CN116047058A - Insect-resistant protein Cry1F colloidal gold immunochromatographic rapid test strip and application method thereof - Google Patents

Insect-resistant protein Cry1F colloidal gold immunochromatographic rapid test strip and application method thereof Download PDF

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CN116047058A
CN116047058A CN202211329916.2A CN202211329916A CN116047058A CN 116047058 A CN116047058 A CN 116047058A CN 202211329916 A CN202211329916 A CN 202211329916A CN 116047058 A CN116047058 A CN 116047058A
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pad
sample
test strip
insect
gold
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刘卫晓
金芜军
王磊
宛煜嵩
苗朝华
董美
高进
王迪
孟丽霞
赵伟玲
文婷婷
王占超
陶然
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Biotechnology Research Institute of CAAS
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    • 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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    • G01—MEASURING; TESTING
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Abstract

The invention provides a colloidal gold immunochromatographic rapid test strip, which comprises a bottom plate, a sample pad, a gold label pad, a reaction membrane and a water absorption pad, wherein the sample pad, the gold label pad, the reaction membrane and the water absorption pad are sequentially connected on the bottom plate, the gold label pad is coated with an insect-resistant protein Cry1F monoclonal antibody-colloidal gold conjugate, the test strip has strong specificity and high sensitivity, and the operation is simple, convenient, rapid and accurate, and the test strip is suitable for on-site rapid detection and safety evaluation of the insect-resistant protein Cry1F in transgenic corn and products thereof.

