CN112526133A - Test strip, preparation method thereof and application thereof in transgenic protein AM79EPSPS detection - Google Patents
Test strip, preparation method thereof and application thereof in transgenic protein AM79EPSPS detection Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention relates to the technical field of biochemical detection, in particular to a test strip, a preparation method thereof and application thereof in transgenic protein AM79EPSPS detection. The hand-held colloidal gold rapid test paper for the transgenic protein AM79EPSPS provided by the invention can be widely applied to rapid detection of the transgenic protein AM79EPSPS, and has the following specific advantages: (1) the method is simple to operate, does not need special instruments and equipment, and can be used for directly detecting in fields and the like conveniently. (2) The detection time is short (5-8 min); the result judgment standard is uniform, namely a negative result (-) only has 1C line; positive (+): both T and C lines appear. (3) The test paper has good sensitivity through verification, and the lowest detection limit can reach 0.1 mu g/mL.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a test strip, a preparation method thereof and application thereof in transgenic protein AM79EPSPS detection.
Background
The 5-enol pyruvyl-shikimate-3-phosphatesynthase (EPSPS) which is separated from the glyphosate polluted soil by using a metagenomic method is a key enzyme in the biosynthesis process of aromatic amino acids of tryptophan, tyrosine and phenylalanine in organisms; glyphosate blocks the synthesis of aromatic amino acids by inhibiting the activity of EPSP synthase, which ultimately leads to the death of the test plant. However, the activity of EPSP synthase of some bacteria is not inhibited by glyphosate, so that the expression of the bacterial EPSPS gene can protect plants from herbicide damage. The herbicide resistance gene EPSPS is transferred into soybean, so that the transgenic soybean with the excellent glyphosate resistance is cultured, the field weeding effect in the soybean production is improved, and the production cost is reduced.
A plant constitutive expression vector pSOY19 is constructed by using an AM79EPSPS gene which is obtained by separating a metagenomic method from glyphosate-polluted soil and is resistant to herbicides, is driven by a 35S promoter, and can provide glyphosate resistance for transgenic plants. The pSOY19 plasmid contained only 1 gene that was expressed in plant cells: bacterial EPSPS, under the control of CaMV35S promoter. The expression plasmid of the herbicide resistant gene is 9557 bp. The full length of the insertion sequence AM79EPSPS gene is 1341bp, and a polypeptide containing 440 amino acids is coded. EPSPS exogenous genes have been transformed into soybean variety Huachun No. 3 and Jack by Agrobacterium-mediated method and herbicide-resistant transgenic soybean materials transformed with EPSPS genes are obtained.
The conventional RT-PCR detection method is used for AM79EPSPS detection, and has the defects of complex operation, long time consumption, high cost and incapability of high-throughput detection.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a test strip, a preparation method thereof, and an application thereof in transgenic protein AM79EPSPS detection, wherein the test strip can be used for rapidly and visually realizing AM79EPSPS detection on transgenic protein.
The test strip provided by the invention comprises a bottom plate, and a sample pad, a marking pad, a nitrocellulose membrane and an absorption pad which are sequentially arranged on the bottom plate;
the gold-labeled pad carries a colloidal gold-labeled albumin antibody and a colloidal gold-labeled mouse anti-AM 79EPSPS monoclonal antibody;
the nitrocellulose membrane is provided with a detection area and a control area, and the detection area is coated with a mouse anti-AM 79EPSPS monoclonal antibody; the control region is coated with an anti-mouse IgG secondary antibody;
the mouse anti-AM 79EPSPS monoclonal antibody is derived from a hybridoma cell strain with the preservation number of CCTCCNO: C201937.
In the invention, the mass ratio of the mouse anti-AM 79EPSPS monoclonal antibody marked by colloidal gold on the gold-marked pad to the mouse anti-AM 79EPSPS monoclonal antibody coated on the nitrocellulose membrane is 1: 20.
In the present invention, the size of the sample pad is 4mm × 25 mm; the size of the marking pad is 3mm multiplied by 4 mm; the size of the nitrocellulose membrane is 4mm multiplied by 35 mm; the size of the absorption pad is 4mm multiplied by 30 mm;
the loading capacity of the colloidal gold labeled mouse anti-AM 79EPSPS monoclonal antibody on each gold label pad is 40 ng; the mass of the murine anti-AM 79EPSPS monoclonal antibody coated on each of the nitrocellulose membranes was 0.8. mu.g.
