CN209460270U - Colloidal gold test strip for detection of herbicide-resistant protein CP4-EPSPS - Google Patents

Abstract

A colloidal gold test strip for detecting herbicide-resistant protein CP4-EPSPS, which includes a base plate, a sample pad fixed on the base plate connected successively, a gold standard pad, a nitrocellulose membrane and an absorbent pad, in the gold standard pad The monoclonal antibody mAb1 coupled with colloidal gold is immobilized; the nitrocellulose membrane is provided with a detection line and a quality control line, and the end of the nitrocellulose membrane near the gold label pad is coated with the monoclonal antibody mAb2 as a detection line. The colloidal gold test strip can quickly detect the transgenic herbicide-resistant CP4-EPSPS protein, and the detection result is accurate, with good specificity and high sensitivity. The detection sensitivity of CP4-EPSPS protein can reach 5ng/mL, and the detection of CP4-containing The sensitivity of ‑EPSPS protein in soybean can reach 0.01%, and the sensitivity of detecting CP4‑EPSPS protein in corn can reach 0.1%.

Description

Colloidal gold test strip for detection of herbicide-resistant protein CP4-EPSPS

technical field

The utility model belongs to the detection field of transgenic proteins, in particular to a colloidal gold test strip for detecting herbicide-resistant protein CP4-EPSPS.

Background technique

In 2016, the global planting area of genetically modified crops has continued to grow at a rate of 3% to 4% for 21 years, reaching 185 million hectares. With the rapid development of the genetically modified planting industry, the safety of genetically modified crops and their products is still one of the most concerned issues for consumers. In order to protect consumers' right to know, countries around the world have made clear legal regulations on the labeling of genetically modified products. my country adopts a mandatory labeling system, the supervision and management of genetically modified ingredients testing rooms and the means of correct labeling.

At present, GMO detection technologies are mainly detection at the gene level and detection at the protein level. At the genetic level, the polymerase chain reaction (PCR) is mainly used to identify the transgenic strains by using the specific genes of different transgenic varieties. However, because this detection technology requires expensive PCR thermal cyclers, professional operators, and Taq Due to the characteristics of reagents and consumables such as enzymes, this technology can only detect genetically modified crops in the laboratory, and is not suitable for rapid on-site screening in fields, logistics, storage, processing and other links.

The detection technology of protein level is mainly transgenic gold label test strip, which uses the specific reaction of antibody to transgenic protein (antigen) to realize the detection of genetically modified components. The detection results are visible to the naked eye, and the detection only takes 5 to 10 minutes Professional operation, low environmental requirements, suitable for on-site supervision and testing in various occasions, especially for genetically modified testing of raw material products.

With the continuous development of research and development and application of genetically modified crops in my country, the supervision and monitoring of genetically modified crops is in urgent need of rapid detection technology. The demand for genetically modified immune colloidal gold test strips in my country is increasing year by year. There are very few institutions, most of which are foreign agency products, and the price is expensive. The research and development of the visual rapid detection technology of genetically modified products with independent property rights in China has broad application prospects and practical significance.

Utility model content

The purpose of the utility model is to provide a colloidal gold test strip for detecting the herbicide-resistant protein CP4-EPSPS, which can quickly detect the transgenic herbicide-resistant CP4-EPSPS protein, and the detection result is accurate, with good specificity and high The sensitivity solves the problem of relying on instruments and professionals in the detection of genetically modified herbicide-resistant crops in the existing detection technology. At the same time, it can improve the long-term dependence on imports and foreign agents for test strips.

In order to achieve the above object, the utility model provides the following technical solutions:

A colloidal gold test strip for detecting herbicide-resistant protein CP4-EPSPS, which includes a base plate, a sample pad fixed on the base plate connected in sequence, a gold standard pad, a nitrocellulose membrane and a water-absorbing pad;

Monoclonal antibody mAb1 coupled with colloidal gold is immobilized in the gold label pad, and the monoclonal antibody mAb1 is a capture antibody against herbicide protein CP4-EPSPS;

The nitrocellulose membrane is provided with a detection line and a quality control line, and the end of the nitrocellulose membrane is coated with a monoclonal antibody mAb2 near the gold standard pad, and as a detection line, the detection line also contains BSA; the monoclonal antibody mAb2 is an anti- The detection antibody of herbicide protein CP4-EPSPS; the end of the nitrocellulose membrane near the absorbent pad is coated with goat anti-mouse IgG antibody as a quality control line.

