CN111190011A - Duck tembusu virus colloidal gold rapid detection test paper and preparation method thereof - Google Patents
Duck tembusu virus colloidal gold rapid detection test paper and preparation method thereof Download PDFInfo
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Abstract
The test paper comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is attached to one end of a PVC bottom plate, the water absorption pad is attached to the other end of the PVC bottom plate, the nitrocellulose membrane is attached to the middle of the bottom plate, one end of the nitrocellulose membrane is connected with the colloidal gold pad, the other end of the nitrocellulose membrane is connected with the water absorption pad, the colloidal gold pad is connected with the sample pad, an E protein monoclonal antibody is coated and combined on the colloidal gold pad by colloidal gold, a mouse antibody E protein polyclonal antibody is coated at a marking line of the nitrocellulose membrane to serve as a detection line, and a goat antibody IgG is coated at a quality control line to serve as a secondary antibody. The beneficial effects are that: the colloidal gold has high sensitivity and strong test paper specificity, only shows positive to the tembusu virus, can quickly identify the tembusu virus, can identify initial symptoms, can also be used for screening common duck groups, can greatly shorten the detection time, can find the disease as soon as possible, and reduces the loss of farmers to the minimum.
Description
Technical Field
The invention relates to a poultry epidemic prevention article and a manufacturing method thereof, in particular to a duck tembusu virus colloidal gold rapid detection test paper and a preparation method thereof.
Background
Duck tembusu virus (DTMUV) is a novel infectious virus of ducks. In 2010, the first outbreak of coastal areas such as Shandong Jiangzhe and Zhejiang in China is named after being analyzed by a plurality of laboratories in China. Similar cases occurred in eastern region of Mongolia in 2016, and the duck dying of illness was finally determined to be infected with duck Tembusu virus after a number of experiments conducted in the animal laboratory of the university of Mongolia. The main infection symptoms of the duck Tembusu virus are the reduction of laying rate, growth retardation, ovariohemorrhage and the like of ducks. The virus has high transmission speed and high infection rate, seriously threatens the duck breeding industry in China and urgently needs to be effectively prevented and treated. However, the lack of rapid diagnostic techniques makes the symptoms of the virus difficult to identify, difficult to control early, and prone to spread. Therefore, finding out the technology and equipment for rapid identification is a key link for preventing and treating the virus.
Disclosure of Invention
The invention aims to provide a colloidal gold rapid detection test paper capable of rapidly identifying duck tembusu virus and a preparation method thereof.
The above purpose is realized by the following technical scheme: the quick detection test paper for duck tembusu virus colloidal gold is characterized by comprising the following components in parts by weight: the test paper comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is adhered to one end of a PVC bottom plate, the water absorption pad is adhered to the other end of the bottom plate, the nitrocellulose membrane is adhered to the middle of the bottom plate, one end of the nitrocellulose membrane is connected with the colloidal gold pad, the colloidal gold pad is connected with the sample pad, the other end of the nitrocellulose membrane is connected with the water absorption pad, an E protein monoclonal antibody is coated and combined on the colloidal gold pad by using colloidal gold as a gold-labeled probe, then a rat anti-E protein polyclonal antibody 1:1 dilution is coated on a scribing line of the nitrocellulose membrane to be used as a detection line, and goat anti-mouse IgG is coated on the position of a quality control line as a.
A preparation method of a duck tembusu virus colloidal gold rapid detection test paper comprises the following steps: using envelope protein E protein of DTMUV as core protein, designing E protein primer, upstream primer sequence F is 5/-TTAGGATCCTTCAGCTGTCTGGGGATG-3/(the underlined position is BamH I restriction site), and the downstream primer sequence R is 5/-TGAGAGCTCGGCATTGACATTTACTGC-3/(the underlined is SacI restriction site), and the cloning and expression of the E protein are carried out: the size of the cloned gene fragment is 1500bp, and the size of the expressed protein is 63 ku; separating and purifying the E protein by adopting a urea gradient centrifugation method, wherein the concentration of the purified protein is 4.98 mg/ml; immunizing BALB/c mice with E protein (100 ug) for three times, and then screening the mice with serum titer higher than 1:5000 by an indirect ELISA method to carry out four times of immunization; extracting the mouse splenocyte, fusing with SP2/O cell, and screening two stable cells A1B3 and B2C 8; preparing 25nm wine red colloidal gold solution by a trisodium citrate reduction method, wherein the pH value of the colloidal gold solution is 8, desalting the E protein monoclonal antibody obtained by separation by a dialysis bag treatment method, measuring the optimal labeling concentration of an antibody to be 1:8 by a multiple dilution method, coating a gold-labeled probe optimized according to the conditions on a colloidal gold pad, diluting and coating the mouse anti-E protein polyclonal antibody at a 1:1 ratio on a nitrocellulose membrane detection line, performing the same treatment on goat anti-mouse IgG, coating the goat anti-mouse IgG on a quality control line, and assembling a bottom plate, the colloidal gold pad, a nitrocellulose membrane, a sample pad and a water absorption pad to obtain the colloidal gold detection test paper.
