CN119064578A - A colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA and a preparation method thereof - Google Patents
A colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA and a preparation method thereof Download PDFInfo
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- 241001135549 Porcine epidemic diarrhea virus Species 0.000 title claims abstract description 111
- 238000012360 testing method Methods 0.000 title claims abstract description 63
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 157
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 56
- 239000012528 membrane Substances 0.000 claims abstract description 56
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 56
- 238000003908 quality control method Methods 0.000 claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 43
- 239000011358 absorbing material Substances 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000003317 immunochromatography Methods 0.000 claims abstract description 11
- 239000000523 sample Substances 0.000 claims description 91
- 241000283707 Capra Species 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 6
- 230000001900 immune effect Effects 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 241000700605 Viruses Species 0.000 abstract description 30
- 230000008569 process Effects 0.000 abstract description 4
- 208000003322 Coinfection Diseases 0.000 abstract description 3
- 239000003053 toxin Substances 0.000 description 10
- 231100000765 toxin Toxicity 0.000 description 10
- 230000006872 improvement Effects 0.000 description 8
- 238000003757 reverse transcription PCR Methods 0.000 description 8
- 230000035945 sensitivity Effects 0.000 description 8
- 244000052769 pathogen Species 0.000 description 6
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 238000007397 LAMP assay Methods 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 239000000783 alginic acid Substances 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 239000002274 desiccant Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 208000005156 Dehydration Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241001137860 Rotavirus A Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
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- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000011140 intestinal infectious disease Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
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- 239000002245 particle Substances 0.000 description 1
- 239000012221 photothermal agent Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 239000002534 radiation-sensitizing agent Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000009589 serological test Methods 0.000 description 1
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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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- 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
- G01N33/577—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
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Abstract
The application discloses a colloidal gold immunochromatography test strip for simultaneously detecting PEDV and PoRVA and a preparation method thereof, belonging to the technical field of detection of PEDV and PoRVA. The application comprises a sample pad, a bonding pad, a nitrocellulose membrane, a first detection line, a second detection line and a quality control line, wherein the nitrocellulose membrane is provided with the first detection line, the second detection line and the quality control line, the first detection line is coated with a first specific antibody, the second detection line is coated with a second specific antibody, a water absorbing material, the sample pad, the bonding pad, the nitrocellulose membrane and the water absorbing material are sequentially arranged and connected, wherein one end of the sample pad connected with the bonding pad is positioned right above one end of the bonding pad, one end of the bonding pad connected with the nitrocellulose membrane is positioned right above one end of the nitrocellulose membrane, and one end of the nitrocellulose membrane connected with the water absorbing material is positioned right below the water absorbing material. The application can detect PEDV and PoRVA virus at the same time, and avoids the phenomenon of PEDV or PoRVA false positive caused by mixed infection of PEDV and PoRVA in the detection process.
Description
Technical Field
The invention belongs to the technical field of preparation of PEDV and PoRVA, and particularly relates to a colloidal gold immunochromatography test strip for simultaneously detecting PEDV and PoRVA and a preparation method thereof.
Background
Porcine epidemic diarrhea virus (Porcine EPIDEMIC DIARRHEA VIR mu s, PEDV) and Porcine group A rotavirus (Porcine gro mu p A rotavir mu s, poRVa) are two main pathogens causing Porcine diarrhea, and infection of the Porcine epidemic diarrhea virus can cause the Porcine to have high-contact intestinal infectious diseases mainly comprising vomiting, diarrhea and dehydration, and the Porcine epidemic diarrhea virus has the characteristics of high morbidity and mortality.
The main diagnostic methods for these two pathogens are currently serological and molecular biological assays. Serological tests mainly include immunochromatography and Enzyme-linked immunosorbent assay (Enzyme-LINKED IMM. Mu. nosorbent assay, ELISA). Molecular biological assays typically include methods such as virus isolation, polymerase chain reaction (Polymerase chain reaction, PCR), real-time fluorescent quantitative PCR (Q. Mu. ANTITATIVE REAL-time PCR, RT-qPCR), and Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP). These methods play a very important role in the prevention and control of PEDV and PoRVA. Although the detection results of the methods are accurate, the methods have great dependence on equipment and professionals, and certain timeliness is required in the application of basic farms, and the immunochromatography technology can realize on-site rapid detection, so that the unique advantages of the method for detecting clinical samples are more obvious.