In the invention, the distance between the detection area and the control area is 6-6.5 mm.
The preparation method of the test strip comprises the following steps:
step 1: fixing colloidal gold labeled mouse anti-AM 79EPSPS monoclonal antibody on the gold label pad,
a mouse anti-AM 79EPSPS monoclonal antibody is fixed on a detection zone on a nitrocellulose membrane,
immobilizing an anti-mouse IgG secondary antibody in a control region on the nitrocellulose membrane;
step 2: and (3) sequentially sticking the sample pad, the gold-labeled pad, the nitrocellulose membrane and the absorption pad prepared in the step (1) on the bottom plate, and drying to obtain the test strip.
The test strip is applied to preparation of a transgenic protein AM79EPSPS detection product.
The invention also provides a detection kit for the transgenic protein AM79EPSPS, which comprises the test strip.
The kit also comprises a sample extraction reagent, a sample extraction tube and a rotary pestle.
The invention also provides a detection method of the transgenic protein AM79EPSPS, which is characterized in that the test strip is used for detecting a sample, and whether the sample contains the transgenic protein AM79EPSPS is judged according to the color development results of the detection area and the control area.
In the present invention, the sample to be tested includes leaves and/or seeds of soybean.
The leaves and/or seeds of the soybeans are extracted before detection, and the extraction adopts extraction buffer EB 004. The extraction method comprises the steps of crushing the leaves and/or seeds, mixing the crushed leaves and/or seeds with an extraction buffer EB004, and oscillating for 20-30 seconds.
The invention discloses a specific detection step and a principle of a hand-held colloidal gold rapid detection test paper for transgenic protein AM79EPSPS, which are as follows:
firstly, vertically inserting a test paper into a detection sample without exceeding the position of an arrow; the sample solution permeates to the test strip of the colloidal gold labeled antibody along the sample pad, if the sample solution contains transgenic protein AM79EPSPS, AM79EPSPS protein is combined with the mouse anti-AM 79EPSPS monoclonal antibody on the gold labeled test strip and is combined with the mouse anti-AM 79EPSPS monoclonal antibody on the NC membrane, after 8-10min, the color change of the detection zone can be observed in the observation zone, and a positive strip appears; if the sample solution does not contain the transgenic protein AM79EPSPS, then no positive band is observed at the detection zone. Regardless of whether the sample solution contains the transgenic protein AM79EPSPS, when the sample solution reaches the nitrocellulose membrane, the mouse anti-AM 79EPSPS monoclonal antibody on the gold-labeled test strip can be combined with the anti-mouse IgG secondary antibody coated by the control area, so that the color is developed. Therefore, the result judgment rule of the handheld colloidal gold rapid test paper for the transgenic protein AM79EPSPS is as follows:
negative result (-), only 1C line appeared;
positive result (+): both T and C lines appear.
The hand-held colloidal gold rapid test paper for the transgenic protein AM79EPSPS has good anti-interference effect on complex matrixes such as leaves, seeds, bulk grains and the like, can be widely applied to rapid detection of the transgenic protein AM79EPSPS, and has the following specific advantages: (1) the method is simple to operate, does not need special instruments and equipment, and can be used for directly detecting in fields and the like conveniently. (2) The detection time is short (5-8 min); the result judgment standard is uniform, namely a negative result (-) only has 1C line; positive (+): both T and C lines appear. (3) The test paper has good sensitivity through verification, and the lowest detection limit can reach 0.1 mu g/mL.
Drawings
FIG. 1 shows the schematic structure of the hand-held colloidal gold test paper for rapid detection of transgenic protein AM79EPSPS prepared by the invention; wherein, 1 is a result observation area, 2 is a mark area, 3 is a control area (C), 4 is a detection area (T), and 5 is a sample addition area;
figure 2 shows a transgenic protein AM79EPSPS standard assay graph, samples (from left to right): 1. CK is EB004 buffer solution; 2. 1, standard protein product; 3. 2, standard protein products; 4. 3, standard protein products;
FIG. 3 shows a sample detection map of actual soybean leaves; sample (left to right): 1. blank control (EB004 buffer); 2. positive control (AM79 EPSPS protein standard 1); 3. negative control (wild-type soybean leaf sample) 4, transgenic soybean leaf sample 1; 5. transgenic soybean leaf sample 2; 6. transgenic soybean leaf sample 3;
FIG. 4 shows an actual soybean seed sample detection map; sample (left to right): 1. blank control (EB004 buffer); 2. positive control (AM79 EPSPS protein standard 1); 3. negative control (wild type soybean leaf sample); 4. transgenic soybean seed sample 1; 5. transgenic soybean seed sample 2; 6. transgenic soybean seed sample 3.
