APV1 virus according to an embodiment of the present invention.
Fig. 4 is a result of detecting two meadow bugs and betel nut yellow disease-like leaves by using a colloidal gold immune test strip for detecting betel nut APV1 virus prepared by using an APV1 monoclonal antibody according to an embodiment of the present invention.
Fig. 5 is a result of detecting betel nut yellow disease-like leaves by using a colloidal gold immune test strip for detecting betel nut APV1 virus prepared by using an APV1 polyclonal antibody according to an embodiment of the present invention.
Reference numerals:
1. a sample pad; 2. a colloidal gold bonding pad; 3. a nitrocellulose membrane; 4. a water absorbing pad; 5. a PVC bottom plate; 6. a test line; 7. and a quality control line.
Detailed Description
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, like modules are denoted by like reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, a detailed description thereof will not be repeated.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.
Example 1
The embodiment provides a preparation method of an APV1 monoclonal antibody, which comprises the following specific steps:
1. preparation of purified APV1CP proteins
Constructing a pET30a prokaryotic expression vector pET30a-CP of APV1 virus coat protein (CP protein), and then transforming BL21 escherichia coli strain; selecting a monoclonal from the transformed plate, adding the monoclonal into 1.5ml LB liquid medium with corresponding resistance, and culturing for 9h at 37 ℃ and 200 rpm; transferring the cultured bacterial liquid into 1000ml of LB liquid medium with corresponding resistance, culturing at 37 ℃ and 200rpm until OD=0.6-0.8, and inducing with 0.5mM isopropyl thiogalactoside (IPTG, strong inducer) at 37 ℃ for 4h; and then centrifuged at 8000rpm for 6min. The supernatant was discarded, and the cells were blown off with 20-30ml of 10mM Tris-HCl (pH=8.0) solution and sonicated (500W, 180 times, 5s each time, 5s apart); taking 100 μl of the ultrasonic bacterial suspension, centrifuging at 12000rpm for 10min, and taking 50 μl of supernatant to another EP tube for standby; after the supernatant was completely removed, the pellet was blown off with 50. Mu.l of 10mM Tris-HCl (pH 8.0); performing SDS-PAGE electrophoresis detection on the supernatant and the resuspended sediment respectively; the results show that the expression product exists in the form of insoluble inclusion bodies in the precipitate after ultrasonic cleavage, and the pET30a-CP protein is proved to be expressed in the form of inclusion bodies in the recombinant strain; APV1CP protein is then obtained by protein purification.
2. Immunization of animals
The APV1CP protein is used for subcutaneously immunizing 4 SPF BALB/c female mice for the first time according to the amount of 60ug protein/mouse, and then carrying out 3 times of booster immunization, wherein the immunization amount is 30ug protein/mouse; after the immune titer was detected, mice No. 4 with high titer were selected for cell fusion experiments (as shown in fig. 1).