A method for using the duck tembusu virus colloidal gold rapid detection test paper comprises the steps of placing a detected object on a sample pad, and enabling a sample to move forwards under the capillary action. If the sample contains the target antigen, the target antigen is combined with the specific antibody on the colloidal gold pad to form a compound, then the compound moves forwards continuously, when the compound moves to the detection line, the antigen is combined with the antibody on the detection line in a specific way, the gold-labeled antibody is gathered at the detection line to show red, the remaining gold-labeled antibody which is not combined with the antibody on the detection line moves forwards continuously, and when the gold-labeled antibody reaches a quality control line, the gold-labeled antibody is combined with the secondary antibody in a specific way and gathered at the secondary antibody to show color; when the detection line and the quality control line are both colored, the detection object contains the target antigen, the detection test paper has normal function, and the detection is positive; when only the quality control line is developed, only the test paper is indicated to be normal in function but no target antigen exists in the detected object, and the test paper is negative; the shade degree of the color of the detection line indicates the content of the target detection object.
The invention has the beneficial effects that: the prepared colloidal gold has high sensitivity, the prepared test paper has strong specificity, only shows positive to the tembusu virus, can quickly identify the tembusu virus, can identify initial symptoms, can also be used for screening common duck groups, can greatly shorten the detection time, enables the disease to be discovered as soon as possible, reduces the loss of farmers to the minimum, and is a necessary article for preventing and treating the tembusu disease of ducks.
Drawings
FIG. 1 is a front view of the first embodiment;
FIG. 2 is a top view of the first embodiment;
fig. 3 is a perspective view of the first embodiment.
It can be seen in the figure that: the device comprises a bottom plate 1, nitrocellulose 2, a detection line 3, a quality control line 4, a sample pad 5, a colloidal gold pad 6 and a water absorption pad 7.
Detailed Description
Duck batch death cases first appeared in duck breeding enterprises in eastern Mongolia areas in the year. The affected duck is suspected to be infected with DTMUV according to the clinical symptoms of the affected duck. To exclude the possibility of bacterial infection, the killed ducks were bacteriologically identified, and the results showed that no bacteria were isolated in the body. And then grinding the viscera of the diseased duck, taking the supernatant to perform specific PCR (polymerase chain reaction) tests and PCR product sequence analysis of the DTMUV and other pathogens, and displaying that the diseased duck only contains the DTMUV in the tissues. Then, the biological characteristics of the separated DTMUV, such as hemagglutination, BHK cell infection and the like, are identified, and the result shows that the virus has the same biological characteristics with the DTMUV, so that the separated virus is determined to be duck tembusu virus. Finally, determining that the diseased duck is infected with duck tembusu virus by combining the test results. In order to master a rapid diagnosis method, the virus is analyzed and researched, and a colloidal gold rapid detection test paper is established for the virus.
The envelope protein E protein is the main structural protein of the protein, has good immunogenicity, and the E protein is selected as the core protein for experiments. Designing E protein primer, cloning E proteinSo as to achieve the purpose. By looking up the data, the primer sequence F was determined to be 5/-TTAGGATCCTTCAGCTGTCTGGGGATG-3/(the underlined position is the BamH I site), R is 5/-TGAGAGCTCGGCATTGACATTTACTGC-3/(Sac I restriction enzyme cutting site is marked), the size of gene fragment cloned by ① is 1500bp, the size of ② expression protein is 63 ku., Western blot analysis and detection show that a specific band appears at the position of about 63ku molecular mass, the protein can react with the mouse monoclonal antibody resisting His label, which shows that the expression protein has good reactogenicity.
BALB/c mice were immunized three times with 100ug of E protein, and then four times with indirect ELISA to screen mice with serum titers above 1: 5000. And extracting the mouse splenocytes, fusing the mouse splenocytes with SP2/O cells, and finally screening two stable cells A1B3 and B2C 8. The recognition of the epitope by the obtained monoclonal antibody was determined by the superimposed ELISA method according to the formula AI = (2 xA)1+2/(A1-A2) -1) x 100%) calculated as a superposition AI of A1B3 and B2C8 of 52% > 10%, i.e. it recognizes different epitopes.