Detailed Description
The invention provides a test strip and a preparation method thereof, and application of the test strip in detection of transgenic protein AM79EPSPS, and a person skilled in the art can realize the test by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Wherein, the preparation of the mouse anti-AM 79EPSPS monoclonal antibody comprises the following steps:
1. recovery culture: taking AM79EPSPS monoclonal antibody hybridoma cell strain with preservation number of CCTCC NO: C201937, and carrying out expanded culture in a cell culture bottle;
2. preparing ascites: injecting liquid paraffin into the abdominal cavity of BALB/c mice of 6-8 weeks old, wherein each mouse is 0.2 mL. After 10 days, a certain number of monoclonal antibody hybridoma cells were intraperitoneally inoculated. The abdomen of the mouse is obviously expanded, ascites is collected by an elbow dropper, and the titer of ascites antibody is measured by indirect ELISA.
3. Monoclonal antibody purification: centrifuging ascites of mouse for 15min (2000rpm, room temperature), removing upper oil, adding saturated ammonium sulfate dropwise slowly at 4 deg.C under stirring to half-saturation, stirring for 30min, centrifuging for 30min (13000rpm, 4 deg.C), and removing supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M pH7.4); slowly adding saturated ammonium sulfate dropwise to 33% under stirring at 4 deg.C, stirring for 30min, centrifuging for 30min (13000rpm, 4 deg.C), and removing supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M, pH7.4), dialyzed overnight at 4 ℃ to determine the protein content, and frozen at-20 ℃ for use.
4. Purifying with Protein G column after ammonium sulfate precipitation, passing 5ml ultrapure water through the new column, and balancing the purified column with 5ml0.4M PB buffer solution (pH7.0); the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the binding site; the purification column was equilibrated with 10ml0.4M PB buffer (pH7.0); eluting the antibody on the binding site with 5ml0.1M glycine-hydrochloric acid buffer solution (pH7.0), and adding 1M Tris-HCI (pH2.7) to neutralize glycine, so that the pH is kept neutral suitable for antibody preservation; equilibrating the purification column with 10mL0.4M PB buffer (pH7.0); 5mL of 20% ethanol solution was passed through the column, and the column was kept at 4 ℃.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of transgenic protein AM79EPSPS Immunity detection card
In each test card, the size of the sample pad was 4mm × 15mm, the size of the label pad was 3mm × 4mm, the size of the nitrocellulose membrane was 4mm × 28mm, and the size of the absorbent pad was 4mm × 19 mm.
40ng of colloidal gold-labeled mouse anti-AM 79EPSPS monoclonal antibody was immobilized on the gold-labeled pad of each test card, 0.8. mu.g of mouse anti-AM 79EPSPS monoclonal antibody was immobilized on the detection zone on nitrocellulose membrane, and 1.0. mu.g of anti-mouse IgG secondary antibody was immobilized on the control zone.
A sample pad, a gold-labeled pad fixed with a colloidal gold-labeled specific monoclonal antibody, a nitrocellulose membrane and an absorption pad are sequentially adhered to the bottom plate. Then dried in vacuum at 37 ℃ for 2 h.
Example 2 evaluation of Effect of transgenic protein AM79EPSPS immunodetection card on detection of protein Standard
200 mu L of blank sample solution EB004 buffer solution (pH7.4, 0.2mol/LPBS:8g of sodium chloride, 3.35g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium dihydrogen phosphate and 0.2g of potassium chloride are added into a sample cup, and double distilled water is dissolved to a constant volume of 1L), a rapid test paper is inserted into the sample cup, the reaction is carried out for 8 minutes under the condition of room temperature, and then the color development result is observed in a result observation area. The results show that the T line is not colored and the C line is colored in the observation area.
② 100 μ L of 0.1 μ g/ml AM79EPSPS protein standard 1 (prepared by diluting with the PBS solution) is added into the sample cup, and the same operation steps are carried out as the blank sample. The results showed that the C line (control zone) and the T line (detection zone) were developed simultaneously in the observation zone.