3. Preparation of APV1 monoclonal antibodies
The preparation method comprises the following steps:
(1) The sp2/0 cells (mouse myeloma cells) with good state are gently blown off from the wall of a culture flask and sucked into a 50ml centrifuge tube;
(2) Collecting blood from the eyeballs of mice, pulling the neck, killing the mice, and soaking the mice in 75% alcohol for 5min;
(3) Pouring a small amount of serum-free IMDM complete culture medium into a plate, and placing a cell sieve and an inner core of a syringe into the plate; taking down spleen of the mouse with scissors and forceps, and placing the spleen on a cell sieve; lightly grinding the spleen with the inner core of a syringe, sucking the ground cells into a centrifuge tube filled with sp2/0, and centrifuging at 1500rad/min for 5min;
(4) Taking down thymus of the mice with scissors and forceps, and grinding; adding the crushed thymic cells into a 15ml centrifuge tube, adding 2ml of myeloma cell selective medium (HAT) medium and 2ml of HT (hypoxanthine and thymus additive) medium, and placing in a incubator for standby;
(5) The centrifuged cells were carefully and gently blown up with serum-free IMDM and centrifuged (1500 rad/min,5 min);
(6) Pouring out the centrifuged cell supernatant as much as possible; beating the bottom of the centrifugal tube to fully suspend cells, putting the centrifugal tube into warm water at 37 ℃, slowly adding 1ml of PEG within 1 minute, and standing in the warm water for 1 minute after the addition is finished; then slowly adding 2ml of serum-free IMDM within 2min, slowly adding 8ml of serum-free IMDM within 2min, and centrifuging at 1000rad/min for 5min;
(7) Pouring out the supernatant, adding 10ml of serum, carefully blowing the cells evenly, and pouring the cells into the prepared thymus cells; then 25ml of sterilized semi-solid culture medium is added and fully and uniformly mixed; then evenly pouring the cells into 30 cell culture dishes, putting the cell culture dishes into a wet box, and then putting the wet box into an incubator for culture;
(8) 10 plates×93 cells were selected for monoclonal and cultured in 96-well cell culture plates (pre-plated with thymocytes, 100 ul/well);
(9) Adopting an ELISA method, and obtaining 10 positive hybridoma cell strains secreting high-titer antibodies through secondary screening;
(10) Finally inoculating the hybridoma cells into the abdominal cavity of the mouse for proliferation, and purifying the ascites to obtain the APV1 monoclonal antibody.
Example 2
The present example provides a method for preparing an APV1 polyclonal antibody, comprising the following steps:
1. isolation and purification of APV1 virus
The steps of separating and purifying APV1 virus are as follows:
(1) Cutting tobacco sample (80-100 g) infected with APV1 virus with scissors, and grinding into powder with liquid nitrogen;
(2) Immediately adding extraction buffer (0.1M-Tris-HCl, 0.5% (v/v) Na to 5 times the volume of the ground sample 2 SO 3 0.5% (v/v) 2-mercaptoethanol, pH 7.4) and stirring on ice;
(3) Filtering the uniformly mixed sample with gauze (three layers);
(4) Adding 2% (v/v) Triton X-100, stirring on ice for 2h, centrifuging 10000g for 10min;
(5) The supernatant was aspirated into a super-isolation tube, 40ml of supernatant per tube, and 2ml of TE (10 mM-Tris-HCl,1mM-EDTA,20% -sucrose (now added), pH 7.4) was added thereto, and centrifuged at 93000g for 1 hour;
(6) Discarding the supernatant, re-suspending the precipitate with 2 ml/tube TE, mixing, and standing in a refrigerator at 4deg.C overnight;
(7) Adding 40ul Triton X-100 into each tube, pouring into a small beaker, placing on ice, stirring for 2 hours, centrifuging 1000g for 10min;
(8) The supernatant was left and centrifuged at 4ml TE,93000g for 2h per tube;
(9) Removing supernatant, adding 3ml TE into each tube, mixing, and standing in a refrigerator at 4deg.C overnight;
(10) Leaving supernatant, centrifuging for 10min at 8000 g;
(11) Leaving supernatant, adding 25% of a cross solution (accounting for about 40% of the total volume of the centrifuge tube), adding the supernatant, and centrifuging for 5h at 14000 g;
(12) The supernatant was discarded to obtain APV1 virus particles, which were used as antigens.
2. Preparation of APV1 polyclonal antibodies
Injecting the separated and purified APV1 virus as antigen into New Zealand white rabbits, and extracting serum to prepare APV1 polyclonal antibody; the preparation process of the antibody is as follows:
1. immunization
The number of the rabbits used for immunization is about 2.0kg of New Zealand white rabbits; taking negative serum from the auricular vein before immunization; taking 400ug or 200ug immunogen (separated and purified APV1 virus), diluting to 200-500ul by using normal saline, and adding an equal volume of Freund's adjuvant (Freund's complete adjuvant for primary immunization and Freund's incomplete adjuvant for booster immunization); mixing the solution and the adjuvant uniformly by using a mixing instrument to form water-in-oil; the well mixed immunogen is subjected to subcutaneous injection for immunization at the back, and 8-10 spots are injected.