A25 nm size wine red colloidal gold solution was prepared by the trisodium citrate reduction method. And desalting the E protein monoclonal antibody obtained by separation by using a dialysis bag treatment method for later use. Adjusting pH of 8 groups of colloidal gold solution to 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10, adding the monoclonal antibodies into the above solutions, reacting, adding 10% NaCl, and mixing. After 2h, the solution color is most similar to the original solution color when the pH is 8, namely the optimal binding pH value of the monoclonal antibody and the colloidal gold particles is 8. And (3) taking 9 parts of the colloidal gold solution with the pH value of 8, and then respectively adding monoclonal antibodies and 10% NaCl with the dilution ratios of 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11 and 1:12 to mix evenly. After 2h, the E protein monoclonal antibody concentration is kept red in the colloidal gold solution of 1:4 to 1:8 groups, and the other groups are blue-green, so that the optimal labeling concentration of the antibody is 1: 8. Coating the gold-labeled probe optimized according to the conditions on a colloidal gold pad 6, then diluting and coating the mouse-anti-E protein polyclonal antibody 1:1 on a detection line 3 on a nitrocellulose membrane, performing the same treatment on the goat-anti mouse IgG, then coating the goat-anti mouse IgG on a quality control line 4 as a second antibody, and then assembling a PVC bottom plate 1, the colloidal gold pad 6, the nitrocellulose membrane 2, a sample pad 5, a water absorption pad 7 and the like as shown in figures 1, 2 and 3: the method comprises the steps of paving a nitrocellulose membrane 2 on the middle upper part of a PVC (polyvinyl chloride) bottom plate 1, arranging a detection line 3 and a quality control line 4 on the nitrocellulose membrane 1, diluting and coating a mouse anti-E protein polyclonal antibody 1:1 on the detection line 3, performing the same treatment on a goat anti-mouse IgG to serve as a secondary antibody to coat the quality control line 4, coating an E protein monoclonal antibody on a colloidal gold pad 6 as a gold-labeled probe, pressing the end part of the colloidal gold pad 6 above one side of the test line of the nitrocellulose membrane 2, pressing the end part of a sample pad 5 on the colloidal gold pad 6, and pressing a water absorption pad 7 on the other end of the nitrocellulose membrane 2 to prepare the colloidal gold test paper shown in figure 1.
The prepared colloidal gold test paper is clinically tested, the allantoic fluid of the virus-injected chick embryo is diluted by 1:100 and then is detected by using the colloidal gold test paper, a positive result is still displayed, and the sensitivity is high. The specificity of the test paper is identified, 20 cases of ducks which are died of diseases and are respectively diagnosed to be infected with the tembusu virus, the newcastle disease virus, the duck plague virus and the H9 avian influenza virus and stored in a laboratory are detected by using the prepared colloidal gold test paper, and a PCR control experiment is set up at the same time. The experimental results are shown in table 1, the PCR detection result completely accords with the original diagnosis result, the detection result of the colloidal gold test paper shows that only the cases infected with the tembusu virus show positive results, the cases infected with other viruses show negative results, and the experimental results show that the prepared test paper has stronger specificity.
The establishment of the detection method provides great convenience for clinical detection of DTMUV, and the strain used by the detection test paper is an inner Mongolia isolate, but the detection test paper is not only suitable for detecting DTMUV in inner Mongolia areas. In table 1, 5 DTMUV cases come from Shandong and Zhejiang, respectively, and the results of the detection using the prepared colloidal gold test paper show that 5 DTMUV cases all show positive detection results. Therefore, the test paper is also suitable for detecting strains in other regions. The detection test paper takes 5-15 min from sample adding to result displaying, greatly shortens the detection time compared with other detection methods, enables the disease to be found early, and reduces the loss of farmers to the minimum.