③ adding 100 μ L of 0.1 μ g/ml AM79EPSPS protein standard 2 (prepared by diluting with the same PBS solution) into the sample cup, and performing the same operation steps as the blank sample. The results show that C and T lines in the observation area are developed simultaneously.
And fourthly, 100 mu L of 0.1 mu g/ml AM79EPSPS protein standard product 3 (prepared by diluting the PBS solution) is added into the sample cup, and the same operation steps as the blank sample are carried out. The results show that C and T lines in the observation area are developed simultaneously.
Example 3 evaluation of Effect of transgenic protein AM79EPSPS ImmunoSoy detection card on detection of actual transgenic Soybean leaf samples
A method for extracting plant leaf tissues comprises the following steps:
1. the plant leaf tissue is placed between the cover and the tube body of the disposable tissue extraction tube, the cover is quickly covered, and the process is repeated for 2 times to obtain 2 pieces of circular leaf tissue. The leaf was placed on the bottom of the extraction tube with a pestle. Marking on the tube wall with a marker pen.
2. And (3) inserting the pestle into the tube, rotating the pestle to grind the crushing blade, and continuously pressing for 20-30 seconds.
3. 0.2mL (about 8 drops) of extraction buffer EB004 was added.
4. The crushing step is repeated to bring the sample into intimate contact with the buffer. The pestle is removed (please note that the pestle is disposable to avoid cross contamination between different samples) to obtain the sample to be tested.
Second, sample detection
1) 200 mu L of blank sample solution EB004 buffer solution is added into the sample cup, the rapid test paper is inserted into the sample cup, and the color development result is observed in the result observation area after the reaction is carried out for 8 minutes under the condition of room temperature. The results show that the T line is not colored and the C line is colored in the observation area.
2) 100200. mu.L of 0.1. mu.g/ml standard AM79EPSPS protein was added to the sample cup, and the same procedure was followed as for the non-transgenic soybean leaf samples. The results show that C and T lines in the observation area are developed simultaneously.
3) 200. mu.L of the non-transgenic soybean leaf sample extract was added to the sample cup, and the same procedure as that of the standard sample was followed. The result shows that only the control line C in the observation area is colored, and the detection line T is not colored.
4) 200. mu.L of AM 79-transferred EPSPS gene soybean leaf sample 1 extract was added to the sample cup, and the same procedure as that for the non-transgenic soybean leaf sample was followed. The result shows that the control line C line and the detection line T line in the observation area are both colored.
5) 200. mu.L of AM 79-transferred EPSPS gene soybean leaf sample 2 extract was added to the sample cup, and the same procedure as that for the non-transgenic soybean leaf sample was followed. The result shows that the control line C line and the detection line T line in the observation area are both colored.
6) 200. mu.L of AM 79-transferred EPSPS gene soybean leaf sample 3 extract was added to the sample cup, and the same procedure as that for the non-transgenic soybean leaf sample was followed. The result shows that the control line C line and the detection line T line in the observation area are both colored.
Example 4 evaluation of Effect of transgenic protein AM79EPSPS ImmunoSoy seed sample detection
A method for extracting a plant seed sample comprises the following steps:
1. 0.25g of the crushed plant seeds were taken and transferred to a labelled extraction tube. Note that: sufficient crushing can improve the test accuracy.
2. Add 0.75mL of extraction buffer EB004 to dissolve.
3. And covering the cover of the extraction tube, forcibly oscillating the extraction tube up and down for 20-30 seconds to ensure that the sample and the buffer solution are fully mixed, and standing for waiting for the solid matter to be precipitated at the bottom of the tube.
4. Note that no cross-contamination was required and a separate extraction tube was used for each sample.
Second, sample detection
1) 200 mu L of blank sample solution EB004 buffer solution is added into the sample cup, the rapid test paper is inserted into the sample cup, and the color development result is observed in the result observation area after the reaction is carried out for 8 minutes under the condition of room temperature. The results show that the T line is not colored and the C line is colored in the observation area.
2) mu.L of 0.1. mu.g/ml standard AM79EPSPS protein was added to the sample cup, and the same procedure was followed as for the non-transgenic soybean leaf samples. The results show that C and T lines in the observation area are developed simultaneously.
3) 200. mu.L of wild type non-transgenic soybean seed sample extract was added to the sample cup, and the same procedure as that of the standard sample was followed. The result shows that only the control line C in the observation area is colored, and the detection line T is not colored.