2. Negative blood is taken from the auricular vein
Wiping the ear vein of the rabbit with 75% alcohol until the vein is fully dilated; inserting a syringe into a vein to extract 1-2ml of negative blood; standing whole blood at room temperature for 30min-120min, centrifuging at 5000rpm for 10min, and collecting serum.
3. Carotid artery positive blood
Rabbits (sodium pentobarbital, 30mg/kg, all by intravenous, intraperitoneal, intramuscular injection) were anesthetized with anesthetic. After the rabbit is anesthetized, fixing the rabbit by a fixing frame; cutting the outer fur of the neck by using a surgical instrument, finding the carotid artery below the side surface of the trachea, clamping the arterial vessel by using a hemostatic forceps, cutting off and bleeding, collecting, centrifuging at 5000rpm for 10min, and collecting serum after two times of centrifugation, thus obtaining the APV1 polyclonal antibody.
4. ELISA titer assay
(1) Reagent preparation
Coating liquid: sodium carbonate-sodium bicarbonate buffer, ph9.6;
PBS buffer pH7.4;
sealing liquid: PBS containing 2% nonfat dry milk;
washing liquid: PBS-T (0.05% Tween, PBS);
color development liquid 1%A liquid+10% B liquid (A liquid: DMSO containing 1% TMB; B liquid: 0.1% H) 2 O 2 Is added to the solution of (2) in citric acid buffer solution);
stop solution: 2M sulfuric acid;
and (2) secondary antibody: goat anti-rabbit IgG/HRP;
(2) Experimental procedure
a. Diluting the antigen (isolated and purified APV1 virus) with a coating solution to a final concentration of 2ug/ml,100 ul/well, 4℃overnight; washing with washing liquid for 2 times;
b. blocking with blocking solution, 200 ul/well, incubating in incubator at 37deg.C for 2h; then washing with washing liquid for 1 time;
c. multiple antisera were diluted 2-fold from 200-fold (using PBS), blank (blank) PBS, negative (negative) 200-fold negative serum (using PBS); each sample was 100 ul/well and incubated in a 37℃incubator for 1h; washing with washing liquid for 3 times;
d. adding PBS to dilute 20000 times of secondary antibody, 100 ul/hole, incubator at 37 ℃ for 1h; taking out, and washing with washing liquid for 3 times;
e. developing color with a developing solution of 100 ul/hole for 5-15min;
f. adding 50ul of stop solution into each hole for stopping;
g. measuring light absorption values of dual wavelengths (450 nm and 630 nm), recording and storing data, and performing graph analysis; the titer was 102400 (FIG. 2) at a dilution factor corresponding to a maximum OD of 1/2.
Example 3
The present embodiment provides a method for preparing a colloidal gold-labeled APV1 antibody solution, specifically comprising:
1. preparing a colloidal gold particle solution by using a physical method and a chemical method;
the specific method comprises the following steps:
(1) 100.0mL of 0.01% (w/v) chloroauric acid solution was placed in a conical flask, placed on a stirrer, and stirred at 500rpm for 10min at room temperature;
(2) Then heating while stirring to boiling, rapidly adding 1mL1% trisodium citrate under high-speed stirring, reducing stirring speed after the color of the solution becomes purplish red, continuing stirring for 30min, and stopping heating;
(3) Cooling to room temperature, fixing volume to 100mL with deionized water, filtering with 0.22 μm microporous membrane, and storing filtrate (colloidal gold granule solution) at 4deg.C.
The average size of the prepared colloidal gold particles is 40+/-2 nm, and the colloidal gold particle solution with uniform shape and size and 40nm diameter is preferable.