Further, in the gold standard pad, the particle size of the colloidal gold is 20-40nm.

Also, the nitrocellulose membrane has a pore size of 10-15 μm.

Preferably, the amount of the colloidal gold-coupled monoclonal antibody mAb1 is 1.2-2.4 μg, and the amount of the detection line-coated monoclonal antibody mAb2 is 1.5-2 mg/mL.

Preferably, a support backboard is provided behind the bottom plate of the colloidal gold test strip.

Further, the colloidal gold test strip also includes a casing sleeved outside the base plate; a sample injection hole and an observation window are provided on the shell of the casing, and the sample injection hole is arranged on the shell corresponding to the top of the sample pad. body, the observation window is located at the housing corresponding to the top of the cellulose film.

Also, an overlapping area of 1-2 mm is provided among the sample pad, the gold standard pad, the nitrocellulose membrane and the water-absorbing pad.

Preferably, both the sample pad and the gold standard pad are made of glass fiber, and the water-absorbing pad is made of cellulose fiber.

The two strains of monoclonal antibody mAb1 and monoclonal antibody mAb2 used in the utility model are paired antibodies of CP4-EPSPS, both of which are obtained by traditional hybridoma technology. After cell fusion, the positive clones screened by ELISA method are expanded and cultivated, and small Mouse ascites, ascites antibodies were purified by protein-G column affinity chromatography to obtain monoclonal antibody mAb1 and monoclonal antibody mAb2, the subtype of monoclonal antibody mAb1 was IgG1, and the subtype of monoclonal antibody mAb2 was IgG2a.

The detection principle and result interpretation process of the utility model are as follows: if there is protein to be measured in the sample, the sample is dropped onto the sample pad during measurement, and the sample moves along the direction of the sample pad, gold standard pad, nitrocellulose membrane, and water-absorbing pad. When flowing through the gold-labeled pad, the monoclonal antibody coupled with the colloidal gold on the gold-labeled pad is firstly reconstituted, and secondly, the transgenic specific protein in the sample combines with the gold-labeled antibody mAb1 to form an immune complex, and the immune complex passes through capillary action When it flows through the nitrocellulose membrane and reaches the detection line, it is captured by the monoclonal antibody mAb2 of the detection line, forming a double-antibody sandwich mode of gold-labeled antibody + protein + antibody, and the colloidal gold gathered on the detection line appears visible to the naked eye. Red; excess gold-labeled antibodies and immune complexes flow through the quality control line, are captured by the solid-phase antibody of the quality control line, and red quality control lines appear at the position of the quality control line on the nitrocellulose membrane. If there is no protein antigen in the sample, the gold-labeled antibody will not accumulate on the test line, and the test strip has only one quality control line.

Therefore, for a positive sample, both the test line and the quality control line are displayed during the test; while for the negative sample, there is no test line during the test, and only the quality control line is displayed. If only one red line appears near the test line or there is no red line on the test strip, the result is considered invalid.

Compared with the prior art, the utility model has the following beneficial effects:

In the colloidal gold test strip of the present utility model, its monoclonal antibody mAb1 has a higher ability to couple to colloidal gold, and 1mL of colloidal gold can couple 60 μg of monoclonal antibody mAb1, and the test strip has higher sensitivity, and can detect transgenic anti-weeding The sensitivity of the reagent CP4-EPSPS protein can reach 5ng/mL, the sensitivity of detecting soybean containing CP4-EPSPPS protein can reach 0.01%, the sensitivity of detecting CP4-EPSPPS protein corn can reach 0.1%, and it has good specificity.