In recent years, an immunochromatography technology for separately detecting PEDV or PoRVA has appeared, but since the disease symptoms of PEDV and PoRVA infected pigs are similar, and mixed infection of PEDV and PoRVA often occurs in many areas of the whole country, the pathogen is difficult to effectively determine only by clinical experience, and targeted prevention and treatment measures are difficult to formulate. However, the current test strip based on the immunochromatography principle cannot detect PEDV and PoRVA simultaneously, and the problem of false positive caused by mutual infection of the test strips for jointly detecting multiple pathogens often occurs, which is not solved yet.
Disclosure of Invention
In order to achieve the above purpose, the invention adopts the following technical scheme:
The colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA comprises a sample pad, a binding pad, a nitrocellulose membrane, a first detection line, a second detection line and a quality control line, wherein the nitrocellulose membrane is provided with the first detection line, the second detection line and the quality control line, the first detection line is coated with a first specific antibody, the second detection line is coated with a second specific antibody, a water absorbing material, the sample pad, the binding pad, the nitrocellulose membrane and the water absorbing material are sequentially connected in an arrayed mode, wherein one end of the sample pad, which is connected with the binding pad, is located right above one end of the binding pad, which is connected with the nitrocellulose membrane, is located right above one end of the nitrocellulose membrane, which is connected with the water absorbing material, is located right below the water absorbing material, the quality control line is close to the water absorbing material, and the first detection line and the second detection line are located between the quality control line and the binding pad.
As a further improvement of the technical scheme, the binding pad further comprises an immune probe, and the immune probe is arranged at one end of the binding pad, which is close to the nitrocellulose membrane.
As a further improvement of the technical scheme, the immune probe is made of AuNPs-mAb.
As a further improvement of the technical scheme, the first specific antibody is PEDV-mAb.
As a further improvement of the technical scheme, the second specific antibody is PoRVA-mAb.
As a further improvement of the technical scheme, the quality control line is coated with goat anti-mouse IgG polyclonal antibody.
As a further improvement of the technical scheme, the colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA also comprises a bottom plate, wherein the sample pad, the binding pad, the nitrocellulose membrane and the water absorbing material are all arranged on the surface of the bottom plate.
The application also provides a preparation method of the colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA, which comprises the following steps:
Spraying an immune probe on a bonding pad;
Spraying a first specific antibody, a second specific antibody and a goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane to form a first detection line, a second detection line and a quality control line;
Step three, closing the other protein binding sites of the nitrocellulose membrane, and then drying for later use;
And fourthly, sequentially adhering a sample pad, a bonding pad, a nitrocellulose membrane and a water absorbing material to the top surface of the bottom plate, and sequentially arranging and connecting the sample pad, the bonding pad, the nitrocellulose membrane and the water absorbing material, wherein one end of the sample pad connected with the bonding pad is positioned right above one end of the bonding pad, one end of the bonding pad connected with the nitrocellulose membrane is positioned right above one end of the nitrocellulose membrane, and one end of the nitrocellulose membrane connected with the water absorbing material is positioned right below the water absorbing material to obtain the test paper strip.
As a further improvement of the technical solution, in the fourth step, the quality control line is close to the water absorbing material, and the first detection line and the second detection line are located between the quality control line and the bonding pad.
As a further improvement of the technical solution, the immunological probe is located at an end of the binding pad near the nitrocellulose membrane.
Compared with the prior art, the invention has the following beneficial effects:
1. The test strip has strong specificity and high sensitivity, can detect PEDV and PoRVA viruses simultaneously, and avoids the phenomenon of PEDV or PoRVA false positive caused by mixed infection of PEDV and PoRVA in the detection process.