Description

Insect-resistant protein Cry1F colloidal gold immunochromatographic rapid test strip and application method thereof
Technical Field
The invention relates to the field of bioengineering, in particular to a colloidal gold immunochromatography rapid test strip for transgenic corn insect-resistant protein Cry1F and a use method thereof.
Background
Bacillus thuringiensis (Bacillus thuringiensis, bt) is a widely occurring gram-positive bacterium, whose secreted anti-insect crystal protein is the current major biopesticide; secreted insect-resistant proteins are classified into two classes according to amino acid sequence similarity: cry and Cry delta-endotoxins. Wherein the Cry proteins are toxic to larvae of a variety of insect pests (e.g., lepidoptera, diptera, coleoptera, nematodes, protozoa, etc.). Cry toxins have been transformed into a variety of crops to render them insect resistant. The Cry1F protein is one of the Cry toxins. At present, crops transformed with Cry genes mainly comprise corn, potato, rice, cotton and the like.
Advances and developments in transgenic technology have driven the development of biology. Although the transgenic food can meet the requirements of people on yield, insect resistance and the like, the transgenic food brings potential threats to the life of people, for example, certain genes can cause toxicity to the food after being introduced into a host, the transgenic food generates allergen, the people generate drug resistance, the nutritional value of the food is changed and the like. In order to comprehensively evaluate the safety of the transgenic food while researching, developing and commercializing the transgenic food, consumers can rapidly distinguish the transgenic food from natural food, and a proper method is established for identifying and detecting the transgenic ingredients in the transgenic food, so that the safety management of agricultural transgenic organisms can be promoted, the safety of people, animals and microorganisms can be ensured, the ecological environment can be protected, and the further research of agricultural transgenic biotechnology can be promoted. In order to carry out rapid qualitative analysis on Cry1F proteins in transgenic crops or derivatives thereof, the development of Cry1F colloidal gold immunochromatography test strips has great significance. The immune colloidal gold technology (Immune colloidal gold technique) is a novel immune labeling technology which uses colloidal gold as a tracer marker to be applied to antigen and antibody. The colloidal gold is prepared by polymerizing chloroauric acid (HAuCL 4) into gold particles with a specific size under the action of a reducing agent such as ascorbic acid, sodium citrate, etc., and becomes a stable colloidal state due to electrostatic action, which is called colloidal gold. Colloidal gold is negatively charged in a weak alkaline environment and can form firm combination with positive charge groups of protein molecules, and the biological properties of the protein are not affected because the combination is electrostatic combination. The colloidal gold labeling technology is a core technology of immunochromatography rapid diagnosis, and is characterized in that colloidal gold is used as a tracer marker for antigen-antibody reaction, and the substance is a coating process that high molecules such as protein are adsorbed on the surfaces of colloidal gold particles. According to some physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, and the immunological and biological properties of the conjugate, colloidal gold has been widely used in the fields of immunology, histology, pathology, cell biology, etc.
Disclosure of Invention
The invention aims to provide a colloidal gold immunochromatographic rapid test strip for a transgenic corn insect-resistant protein Cry1F and a use method thereof, so that the colloidal gold immunochromatographic rapid test strip is suitable for on-site rapid detection and safety evaluation of the insect-resistant protein Cry1F in transgenic corn and products thereof.
In order to achieve the above purpose, the invention provides a colloidal gold immunochromatographic rapid test strip, which comprises a bottom plate, and a sample pad, a gold label pad, a reaction membrane and a water absorption pad which are adhered on the bottom plate and are sequentially connected.
In another embodiment, the colloidal gold immunochromatographic rapid test strip comprises a sample pad, a gold-labeled pad, a reaction membrane, a water-absorbing pad and a reaction membrane, wherein the width of the overlapping part is 1-2mm, the sample pad is overlapped on the gold-labeled pad by the sample pad and the gold-labeled pad, the gold-labeled pad is overlapped on the reaction membrane by the gold-labeled pad and the reaction membrane, and the water-absorbing pad is overlapped on the reaction membrane by the reaction membrane and the water-absorbing pad.
In another embodiment, the colloidal gold immunochromatographic rapid test strip is obtained by secreting an anti-insect protein Cry1F monoclonal antibody from a hybridoma cell strain 1F 7D 8 with a preservation number of CGMCC No. 45009.
In another embodiment, the colloidal gold immunochromatographic rapid test strip is provided with a detection line for coating the anti-insect protein Cry1F monoclonal antibody and a quality control line for coating the goat anti-mouse IgG on the reaction film.
In another embodiment, the coated anti-insect protein Cry1F monoclonal antibody provided on the reaction membrane is secreted by hybridoma cell strain 1D7 1C6 with a preservation number of CGMCC No.45008.
In another embodiment, the separation between the detection line and the quality control line is 0.5-1.0cm.
In another embodiment, the colloidal gold immunochromatographic rapid test strip is characterized in that the reaction membrane is a nitrocellulose membrane, the sample pad is made of a glass fiber membrane or filter paper, and the water absorbing pad is made of filter paper.
The invention also provides a detection method of the colloidal gold immunochromatography speed test strip, which comprises the following steps: the method comprises the following steps:
pretreatment of a sample to be tested: if the sample to be detected is liquid substances such as plant tissue extract, 40-60 mu L of the sample to be detected can be directly taken, if the sample to be detected is non-liquid substances such as plant leaves, stems, seeds, and the like, 90-110mg of the sample to be detected is required to be ground, 0.5-1.5mL of sample treatment liquid is added for fully and uniformly mixing, and the supernatant is taken for 40-60 mu L for detection after standing on ice for 25-35 min;
table 1 sample treatment fluid formulations
Figure SMS_1
Figure SMS_2
2) And (3) detection: immersing the test strip into a sample to be tested, allowing the test strip to act for 5-10min at room temperature, and observing the result;
3) Analysis of detection results:
positive: the quality control line and the detection line are both in strips, which indicates that Cry1F proteins exist in the sample to be detected;
negative: the detection line is not provided with a strip, and the quality control line is provided with a strip, which indicates that no Cry1F protein exists in the sample to be detected;
failure: if the detection line and the quality control line are not provided with strips or only the detection line is provided with strips, the test strip is proved to be invalid.
Compared with the prior art, the invention has the following beneficial effects: the test strip is high in specificity and sensitivity, simple, convenient, quick and accurate to operate, and suitable for on-site quick detection and safety evaluation of the insect-resistant protein Cry1F in transgenic corn and products thereof.