In the utility model, the detection result is accurate. In order to avoid false positives in the detection results, 1% BSA is added to the buffer solution coated with the detection line, and the sensitivity is high when detecting actual samples without false positives.

Description of drawings

Fig. 1 is the structural representation of the immune colloidal gold detection test strip in the utility model embodiment.

Fig. 2 is the colloidal gold test strip detection protein sensitivity result of immune colloidal gold detection test strip in the utility model embodiment.

Fig. 3 is the colloidal gold test strip detection specificity result of immune colloidal gold detection test strip in the utility model embodiment, wherein, P is positive control; N is blank corn flour, as negative control.

Fig. 4 is the sensitivity result that the colloidal gold test strip of immune colloidal gold detection test strip detects actual sample soybean in the utility model embodiment.

Fig. 5 is the sensitivity result that the colloidal gold test strip of immune colloidal gold detection test strip detects actual sample corn in the utility model embodiment.

Detailed ways

The present invention will be further described below in conjunction with specific examples.

Trisodium citrate and Tween-20 used in the examples were purchased from Shanghai Sangon Bioengineering Co., Ltd.; BSA and chloroauric acid were purchased from Sigma, USA; nitrocellulose membrane (NC membrane) CN 95 was purchased from Sartorius, Germany; Mouse IgG antibody, paired antibodies mAb1 and mAb2 of CP4-EPSPS protein were purchased from Canada Altron Biotechnology Company; Nanodrop micro-spectrophotometer was purchased from American Thermo Company; spotting instrument and microcomputer cutting machine were purchased from Hangan Technology Company; magnetic stirrer Purchased from Shanghai Sile Instrument Company.

Example

As shown in Fig. 1, a kind of colloidal gold test strip of detecting herbicide-resistant protein CP4-EPSPS of the present utility model, it comprises, PVC bottom plate A, be fixed on the sample pad 1 that is connected successively on PVC bottom plate A, gold standard pad 2, Nitrocellulose membrane 3 and absorbent pad 4; the sample pad 1 and the gold standard pad 2 are both glass fibers, and the absorbent pad 4 is cellulose fiber.

A monoclonal antibody mAb1 coupled to colloidal gold is immobilized in the gold label pad 2, wherein the amount of the monoclonal antibody mAb1 is 2.4 μg;

The nitrocellulose membrane 3 is provided with a detection line 31 and a quality control line 32, and the nitrocellulose membrane 3 is coated with a monoclonal antibody mAb2 near the gold standard pad 2 as the detection line 31; the nitrocellulose membrane 3 is near the water-absorbing pad 4 The end was coated with goat anti-mouse IgG antibody as the quality control line32.

The concentration of monoclonal antibody mAb2 of the detection line is 1.5 mg/mL, and 1% BSA is added to the buffer solution of the coating detection line; the concentration of goat anti-mouse IgG antibody of the quality control line is 1 mg/mL; The nitrocellulose membrane is a CN95 membrane with a pore size of 15 μm.

The sensitivity determination of the colloidal gold test strip of the detection herbicide-resistant protein CP4-EPSPS of the present embodiment:

The transgenic protein CP4-EPSPS was diluted with PBS into different concentration gradients, respectively 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 5000ng/mL, each Take 70 μL of each sample and drop it on the sample pad of the colloidal gold test strip, observe the results after 5-10 minutes, add the same volume of PBS dropwise to the sample pad as a negative control, the results are shown in Figure 2.

It can be seen from FIG. 2 that the detection sensitivity of the herbicide-resistant protein CP4-EPSPS colloidal gold test strip for CP4-EPSPS protein in this embodiment is 5 ng/mL.

The specificity determination of the colloidal gold test strip of the detection herbicide-resistant protein CP4-EPSPS of the present embodiment:

Transgenic test strips were used to detect soybeans and corn with different genes in the laboratory. The above-mentioned crop seed samples were ground into powder, and added to double distilled water for water extraction according to the ratio of 1:5, shaken vigorously for 20 seconds, and then separated by standing. layer, and the supernatant was taken for detection, and the specificity of the test strip was judged by the detection results.