2. The test strip disclosed by the application does not need to adopt a special container or instrument, is convenient to carry, is easy to operate, is short in detection time consumption and is low in detection cost, and detection is carried out at any time and any place.
Drawings
FIG. 1 is a schematic diagram showing the structure of a colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA according to the present invention;
FIG. 2 is a schematic diagram of a second embodiment of the present invention;
FIG. 3 is a diagram of a use process according to the present invention;
FIG. 4 is a second view of the use process according to the present invention;
Reference numerals 1-sample pad, 2-conjugate pad, 21-immuno probe, 3-nitrocellulose membrane, 31-first detection line, 32-second detection line, 33-quality control line, 4-water absorbent material, 5-base plate,
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental procedure for specific adjustment was not noted in the examples below and was selected according to the commercial instructions. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention. Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
Example 1:
As shown in FIGS. 1 to 4, the colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA comprises a sample pad 1, a bonding pad 2, a nitrocellulose membrane 3 and a water absorbing material 4, wherein a first detection line 31, a second detection line 32 and a quality control line 33 are arranged on the nitrocellulose membrane 3, a first specific antibody is coated on the first detection line, a second specific antibody is coated on the second detection line 32, the first specific antibody is PEDV-mAb, the second specific antibody is PoRVA-mAb2, a goat anti-mouse IgG polyclonal antibody is coated on the quality control line 33, the length direction of the first detection line 31, the second detection line 32 and the quality control line 33 is consistent with the width direction of the nitrocellulose membrane 3, the sample pad 1, the bonding pad 2, the nitrocellulose membrane 3 and the water absorbing material 4 are sequentially connected, one end of the sample pad 1, the bonding pad 2 is positioned right above one end of the nitrocellulose membrane 3, one end of the bonding pad 2, which is connected with the nitrocellulose membrane 3, the other end of the bonding pad 2 is positioned right above one end of the nitrocellulose membrane 3, the goat anti-mouse IgG polyclonal antibody is coated on the quality control line 33, the first detection line 32 is positioned between the water absorbing material and the first detection line 33 and the water absorbing material 33, the first detection line 32 is positioned between the first detection line and the water absorbing material 33 and the first detection line is positioned between the first detection line and the water absorbing material 33 and the first detection line and the water absorbing material 32.
As shown in fig. 1 to 4, the preferred binding pad 2 further includes an immune probe 21, wherein the immune probe 21 is disposed at one end of the binding pad 2 near the nitrocellulose membrane 3, the immune probe 21 is made of AuNPs-mAb, wherein AuNPs is nano-gold particles with a diameter in a range of 1-100nm, which are also called gold nanoparticles, have high electron density, dielectric property and catalysis, can be combined with various biological macromolecules, do not affect the biological activity, and can be used as an immune marker, a tumor imaging material, a drug delivery system, a radiosensitizer, a photothermal agent and the like for disease diagnosis and treatment, mAb is a monoclonal antibody, and AuNPs-mAb is made of gold nanoparticles.
As shown in fig. 1 to 4, the present application preferably further comprises a base plate 5, and the sample pad 1, the bonding pad 2, the nitrocellulose membrane 3 and the water absorbing material 4 are all disposed on the plate surface of the base plate 5 to support and connect the sample pad 1, the bonding pad 2, the nitrocellulose membrane 3 and the water absorbing material 4, and the base plate 5 may be a PVC base plate.
The colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA can be prepared according to the following method, and comprises the following steps:
step one, preparing an immune probe 21AuNPs-mAb, and spraying the immune probe 21 on one end of a binding pad 2;
Spraying a first specific antibody PEDV-mAb, a second specific antibody PoRVA-mAb2 and a goat anti-mouse IgG polyclonal antibody on the nitrocellulose membrane 3 to form a first detection line 31, a second detection line 32 and a quality control line 33
Step three, closing the rest protein binding sites of the nitrocellulose membrane 3, and then drying for later use;
And fourthly, sequentially adhering a sample pad 1, a bonding pad 2, a nitrocellulose membrane 3 and a water absorbing material 4 on the top surface of the bottom plate 5, sequentially arranging and connecting the sample pad 1, the bonding pad 2, the nitrocellulose membrane 3 and the water absorbing material 4, wherein one end of the sample pad 1 connected with the bonding pad 2 is positioned right above one end of the bonding pad 2, one end of the bonding pad 2 connected with the nitrocellulose membrane 3 is positioned right above one end of the nitrocellulose membrane 3, an immune probe 21 is positioned at one end of the bonding pad 2 close to the nitrocellulose membrane 3, one end of the nitrocellulose membrane 3 connected with the water absorbing material 4 is positioned right below the water absorbing material 4, a quality control line 33 is positioned close to the water absorbing material 4, a first detection line 31 and a second detection line 32 are positioned between the quality control line 33 and the bonding pad 2, and then cutting to obtain a test strip.
The application provides a detection principle for simultaneously detecting PEDV and PoRVA colloidal gold immunochromatographic test strips, which comprises the following specific steps:
The sample to be detected (sample dilution containing pig manure) is dripped onto the sample pad 1, the liquid moves forwards under capillary action to reach the binding pad 2, if virus PEDV or PoRVA virus exists in the liquid, specific antigen-antibody interaction occurs with the immune probe 21 on the binding pad 2 to form an AuNPs-mAb-virus complex, the complex continues to flow to the direction of absorbent paper, then is captured and trapped by the first specific antibody on the first detection line 31 and/or the second specific antibody on the second detection line 32, and is accumulated on the first detection line 31 and/or the second detection line 32 to form an AuNPs-labeled mAb-virus-mAb virus complex, the first detection line 31 is positive if the virus PEDV and/or the second detection line 32 are colored, the first detection line 31 is colored, the first detection line is negative if the virus is not colored, the second detection line 32 is colored, the PoRVA of the sample is positive, the negative if the quality control line 33 captures and the excessive virus complex or the immune probe is captured and the quality control line is colored, and the quality control line is not colored, if the test strip of the sample is required to be successfully colored, and the test strip is not shown in fig. 2 shows that the sample is not colored.
Example 2:
the difference compared to example 1 is that the second specific antibody is PoRVA-mAb1.
Example 3:
Application method of colloidal gold immunochromatography test strip for simultaneously detecting PEDV and PoRVA
Sample treatment, namely collecting pig manure by using a sample collecting cotton swab, inserting the sampled cotton swab into a sample buffer tube, stirring for 10-15 seconds, taking out, turning over for 8-10 times, standing for 1 minute, and precipitating an undissolved sample, wherein the treated sample is a sample to be detected.
Sample detection, namely detecting by adopting the test strip in the embodiment 1, sucking a sample to be detected by using a dropper, enabling the dropper to be vertical, gradually dripping the sample to be detected into the sample pad 1 of the test strip in the embodiment 1, starting timing, standing for 10-20 minutes, and observing the result.
As shown in FIG. 2, the result is determined that if the first detection line 31 and the second detection line 32 of the sample to be tested are color-free and color-free, and a red stripe appears on the quality control line 33, then the sample is determined to be negative, namely the sample does not contain PEDV and PoRVA virus, if the first detection line 31 of the sample to be tested is color-changed and the second detection line 32 is color-free and color-free, and a red stripe appears on the quality control line 33, then the sample is determined to be positive, poRVA is negative, namely the sample contains PEDV virus and PoRVA virus, if the second detection line 32 of the sample to be tested is color-changed and the red stripe appears, the first detection line 31 is color-free and color-free, and the quality control line 33 is color-free, then the sample is determined to be positive PoRVA, the sample is determined to be negative, namely the sample does not contain PEDV virus, and PoRVA virus appears on the quality control line 33, and the sample is determined to be positive, namely the sample PoRVA and the quality control line is color-free, and the sample is not effective.