2. Preparation of colloidal gold-labeled APV1 antibody solution
The specific method comprises the following steps:
(1) Taking 10ml of colloidal gold particle solution, using 0.02mol/l K 2 CO 3 The pH of the solution was adjusted to 8.0;
(2) Stirring the colloidal gold particle solution, dropwise adding the purified APV1 virus monoclonal or APV1 virus polyclonal antibody solution (40 ul,1 mg/ml) prepared in example 1 or example 2 while stirring, and stirring for 20min;
(3) 2.5ml of 20mg/ml polyethylene glycol 2000 (PEG 2000) was added dropwise with stirring, and stirring was continued for 20min;
(4) Centrifuging at 7830r/min at 4deg.C for 40min;
(5) The supernatant was discarded, and the pellet was resuspended in 0.5ml of PBS buffer, pH7.4, containing 2% BSA, and filtered through a 0.22 μm sterile filter to obtain a colloidal gold-labeled APV1 antibody solution, which was stored at 4 ℃.
Example 4
The embodiment provides a colloidal gold immune test strip for detecting betel nut APV1 virus, as shown in fig. 3, the colloidal gold test strip mainly comprises a sample pad 1, a colloidal gold binding pad 2, a nitrocellulose membrane 3 (NC membrane), a water absorbing pad 4 and a PVC bottom plate 5;
the preparation method comprises the following steps:
s1, treating a colloidal gold bonding pad: firstly, treating a colloidal gold binding pad 2 by using PBS buffer solution containing 1% of sucrose and 1% of BSA, and drying at 37 ℃;
s2, preparing a gold mark pad: drying the colloidal gold bonding pad 2, diluting the colloidal gold-labeled APV1 antibody solution by 4 times, spraying the diluted solution on the colloidal gold bonding pad by using a film spraying instrument according to a spraying amount (spraying rate) of 1.0 mu L/cm, and drying the solution in a drying oven at 37 ℃ for 2 hours;
s3, preparing a detection line and a quality control line: the nitrocellulose membrane 3 (NC membrane) part includes a detection line 6 (T line) and a quality control line 7 (C line); spraying a mouse anti-APV 1 monoclonal antibody (1 mg/ml) onto the nitrocellulose membrane 3 with a spraying amount of 1.0 mu L/cm by using a film spraying instrument to form a T line, spraying a sheep anti-mouse IgG antibody (1 mg/ml) onto the nitrocellulose membrane 3 with a spraying amount of 1.0 mu L/cm to form a C line, and drying the nitrocellulose membrane 3 in a blast drying oven at 37 ℃ for 8 hours;
s4, assembling a test strip: the dried nitrocellulose membrane 3 is stuck to the middle section of the surface of the PVC bottom plate 5, a water absorption pad 4 and a gold mark pad are respectively stuck to the two ends of the nitrocellulose membrane 3, the water absorption pad 4 and the gold mark pad are overlapped above the nitrocellulose membrane 3 by about 2mm, a sample pad is stuck to the gold mark pad, after assembly, a test strip with the width of 4mm is cut, and the test strip can be used after being mounted with a clamping shell;
the length of the colloidal gold bonding pad 2 is 8mm;
the length of the nitrocellulose membrane 3 is 14mm;
the length of the water absorption pad 4 is 32mm, and the material is water absorption filter paper;
the distance between the detection line 6 and the quality control line 7 is about 7mm;
the colloidal gold-labeled APV1 antibody solution is colloidal gold-labeled APV1 virus monoclonal antibody solution or colloidal gold-labeled APV1 virus polyclonal antibody solution.
Example 5
Field detection application of colloidal gold immune test strip for detecting betel nut APV1 virus
1. Colloidal gold immune test strip prepared by colloidal gold marked APV1 virus monoclonal antibody solution
Collecting 3 parts of double-strip mealy bugs samples, 1 plant of healthy betel nut leaves and 3 plant of betel nut yellow disease leaves from the field, adding 2mL of 0.01mol/l PBS buffer solution, grinding into homogenate, transferring the homogenate into a 1.5mL centrifuge tube, standing for 5min for clarification; taking 1 colloidal gold immune test strip, flatly placing, sucking the supernatant liquid of the sample by using a sample suction pipe, dripping 3 drops (about 150-200 mu l) into a sample adding hole of the test strip, and standing at room temperature for 5min to observe the result. In the results of 3 double mealy bugs, both the T line and the C line simultaneously appear obvious red stripes, and the T line stripes of leaf samples (health samples) of healthy betel nuts are not shown; the test strips T line and C line of the disease samples 1-3 have red strips at the same time, which indicates that the samples are positive to APV 1; the healthy and water control C-line showed a red band and T-line did not show a red band, indicating that the sample was APV1 negative (fig. 4). The test result simultaneously verifies that the test result of the test strip is accurate.