Preservation information
The Cry1Fa monoclonal antibody hybridoma cell strain 1F7 1D8 and the Cry1Fa monoclonal antibody hybridoma cell strain 1D7 1C6 are sequentially preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 12 months and 10 days of 2021, and the preservation addresses are as follows: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Post code: 100101, and the preservation numbers are CGMCC No.45009 and CGMCC No.45008 in sequence.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold immunochromatographic test strip according to the present invention;
FIG. 2 is a schematic diagram showing the detection results of a colloidal gold immunochromatographic rapid test strip according to the present invention; positive, the quality control line (namely the C line) develops color, the detection line (namely the T line) is visible to naked eyes, and no matter the color depth is positive; negative, the color of the C line is developed, the color of the T line is not developed, and the judgment is negative; the color of the T line is invalid whether the color is developed or not;
FIG. 3 is a graph showing the results of testing transgenic corn positive leaf samples at room temperature using the colloidal gold immunochromatographic rapid test strip according to the present invention.
Detailed Description
The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 colloidal gold immunochromatographic test strip
The utility model provides an insect-resistant protein Cry1F colloidal gold immunochromatographic rapid test strip, includes bottom plate 1, be sample pad 2, gold mark pad 3, reaction membrane 6, water absorption pad 7 along sample flow direction on the bottom plate in proper order, be equipped with detection line 4 and quality control line 5 on the reaction membrane 6, the gold mark pad coats insect-resistant protein Cry1F monoclonal antibody-colloidal gold conjugate.
The sample pad 2 overlaps with the adjacent end of the gold mark pad 3, the adjacent end of the gold mark pad 3 and the adjacent end of the reaction membrane 6 and the adjacent end of the water absorption pad 7, the width of the overlapping part is 1-2mm, the adjacent end of the sample pad 2 and the gold mark pad 3 is the sample pad 2 which is overlapped on the gold mark pad, the adjacent end of the gold mark pad 3 and the reaction membrane 6 is the gold mark pad 3 which is overlapped on the reaction membrane 6, and the adjacent end of the reaction membrane 6 and the water absorption pad 7 is the water absorption pad 7 which is overlapped on the reaction membrane 6.
In another embodiment, the colloidal gold immunochromatographic rapid test strip is obtained by secreting an anti-insect protein Cry1F monoclonal antibody from a hybridoma cell strain 1F 7D 8 with a preservation number of CGMCC No. 45009.
In another embodiment, the colloidal gold immunochromatographic rapid test strip is provided with a detection line for coating the anti-insect protein Cry1F monoclonal antibody and a quality control line for coating the goat anti-mouse IgG on the reaction film.
In another embodiment, the coated anti-insect protein Cry1F monoclonal antibody provided on the reaction membrane is secreted by hybridoma cell strain 1D7 1C6 with a preservation number of CGMCC No.45008.
In another embodiment, the separation between the detection line and the quality control line is 0.5-1.0cm.
In another embodiment, the colloidal gold immunochromatographic rapid test strip is characterized in that the reaction membrane is a nitrocellulose membrane, the sample pad is made of a glass fiber membrane or filter paper, and the water absorbing pad is made of filter paper.
Example 2 detection of transgenic maize leaves with colloidal gold immunochromatographic test strip
Taking 100mg of transgenic corn leaves, grinding the transgenic corn leaves by liquid nitrogen, fully mixing the transgenic corn leaves with a sample extracting solution, standing the transgenic corn leaves on ice for 30min, and taking 50 mu L of supernatant for detection. The detection result was observed at room temperature for 5 min. The specific results are shown in fig. 3, and it can be seen from fig. 3 that the Cry1F protein contained in the stock solution can be specifically detected by the colloidal gold immunochromatographic rapid test strip related to the application.
Example 2 detection of colloidal gold immunochromatographic quick test strip
1. The content is as follows:
1.1 preparation: ( The sampling amount is not less than 20 parts for inspection; rechecking, reserving samples and other special cases can be extracted separately )
1.1.1 checking, adjusting and, if appropriate, approving the instruments or related devices involved in the test item.
1.1.2 checking and checking whether the information of the product name, the lot number, the batch and the like is consistent with the instruction;
1.2 appearance: whether each component of the reagent (kit) is complete, complete or whether liquid leaks or not; whether the label is clear and easy to identify.
1.3 film strip width: 2 test papers are randomly detected by using a vernier caliper (the precision is 0.02 mm), the average value of 2 measurement results is calculated, and the results meet the requirements of Cry1F colloidal gold finished product quality standard.
1.4 liquid movement speed: operating according to the instruction, starting timing after adding the sample, and recording the time (t) from the sample adding to the complete flow through the detection window; and measuring the upper edge distance (L) from the sample adding window to the detection window by using a vernier caliper, wherein L/t is calculated to be the liquid moving speed, and the result meets the requirements of Cry1F colloidal gold finished product quality standard.
1.5 net content: and taking a sample diluent component in the kit, and measuring by using a universal measuring tool, wherein the detection result meets the requirement of Cry1F colloidal gold finished product quality standard.
1.6 Properties: detecting enterprise references, the following criteria should be met:
1.6.1 minimum detection limit: the lowest limit of detection reference was repeatedly tested 10 times, and the results were calculated. The result meets the requirements of Cry1F colloidal gold finished product quality standard;
the calculation method comprises the following steps:
Figure SMS_3
LLD＝ X +3×S
wherein: x-series of measurements;
X -determining a mean value;
n is the number of measurements;
s is standard deviation;
LLD-minimum detection limit
1.6.2 accuracy: with not less than 40 samples of different concentrations in the measurement range, each sample was measured separately by the test kit procedure and the comparison method using the manufacturer-specified analysis system as the comparison method.
The calculation method comprises the following steps:
Figure SMS_4
wherein: bias-relative deviation;
x is the measurement mean value;
x-calibrator, standard or reference substance.
1.6.3 repeatability: detecting repetitive references CV1 and CV2, wherein each repetitive detection is not less than 10 times, and each horizontal variation Coefficient (CV) is calculated respectively, so that the result meets the requirements of Cry1F colloidal gold finished product quality standard:
the calculation method comprises the following steps:
Figure SMS_5
Figure SMS_6
wherein: x-series of measurements;
x is the measurement mean value;
n is the number of measurements;
s is standard deviation;
CV-intra-batch coefficient of variation.
Through the above-mentioned inspection, the colloidal gold immunochromatography test strip that obtains of this application detects limit: 5ppb (ng/mL).