If the first detection line 31 and the second detection line 32 of the sample to be tested are not colored, and a red stripe appears on the quality control line 33, the sample is judged to be a positive sample, namely, no PEDV and PoRVA virus exist in the sample, if the detection line of the sample to be tested is not colored, and a red stripe appears on the quality control line, the sample is judged to be a positive sample, namely, the concentration of the alginic acid in the sample is greater than or equal to 20ng/mL, if the detection line of the sample to be tested is consistent with the detection line of the negative blank test strip, and a red stripe appears on the quality control line, the sample is judged to be a negative sample, namely, no alginic acid is contained in the sample, and if the sample is not colored on the quality control line, the test strip is invalid.
Example 4:
Sensitivity and specificity detection of colloidal gold immunochromatography test strip for simultaneously detecting PEDV and PoRVA
According to the method of example 3, the test strips of example 1 were used to detect PEDV and PoRVA respectively, PEDV and PoRVA respectively, poRVA and PEDV respectively, and PoRVA respectively, and the samples were repeated 4 times, and the RT-PCR test was performed simultaneously (the samples were tested by the RT-PCR tester) as a control, wherein the samples positive and PoRVA for PEDV and the samples positive for PEDV and negative for PEDV were each 2.5×103、1.25×102、6.25×102、3.13×102、1.56×102、7.81×101、3.90×101TICD50/mL,PoRVA and 9.5×104、4.75×104、2.38×104、1.19×104、5.94×103、2.97×103、1.48×103copies/μL,PEDV and PoRVA were each positive for PEDV, 9.5×104、4.75×104、2.38×104、1.19×104、5.94×103、2.97×103、1.48×103copies/μL; for PEDV and 2.5×103、1.25×102、6.25×102、3.13×102、1.56×102、7.81×101、3.90×101TICD50/mL,PoRVA were each positive for PEDV, and the test results were determined by visual inspection of the first test line 31 and the second test line 32, and the test results are shown in tables 1 to 4.
Tables 1 and 2, As shown in tables 4 to 6, when the toxin carrying amount is 0, the first detection line and the second detection line are not discolored, the quality control line is discolored, and a red strip appears, so that the test strip is successfully detected; detecting samples with positive PEDV and negative PEDV PoRVA, wherein when the PEDV virus carrying amount is 3.90 multiplied by 10 1TICD50/mL, the first detection line and the second detection line are not discolored, only the quality control line is discolored, a red strip appears, when the PEDV virus carrying amount is more than 7.81 multiplied by 10 1TICD50/mL, the first detection line is discolored, the red strip appears, the PEDV virus cannot be detected when the PEDV virus carrying amount is lower than 7.81 multiplied by 10 1TICD50/mL, and when the PEDV virus carrying amount is 2.5 multiplied by 10 3ICD50/mL, the second detection line is still not discolored, so that the PEDV virus carrying amount cannot influence the second detection line; when PoRVA is positive and PEDV is a negative sample, the first detection line and the second detection line are not discolored when the PoRVA virus carrying amount is 1.48 multiplied by 10 3 copies/. Mu.L, only the quality control line is discolored, a red strip appears, when the PoRVA virus carrying amount is more than 2.97 multiplied by 10 3 copies/. Mu.L, the second detection line begins to discolor, the red strip appears, the PoRVA virus carrying amount is lower than 2.97 multiplied by 10 3 copies/. Mu.L, poRVA virus cannot be detected, when the PoRVA virus carrying amount is 9.5 multiplied by 10 4 copies/. Mu.L, the first detection line remains uncolored, which shows that the PoRVA virus carrying amount does not affect the first detection line, and when the PEDV and PoRVA positive samples are detected, the PEDV virus carrying amount is 3.90 multiplied by 10 1TICD50/mL, When the virus carrying amount of PoRVA is 1.48X10. 10 3 copies/. Mu.L, the first detection line and the second detection line are not discolored, only the quality control line is discolored, a red strip appears, which indicates that after PEDV and PoRVA are mixed, the detection results of the first detection line and the second detection line are not affected, when the virus carrying amount of PEDV is above 7.81X 10 1TICD50/mL, and the virus carrying amount of PoRVA is above 2.97X 10 3 copies/. Mu.L, the first detection line and the second detection line are discolored, and the red strip appears, so that the test strip disclosed by the application has strong specificity on the detection of PEDV and PoRVA, and can detect whether PEDV and PoRVA are detected simultaneously.