TABLE 1 RT-PCR and colloidal gold assay results
| Detection method | Tembusu virus | Newcastle disease virus | Duck plague virus | Subtype H9 avian influenza virus |
| Number of positive detections in RT- |
5 are | 7 are | 3 pieces of | 5 are |
| Positive detection quantity of prepared |
5 are | 0 is | 0 is | 0 is |
Claims (3)
1. The utility model provides a duck tembusu virus colloidal gold short-term test paper which characterized in that: the test paper comprises a bottom plate (1), a sample pad (5), a colloidal gold pad (6), a nitrocellulose membrane (2) and a water absorption pad (7), wherein the sample pad (5) is adhered to one end of a PVC bottom plate (1), the water absorption pad (7) is adhered to the other end of the bottom plate (1), the nitrocellulose membrane (2) is adhered to the middle of the bottom plate (1), one end of the nitrocellulose membrane (2) is connected with the colloidal gold pad (6), the colloidal gold pad (6) is connected with the sample pad, the other end of the nitrocellulose membrane is connected with the water absorption pad (7), an E protein monoclonal antibody is coated and combined on the colloidal gold pad (6) by colloidal gold as a gold-labeled probe, then a mouse-resistant E protein polyclonal antibody 1:1 is diluted and coated on a scribing line of the nitrocellulose membrane (2) as a detection line (3), and a goat-resistant mouse IgG is coated on a quality control line (4) as a second antibody after the same treatment.
2. The preparation method of the duck tembusu virus colloidal gold rapid detection test paper according to claim 1, which comprises the following steps: the method is characterized in that: an E protein primer is designed by taking an envelope protein E protein of duck tembusu virus DTMUV as a core protein, and an upstream primer sequence F is 5/-TTAGGATCCTTCAGCTGTCTGGGGATG-3/(the underlined position is BamH I restriction site), and the downstream primer sequence R is 5/-TGAGAGCTCGGCATTGACATTTACTGC-3/(the underlined is SacI restriction site), and the cloning and expression of the E protein are carried out: the size of the cloned gene fragment is 1500bp, and the size of the expressed protein is 63 ku; separating and purifying the E protein by adopting a urea gradient centrifugation method, wherein the concentration of the purified protein is 4.98 mg/ml; immunizing BALB/c mice with E protein (100 ug) for three times, and then screening the mice with serum titer higher than 1:5000 by an indirect ELISA method to carry out four times of immunization; extracting the mouse splenocyte, fusing with SP2/O cell, and screening two stable cells A1B3 and B2C 8; preparing 25nm wine red colloidal gold solution by a trisodium citrate reduction method, wherein the pH value of the colloidal gold solution is 8, desalting the E protein monoclonal antibody obtained by separation by a dialysis bag treatment method, measuring the optimal labeling concentration of an antibody to be 1:8 by a multiple dilution method, coating a gold-labeled probe optimized according to the conditions on a colloidal gold pad, diluting and coating the mouse anti-E protein polyclonal antibody at a 1:1 ratio on a nitrocellulose membrane detection line, performing the same treatment on goat anti-mouse IgG, coating the goat anti-mouse IgG on a quality control line, and assembling a bottom plate, the colloidal gold pad, a nitrocellulose membrane, a sample pad and a water absorption pad to obtain the colloidal gold detection test paper.
3. The use method of the colloidal gold rapid detection test paper for duck tembusu virus according to claim 1 or 2, which is characterized in that: placing the detected object on a sample pad, enabling the sample to move forward under the capillary action, if the sample contains a target antigen, combining the target antigen with a specific antibody on a colloidal gold pad to form a compound, then continuing to move forward, enabling the antigen to be specifically combined with the antibody on a detection line when the compound moves to the detection line, enabling the gold-labeled antibody to gather at the detection line and display red, enabling the rest gold-labeled antibody which cannot be combined with the antibody on the detection line to continue to move forward, and enabling the gold-labeled antibody to be specifically combined with the secondary antibody at the position and gather at the position after the gold-labeled antibody reaches a quality control line so as to display color; when the detection line and the quality control line are both colored, the detection object contains the target antigen, the detection test paper has normal function, and the detection is positive; when only the quality control line is developed, only the test paper is indicated to be normal in function but no target antigen exists in the detected object, and the test paper is negative; the shade degree of the color of the detection line indicates the content of the target detection object.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749368A (en) * | 2015-04-20 | 2015-07-01 | 武汉科缘生物发展有限责任公司 | Monoclonal antibody for duck tembusu virus and application |
| CN105203757A (en) * | 2015-09-21 | 2015-12-30 | 山东农业大学 | Colloidal gold test strip for fast diagnosing tembusu viruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104749368A (en) * | 2015-04-20 | 2015-07-01 | 武汉科缘生物发展有限责任公司 | Monoclonal antibody for duck tembusu virus and application |
| CN105203757A (en) * | 2015-09-21 | 2015-12-30 | 山东农业大学 | Colloidal gold test strip for fast diagnosing tembusu viruses |
Non-Patent Citations (2)
| Title |
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| 顾玲玲 等: "鸭坦布苏病毒 E 蛋白的原核表达" * |
| 顾玲玲;朱善元;王安平;: "鸭坦布苏病毒E蛋白的原核表达" * |
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