4) 200 mu L of AM79EPSPS transgenic soybean seed sample 1 extracting solution is added into the sample cup, and non-transgenic soybean sample extracting solution is adopted. The result shows that the control line C line and the detection line T line in the observation area are both colored.
5) 200 mu L of AM79EPSPS transgenic soybean seed sample 2 extracting solution is added into the sample cup, and non-transgenic soybean sample extracting solution is adopted. The result shows that the control line C line and the detection line T line in the observation area are both colored.
6) 200 mu L of AM79EPSPS transgenic soybean seed sample 3 extracting solution is added into the sample cup, and non-transgenic soybean sample extracting solution is adopted. The result shows that the control line C line and the detection line T line in the observation area are both colored.
Example 5 sensitivity of transgenic protein AM79EPSPS Immunodetection card
Diluting the transgenic protein to 10. mu.g/mL, 5. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.2. mu.g/mL, 0.1. mu.g/mL, 0.05. mu.g/mL, 0.02. mu.g/mL, 0.01. mu.g/mL; the detection was carried out using the detection cards obtained in example 1, respectively, wherein the T-line color development at a concentration of 0.1. mu.g/mL or more was visible to the naked eye, and the color development at 0.05. mu.g/mL or less was not evident or did not develop, and therefore, it was determined that the detection card sensitivity reached 0.1. mu.g/mL.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The test strip is characterized by comprising a bottom plate, and a sample pad, a marking pad, a nitrocellulose membrane and an absorption pad which are sequentially arranged on the bottom plate;
the gold-labeled pad carries a colloidal gold-labeled albumin antibody and a colloidal gold-labeled mouse anti-AM 79EPSPS monoclonal antibody;
the nitrocellulose membrane is provided with a detection area and a control area, and the detection area is coated with a mouse anti-AM 79EPSPS monoclonal antibody; the control region is coated with an anti-mouse IgG secondary antibody;
the mouse anti-AM 79EPSPS monoclonal antibody is derived from a hybridoma cell strain with the preservation number of CCTCC NO: C201937.
2. The test strip of claim 1, wherein the mass ratio of the colloidal gold-labeled mouse anti-AM 79EPSPS monoclonal antibody on the gold label pad to the mouse anti-AM 79EPSPS monoclonal antibody coated on the nitrocellulose membrane is 1: 20.
3. The test strip of claim 1 or 2,
the sample pad is 4mm × 25mm in size;
the size of the marking pad is 3mm multiplied by 4 mm;
the size of the nitrocellulose membrane is 4mm multiplied by 35 mm;
the size of the absorption pad is 4mm multiplied by 30 mm;
the loading capacity of the colloidal gold labeled mouse anti-AM 79EPSPS monoclonal antibody on each gold label pad is 40 ng; the mass of the murine anti-AM 79EPSPS monoclonal antibody coated on each of the nitrocellulose membranes was 0.8. mu.g.
4. The strip of any one of claims 1 to 3, wherein the distance between the detection zone and the control zone is 6 to 6.5 mm.
5. The method for preparing the test strip of any one of claims 1 to 4, comprising:
step 1: fixing colloidal gold labeled mouse anti-AM 79EPSPS monoclonal antibody on the gold label pad,
a mouse anti-AM 79EPSPS monoclonal antibody is fixed on a detection zone on a nitrocellulose membrane,
immobilizing an anti-mouse IgG secondary antibody in a control region on the nitrocellulose membrane;
step 2: and (3) sequentially sticking the sample pad, the gold-labeled pad, the nitrocellulose membrane and the absorption pad prepared in the step (1) on the bottom plate, and drying to obtain the test strip.
6. Use of the test strip of any one of claims 1 to 5 in the preparation of a transgenic protein AM79EPSPS detection product.
7. A kit for detecting transgenic protein AM79EPSPS, which is characterized by comprising the test strip of any one of claims 1-5.
8. The kit of claim 7, further comprising a sample extraction reagent, a sample extraction tube, and a rotary pestle.
9. A method for detecting transgenic protein AM79EPSPS, which is characterized in that a sample is detected by the test strip of any one of claims 1-5, and whether the sample contains the transgenic protein AM79EPSPS is judged according to the color development results of the detection area and the control area.
10. The assay of claim 9, wherein the sample comprises leaves and/or seeds of soybean.
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