As shown in tables 2 to 7, the test strips of the present application have almost the same detection results compared with RT-PCR, only have differences in detecting 3.90X10 1TICD50/mL of PEDV virus, the test strips of the present application are all negative in detection results, the RT-PCR is negative in detection results three times, and once positive in detection results, and the main reason is that the RT-PCR technology is mature, the detection accuracy of the virus is very high, and the test strip of the present application has the detection results similar to the RT-PCR detection results, and the sensitivity of the test strip of the present application is high, and the virus carrying amount of PEDV virus can be 7.81X 10 1TICD50/mL, and the virus carrying amount of PoRVA virus is 2.97X10 3 copies/μL and other low virus carrying amount samples are detected.
Table 1 test strips for detection of negative samples for both PEDV and PoRVA
Table 2 test strips were tested for detection of samples positive for PEDV and negative for PoRVA
TABLE 3 detection results of samples positive for PEDV and negative for PoRVA by RT-PCR
Table 4 test strips positive for PoRVA and negative for PEDV
TABLE 5 detection results of RT-PCR positive for PoRVA and PEDV negative samples
Table 6 test strips for detection results of positive samples for both PEDV and PoRVA
TABLE 7RT-PCR detection results on samples positive for both PEDV and PoRVA
In the table, "+", and "-" respectively indicate that the test result of the test strip is positive and negative.
Example 5:
Sensitivity test of second specific antibodies PoRVA-mAb1 and PoRVA-mAb2
According to the method of example 3, the test was performed using the test strip of example 2, and the test results were determined by visually observing the first detection line 31 and the second detection line 32 for samples (distilled water) negative for PEDV and PoRVA, positive for PEDV and PoRVA, positive for PEDV and negative for PEDV, and positive for PEDV and PoRVA, respectively, for samples positive for PEDV and PoRVA, positive for PEDV and negative for PEDV, for sample 2.5×103、1.25×102、6.25×102、3.13×102、1.56×102、7.81×101、3.90×101TICD50/mL,PoRVA, positive for PEDV, for sample PoRVA, positive for both 9.5×104、4.75×104、2.38×104、1.19×104、5.94×103、2.97×103、1.48×103copies/μL,PEDV and PoRVA, and for sample 9.5×104、4.75×104、2.38×104、1.19×104、5.94×103、2.97×103、1.48×103copies/μL; for PEDV, for example, for 2.5×103、1.25×102、6.25×102、3.13×102、1.56×102、7.81×101、3.90×101TICD50/mL,PoRVA, respectively.
TABLE 8 detection results of negative samples for both PEDV and PoRVA
TABLE 9 detection results of samples positive for PEDV and negative for PoRVA
Table 10PoRVA shows the results of detection of positive and negative samples of PEDV
TABLE 11 detection results of samples positive for both PEDV and PoRVA
In the table, "+", and "-" respectively indicate that the test result of the test strip is positive and negative.