Figure 3 shows the cross-reaction of herbicide-resistant test strips to other herbicide-resistant proteins. BT-11 corn contains PAT protein, and BT-176 corn contains BAR protein, which can make plants resistant to glufosinate-ammonium and phosphorus pesticides. It can be seen from Figure 3 that the CP4-EPSPS test strip has no cross-reaction to PAT and BAR proteins, and the specificity is good.

The colloidal gold test strip of the detection herbicide-resistant protein CP4-EPSPS of the present embodiment is to the detection sensitivity of actual sample and practicability determination:

The components contained in the solution after water extraction of different crops are different, and these matrices may have different matrix effects. Therefore, it is necessary to test different crops containing different transgenic ratios. One is to determine the practicability of test strip detection, and the other is To determine the detection sensitivity of the actual sample.

After the RRS soybeans with 100% transgenic content were extracted with water, the supernatant of blank soybeans was used for gradient dilution, so that the contents of transgenic soybeans were: 0.001%, 0.01%, 0.1%, 1%, and 10%, respectively, and used The herbicide-resistant test strips were tested, and the results are shown in Figure 4. The test strips can detect transgenic soybeans with a content of 0.01%; after the NK603 corn was extracted with water, the supernatant of the blank corn was used for gradient dilution. Make the content of transgenic corn be respectively: 0.05%, 0.1%, 1%, 5%, and detect with herbicide resistance test paper, the result is shown in Figure 5, the detection sensitivity of test paper to NK603 corn is 0.1%, the sensitivity higher.

Through experimental verification, the monoclonal antibodies mAb1 and mAb2 of the present invention can be successfully paired through CP4-EPSPS protein, and can be used to detect transgenic soybeans, corn, rape, cotton, sugar beet, etc., and the detection sensitivity of soybean GTS-40-3-2 0.01%, the detection sensitivity of NK603 corn is 0.1%, and there is no cross-reaction with the transgenic protein PAT and BAR, and the detection sensitivity of CP4-EPSPS pure protein is 5ng/mL, which has high sensitivity, strong specificity, convenient and fast detection Features.

Claims (8)

1. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS comprising, bottom plate is fixed on bottom plate successively phase Sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad even, which is characterized in that

It is anti-weeding that the monoclonal antibody mAb1, the monoclonal antibody mAb1 coupled with colloidal gold is fixed in the gold-labelled pad The capture antibody of agent PROTEIN C P4-EPSPS；

The nitrocellulose filter is equipped with detection line and nature controlling line, and the nearly gold-labelled pad end of nitrocellulose filter is coated with monoclonal antibody MAb2 also contains BSA as detection line in detection line, the monoclonal antibody mAb2 is herbicide resistant protein CP4-EPSPS Detect antibody；The nearly water absorption pad end of nitrocellulose filter is coated with sheep anti-mouse igg antibody, as nature controlling line.

2. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute It states in gold-labelled pad, the partial size of colloidal gold is 20-40nm.

3. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute The aperture for stating nitrocellulose filter is 10-15 μm.

4. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute The dosage for stating the monoclonal antibody mAb1 coupled with colloidal gold is 1.2-2.4 μ g, and detection line is coated with the use of monoclonal antibody mAb2 Amount is 1.5-2mg/mL.

5. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute Supporting back board is equipped with after stating the bottom plate of colloidal gold strip.

6. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute Stating colloidal gold strip further includes the shell being set in outside bottom plate；Well and observation window, institute are provided on the shell of the shell It states well to be set at the shell above the corresponding sample pad, the observation window is set to the shell above the corresponding cellulose membrane At body.

7. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute State the overlay region that 1-2mm is equipped between sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad.

8. detecting the colloidal gold strip of herbicide resistant protein CP4-EPSPS according to claim 1, which is characterized in that institute State sample pad, gold-labelled pad is glass fibre, water absorption pad is cellulose fibre.