2. Colloidal gold immune test strip prepared by using colloidal gold marked APV1 virus polyclonal antibody solution
Sampling 3 healthy betel nut leaves and betel nut yellowing disease sample leaves 0.1g in the field, adding 2ml of 0.01mol/l PBS buffer solution, grinding into homogenate, transferring the homogenate into a 1.5ml centrifuge tube, standing for 5min, and clarifying; taking 1 colloidal gold immune test strip, flatly placing, sucking the sample supernatant by using a sample suction pipe, dripping 3 drops (about 150-200 mu l) into a test strip sample adding hole, and observing the result after 5 min. The test strips of the disease samples showed both lines T and C with a red band, indicating that these samples were both APV1 positive, the healthy samples and the water control showed lines C with a red band and the lines T with no red band, indicating that the samples were APV1 negative (FIG. 5).
The whole detection process takes 15 minutes, the whole reaction process is short in time, and the detection accuracy is high. Solves the problems of complex flow, long time and high loss of the detection method of the APV1 virus such as RT-PCR, ELISA and the like. Besides, the test strip has the characteristics of high specificity, rapid response, simple operation, low cost and the like, and has wide application prospect.
It should be appreciated that various forms of the flows shown above may be used to reorder, add, or delete steps. For example, the steps described in the present disclosure may be performed in parallel, sequentially, or in a different order, provided that the desired results of the technical solutions of the present disclosure are achieved, and are not limited herein.
The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives are possible, depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.
Claims (10)
1. The colloidal gold immune test strip for detecting betel nut APV1 virus is characterized in that: the device comprises a sample pad, a colloidal gold combination pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line;
the nitrocellulose membrane is adhered to the middle section of the surface of the PVC bottom plate, the two ends of the nitrocellulose membrane are respectively overlapped with the colloidal gold bonding pad and the water absorption pad, and the overlapped area is 2mm;
the sample pad is adhered to one end of the colloidal gold bonding pad, which is far away from the nitrocellulose membrane, and is used for dropwise adding a sample to be detected;
the colloidal gold binding pad is coated with 0.6-1 mu L of colloidal gold-labeled APV1 antibody solution, and the concentration of the colloidal gold-labeled APV1 antibody solution is 0.15-0.25 mg/mL;
the detection line is coated with a mouse anti-APV 1 monoclonal antibody, and 0.8-1.5 mu L of the mouse anti-APV 1 monoclonal antibody with the concentration of 0.8-1.2 mg/mL is coated on a test strip with the width of each centimeter;
the quality control line is coated with the goat anti-mouse IgG antibody, and the concentration of the goat anti-mouse IgG antibody is 0.8-1.2 mg/mL per centimeter of wide test strip, and the concentration of the goat anti-mouse IgG antibody is 0.8-1.5 mu L.
2. The colloidal gold immunoassay strip for detecting betel nut APV1 virus according to claim 1, wherein the assay strip is characterized in that: the distance between the detection line and the quality control line is 5-10 mm.
3. The colloidal gold immunoassay strip for detecting betel nut APV1 virus according to claim 2, wherein the assay strip is characterized in that: the length of the colloidal gold bonding pad is 7.5-8.5 mm; the length of the nitrocellulose membrane is 13.5-14.5 mm; the length of the water absorbing pad is 31-33 mm.
4. The colloidal gold immunoassay strip for detecting betel nut APV1 virus according to any one of claims 1 to 3, wherein: the colloidal gold-labeled APV1 antibody solution is colloidal gold-labeled APV1 virus monoclonal antibody solution or colloidal gold-labeled APV1 virus polyclonal antibody solution.