As shown in tables 8 to 11, when the toxin carrying amount is 0, the first detection line and the second detection line are not discolored, the quality control line is discolored, and a red strip appears, so that the test strip is successfully detected; the detection results of the samples positive for PEDV and negative for PoRVA were the same as those shown in example 4; when PoRVA is positive and PEDV is a negative sample, the detection result is stable, when PoRVA is 2.97X10. 3 copies/. Mu.L, the first detection line and the second detection line are not discolored, only the quality control line is discolored, a red strip appears, when PoRVA is 5.94X10. 3 copies/. Mu.L, the second detection line is discolored, pink strip appears, the detection sensitivity is reduced when the PoRVA is unstable and the non-discolored condition appears, when PoRVA is 1.9X10. 4 copies/. Mu.L, the second detection line appears red strip, the detection result is stable, poRVA is lower than 2.97X103 copies/. Mu.L, poRVA virus cannot be detected, when PoRVA is 9.5X10.3995 copies/. Mu.L, the first detection line is still not discolored, the PoRVA is not affected on the first detection line, when PEDV and 2 are detected, and when PEDV and PEDV is detected to be 58595. 1TICD50 mL, the detection result is stable, and PEDV is detected to be 3.9690 mL PoRVA the toxin carrying amount is 1.48X10. 10 3 copies/. Mu.L, the first detection line and the second detection line are not discolored, only the quality control line is discolored, red stripes appear, when the PEDV toxin carrying amount is 7.81X 10 1TICD50/mL, the PoRVA toxin carrying amount is 2.97X10 3 copies/. Mu.L, the first detection line is discolored and the quality control line is discolored, red stripes appear, the second detection line is still not discolored, when the PEDV toxin carrying amount is 1.56X10 2TICD50/mL, And when the PoRVA.94X10. 10 3 copies/. Mu.L of the virus loading amount is achieved, the first detection line, the second detection line and the quality control line are discolored, the first detection line and the quality control line are provided with red strips, and the second detection line is provided with pink strips, which indicates that after PEDV and PoRVA are mixed, the detection results of the first detection line and the second detection line are not affected, and the specificity is strong.
This example is a reduction in detection sensitivity for PoRVA virus compared to example 4, probably because the recognition sites of the AuNPs-mAb of the immuno-probe, the PEDV-mAb of the first detection line and the PEDV-mAb of the second detection line are identical, and during the detection, the AuNPs-mAb, the PEDV-mAb and the PEDV-mAb may compete for the same recognition site of the pathogen, resulting in a reduction in sensitivity, while the PoRVA-mAb2 and the PoRVA-mAb do not compete for the same pathogen, poRVA-mAb2 does not compete with the AuNPs-mAb and the PEDV-mAb, and thus the highest sensitivity can be achieved with PoRVA-mAb 2. It should be noted that only one antibody of PEDV, namely, only two antibodies of PEDV-mAb and PoRVA, namely, two antibodies of PoRVA-mAb and PoRVA-mAb2 are present.
Example 6:
test for simultaneously detecting stability of PEDV and PoRVA colloidal gold immunochromatography test strip
(1) The test strip manufactured in the example 1 is placed in an opaque plastic bag (desiccant is added) to be sealed, the test strip is placed in an environment of normal temperature (25 ℃) to be tested by taking standard samples of distilled water, 3.90 multiplied by 10 1TICD50/mL PEDV virus carrying amount, 2.5 multiplied by 10 3TICD50/mL PEDV virus carrying amount, 1.48 multiplied by 10 3 copies/mu L PoRVA virus carrying amount and 9.5 multiplied by 10 4 copies/mu L PoRVA virus carrying amount after 1,2, 3,4, 5 and 6 months, and stability test results (the existence of a first detection line, a second detection line and a quality control line and the definition of the strip) are observed every 3 times.