Claims (9)

1. The utility model provides an insect-resistant protein Cry1F colloidal gold immunochromatography rapid test strip, includes bottom plate (1), its characterized in that: sample pad (2), gold mark pad (3), reaction membrane (6), pad (7) absorb water are in proper order along sample flow direction on the bottom plate, be equipped with detection line (4) and matter control line (5) on reaction membrane (6), the gold mark pad coats insect-resistant protein Cry1F monoclonal antibody-colloidal gold conjugate.
2. The test strip of claim 1, wherein the sample pad (2) and the gold-labeled pad (3) are adjacent, the gold-labeled pad (3) and the reaction membrane (6) are adjacent, and the reaction membrane (6) and the water-absorbent pad (7) are adjacent, the width of the overlapping part is 1-2mm, the sample pad (2) and the gold-labeled pad (3) are adjacent, the gold-labeled pad (3) and the reaction membrane (6) are adjacent, and the water-absorbent pad (7) is adjacent, and the reaction membrane (6) and the water-absorbent pad (7) are adjacent.
3. The test strip according to claim 1 or 2, characterized in that the anti-insect protein Cry1F monoclonal antibody is secreted by hybridoma cell line 1F7 1D8 with a preservation number of CGMCC No. 45009.
4. The test strip according to claim 1 or 2, characterized in that the reaction membrane is provided with a detection line (4) coated with an anti-insect protein Cry1F monoclonal antibody and a quality control line (5) coated with goat anti-mouse IgG.
5. The test strip of claim 4, wherein the coated anti-insect protein Cry1F monoclonal antibody provided on the reaction membrane is secreted by hybridoma cell line 1D7 1C6 with a preservation number of CGMCC No.45008.
6. The test strip of claim 4, wherein the detection line and the quality control line are spaced apart by 0.5-1.0cm.
7. The test strip of claim 1 or 2, wherein the reaction membrane is a nitrocellulose membrane, the sample pad is made of a glass fiber membrane or a filter paper, and the absorbent pad is made of a filter paper.
8. The method for detecting a test strip according to claim 1 or 2, comprising the steps of:
1) Pretreatment of a sample to be tested: if the sample to be detected is a liquid substance, directly taking 40-60 mu L of the sample to be detected, if the sample to be detected is a non-liquid substance, grinding 90-110mg of the sample to be detected, adding 0.5-1.5mL of sample treatment liquid, fully and uniformly mixing, standing on ice for 25-35min, and taking 40-60 mu L of supernatant for detection;
2) And (3) detection: immersing the test strip into a sample to be tested, allowing the test strip to act for 5-10min at room temperature, and observing the result;
3) Analysis of detection results:
positive: the quality control line and the detection line are both in strips, which indicates that Cry1F proteins exist in the sample to be detected;
negative: the detection line is not provided with a strip, and the quality control line is provided with a strip, which indicates that no Cry1F protein exists in the sample to be detected;
failure: if the detection line and the quality control line are not provided with strips or only the detection line is provided with strips, the test strip is proved to be invalid.
9. The method of claim 8, wherein the sample processing fluid is formulated as follows: 1M Tris,pH 7.5 500uL,1M NaCL 1.5mL,0.5M EDTA 20uL,50% glycerol 2mL,10% SDS 1mL, and 10mL of double distilled water were prepared, and 1 piece of protease inhibitor and 50uL of 1mM phenylmethylsulfonyl fluoride were added at the time of use.
CN202211329916.2A 2022-10-27 2022-10-27 Insect-resistant protein Cry1F colloidal gold immunochromatographic rapid test strip and application method thereof Pending CN116047058A (en)

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