5. The method for preparing the colloidal gold immune test strip for detecting betel nut APV1 virus according to claim 1, which is characterized by comprising the following steps:
s1, treating a colloidal gold bonding pad: firstly, treating a colloidal gold binding pad by using PBS buffer solution containing 0.5-1.5% of sucrose and 0.5-1.5% of BSA, and drying;
s2, preparing a gold mark pad: spraying the APV1 antibody solution marked by the colloidal gold on a dried colloidal gold bonding pad, and drying for 1-3 hours at the temperature of 35-38 ℃ to obtain a gold-marked pad;
s3, preparing a detection line and a quality control line: spraying a mouse anti-APV 1 monoclonal antibody onto a nitrocellulose membrane to serve as a detection line, spraying a sheep anti-mouse IgG antibody onto the nitrocellulose membrane to serve as a quality control line, and drying the nitrocellulose membrane at 35-38 ℃ for 6-10 h;
s4, assembling a test strip: the dried nitrocellulose membrane is stuck to the middle section of the surface of the PVC bottom plate, and then a water absorption pad and a gold mark pad are respectively stuck to the two ends of the nitrocellulose membrane, wherein the water absorption pad and the gold mark pad are overlapped above the nitrocellulose membrane by 2mm; the sample pad is adhered to one end of the gold mark pad far away from the nitrocellulose membrane; cutting into test strips with the width of 3.5-4.5 mm after assembly, mounting a clamping shell, sealing, drying and preserving.
6. The method for preparing the colloidal gold immunoassay test strip for detecting betel nut APV1 virus according to claim 5, wherein the method is characterized in that: the colloidal gold-labeled APV1 antibody solution in the step S2 is colloidal gold-labeled APV1 virus monoclonal antibody solution, and the specific preparation method comprises the following steps:
s201, taking 8-12 ml of colloidal gold particle solution, and using K with the concentration of 0.02mol/l 2 CO 3 The pH of the solution is adjusted to 8;
s202, stirring the colloidal gold particle solution, dropwise adding 35-45 ul of purified APV1 virus monoclonal antibody solution with the concentration of 0.5-1.5 mg/ml while stirring, and continuing stirring for 15-25 min;
s203, adding 2-3 ml of polyethylene glycol 2000 with the concentration of 15-25 mg/ml dropwise, and continuously stirring for 15-25 min;
s204, centrifuging the solution obtained in the step S203 at 7000-8000 r/min for 30-50 min at 0-4 ℃; the supernatant was discarded, and the pellet was resuspended in PBS buffer containing 1.5-2.5% BSA and filtered at 0.22 μm to give a colloidal gold-labeled APV1 virus monoclonal antibody solution.
7. The method for preparing the colloidal gold immune test strip for detecting betel nut APV1 virus according to claim 6, which is characterized in that: the particle size of the colloidal gold particles in the colloidal gold particle solution is 38-42 nm; the colloidal gold particle solution is prepared by adding trisodium citrate into a boiling chloroauric acid solution.
8. The method for preparing the colloidal gold immune test strip for detecting betel nut APV1 virus according to claim 7, which is characterized in that: the concentration of the APV1 virus monoclonal antibody solution purified in the step S202 is 1mg/ml.
9. The method for preparing the colloidal gold immune test strip for detecting betel nut APV1 virus according to claim 8, which is characterized in that: the concentration of the polyethylene glycol 2000 in the step S203 is 20mg/ml, and the continuous stirring time is 20min.
10. The method for detecting the betel nut APV1 virus is characterized by comprising the following steps of: preparing a sample to be detected into a solution, dripping 150-200 μl into a sample-adding hole of the colloidal gold immune test strip for detecting the betel nut APV1 virus according to claim 1, and placing the sample at room temperature for 5-15min, wherein positive reaction shows two red lines of a detection line and a quality control line, and the sample contains betel nut APV1 virus; the negative reaction only shows a red line on the quality control line, which indicates that the betel nut APV1 virus is not present in the sample.
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| CN116676423B (en) * | 2023-06-28 | 2024-04-09 | 中国热带农业科学院热带生物技术研究所 | APV1 detection primer set and application |
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