In the stability test, when a distilled water standard sample is detected, the first detection line and the second detection line are not discolored, a red strip appears, the AuNPs-mAb antibody on the bonding pad is released, the 3.90 multiplied by 10 1TICD50/mL PEDV toxin carrying amount and the 2.5 multiplied by 10 3TICD50/mL PEDV toxin carrying amount standard sample are detected, the first detection line and the quality control line are discolored, the red strip appears, the second detection line is not discolored, the detection result is met, the standard sample with the 1.48 multiplied by 10 3 copies/mu L PoRVA toxin carrying amount and the 9.5 multiplied by 10 4 copies/mu L PoRVA toxin carrying amount is detected, the second detection line and the quality control line are discolored, the red strip appears, the first detection line is not discolored, the detection result is met, and therefore, the storage condition of the PEDV and PoRVA colloidal gold immunochromatographic test strip is sealed in a light-tight plastic bag (desiccant is added), and the test strip is placed in a normal temperature (25 ℃) environment, and the validity period is at least 6 months.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. The utility model provides a detect PEDV and PoRVA colloidal gold immunochromatography test strip simultaneously which characterized in that includes:
A sample pad (1);
A bonding pad (2);
The device comprises a nitrocellulose membrane (3), wherein a first detection line (31), a second detection line (32) and a quality control line (33) are arranged on the nitrocellulose membrane (3), a first specific antibody is coated on the first detection line (31), and a second specific antibody is coated on the second detection line (32);
A water-absorbing material (4);
The sample pad (1), the bonding pad (2), the nitrocellulose membrane (3) and the water absorbing material (4) are sequentially arranged and connected, wherein one end of the sample pad (1) connected with the bonding pad (2) is located right above one end of the bonding pad (2), one end of the bonding pad (2) connected with the nitrocellulose membrane (3) is located right above one end of the nitrocellulose membrane (3), one end of the nitrocellulose membrane (3) connected with the water absorbing material (4) is located right below the water absorbing material (4), the quality control line (33) is close to the water absorbing material (4), and the first detection line (31) and the second detection line (32) are located between the quality control line (33) and the bonding pad (2).
2. The colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA according to claim 1, wherein the binding pad (2) further comprises an immunological probe (21), and the immunological probe (21) is arranged at one end of the binding pad (2) close to the nitrocellulose membrane (3).
3. The colloidal gold immunochromatographic strip for simultaneous detection of PEDV and PoRVA according to claim 2, wherein the immuno-probe (21) is made of AuNPs-mAb.
4. The simultaneous assay PEDV and PoRVA colloidal gold immunochromatographic strip of claim 1, wherein the first specific antibody is PEDV-mAb.
5. The simultaneous detection PEDV and PoRVA colloidal gold immunochromatographic strip of claim 1, wherein the second specific antibody is PoRVA-mAb2.
6. The colloidal gold immunochromatographic strip for simultaneously detecting PEDV and PoRVA according to claim 1, wherein the quality control line (33) is coated with goat anti-mouse IgG polyclonal antibody.
7. The colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA according to claim 1, further comprising a bottom plate (5), wherein the sample pad (1), the binding pad (2), the nitrocellulose membrane (3) and the water absorbing material (4) are all arranged on the surface of the bottom plate (5).
8. The preparation method of the colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA is characterized by comprising the following steps of:
Spraying an immune probe (21) on a bonding pad (2);
Spraying a first specific antibody, a second specific antibody and a goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane (3) to form a first detection line (31), a second detection line (32) and a quality control line (33);
step three, closing the rest protein binding sites of the nitrocellulose membrane (3), and then drying for later use;
The method comprises the steps of sequentially adhering a sample pad (1), a bonding pad (2), a nitrocellulose membrane (3) and a water absorbing material (4) to the top surface of a bottom plate (5), and sequentially arranging and connecting the sample pad (1), the bonding pad (2), the nitrocellulose membrane (3) and the water absorbing material (4), wherein one end of the sample pad (1) connected with the bonding pad (2) is located right above one end of the bonding pad (2), one end of the bonding pad (2) connected with the nitrocellulose membrane (3) is located right above one end of the nitrocellulose membrane (3), and one end of the nitrocellulose membrane (3) connected with the water absorbing material (4) is located right below the water absorbing material (4), so that a test paper strip is obtained.
9. The method for preparing a colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA according to claim 8, wherein in the fourth step, the quality control line (33) is close to the water absorbing material (4), and the first detection line (31) and the second detection line (32) are located between the quality control line (33) and the binding pad (2).
10. The method for preparing the colloidal gold immunochromatographic test strip for simultaneously detecting PEDV and PoRVA according to claim 8, wherein the immunological probe (21) is positioned at one end of the binding pad (2) close to the nitrocellulose membrane (3).
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