CN-121499799-B - Colloidal gold immunochromatography detection test strip for rice flower honey and preparation method and application thereof

CN121499799BCN 121499799 BCN121499799 BCN 121499799BCN-121499799-B

Abstract

The invention relates to the technical field of biotechnology and food detection, and discloses a rice flower honey colloidal gold immunochromatography detection test strip, a preparation method and application thereof. According to proteomics analysis, ycf protein from chloroplast is identified in honey in rice flower as a plant source specific marker, and a rice flower honey colloidal gold immunochromatography detection test strip is developed, wherein the test strip comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped, the gold-labeled pad is coated with a gold-labeled antibody formed by combining a monoclonal antibody resisting Ycf protein specific section with colloidal gold, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a monoclonal antibody resisting Ycf1 protein, and the quality control line is coated with goat anti-mouse IgG. The test strip is simple and convenient to operate, can finish detection within 15 minutes, has high sensitivity, strong specificity and visual and reliable results, and is suitable for on-site rapid screening and identification of the authenticity of the honey in the rice ball.

Inventors

HU HAN
Liu Xizi
DING JIA
XUE XIAOFENG
Yang Suoliang
Nan Liqin
ZHANG GUANGYUN
Cheng Ruhuai
LI YONGCHUN

Assignees

中国农业科学院蜜蜂研究所
沧源佤族自治县地方产业发展服务中心

Dates

Publication Date: 20260508
Application Date: 20260113

Claims (10)

Any of the following applications of the ycf1 protein or a truncate thereof: 1) The method is used for identifying the rice flower honey; 2) As a characteristic marker of the rice flower honey; 3) The fingerprint spectrum is used for constructing the rice flower honey; the amino acid sequence of Ycf protein is shown as SEQ ID NO. 2; wherein, the truncated body of Ycf protein is 255-698 amino acid sequence of Ycf protein.
2. The authenticity identification method of the rice ball honey is characterized by comprising the steps of detecting components in the honey to be detected, and judging that the honey to be detected is the rice ball honey or the rice ball honey mixed with the rice ball honey if the components contain Ycf protein or a truncated body thereof; The Ycf protein or a truncation thereof is identical to the Ycf protein or a truncation thereof of claim 1.
3. The monoclonal antibody of the Ycf protein is characterized in that the monoclonal antibody is secreted by a hybridoma cell line A11 with the preservation number of CGMCC No.46751 or a hybridoma cell line C9 with the preservation number of CGMCC No. 46752.
4. The composition is characterized by comprising a monoclonal antibody secreted by a hybridoma cell strain A11 with the preservation number of CGMCC No.46751 and a monoclonal antibody secreted by a hybridoma cell strain C9 with the preservation number of CGMCC No. 46752.
5. The anti-Ycf protein monoclonal antibody of claim 3 or any one of the following uses of the composition of claim 4: (i) For detecting Ycf protein or a truncate thereof in a sample; (ii) Preparing a reagent or test paper for detecting Ycf protein or a truncated body thereof; (iii) A kit for detecting Ycf protein or a truncate thereof is prepared.
6. The test paper strip for detecting the colloidal gold immunochromatography of the rice flower honey comprises a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are sequentially laminated, The gold-labeled pad is coated with a gold-labeled antibody formed by combining a first antibody with colloidal gold, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a second antibody, and the quality control line is coated with goat anti-mouse IgG; wherein the first antibody is a monoclonal antibody secreted by a hybridoma cell strain A11 with the preservation number of CGMCC No.46751, and the second antibody is a monoclonal antibody secreted by a hybridoma cell strain C9 with the preservation number of CGMCC No.46752, or The first antibody is a monoclonal antibody secreted by a hybridoma cell line C9 with the preservation number of CGMCC No.46752, and the second antibody is a monoclonal antibody secreted by a hybridoma cell line A11 with the preservation number of CGMCC No. 46751.
7. The test strip of claim 6, wherein the sample pad is a glass fiber membrane.
8. The method for preparing the test strip according to claim 6 or 7, comprising the following steps: (1) Preparing a colloidal gold solution; (2) Adding the purified first antibody into a colloidal gold solution, and stirring to enable the first antibody to be stably combined with colloidal gold particles; (3) Adding bovine serum albumin solution for sealing, high-speed centrifugal purification, and re-suspending in a buffer solution containing a stabilizing agent to prepare Cheng Jinbiao pad working solution; (4) Uniformly spraying the gold-labeled pad working solution on a glass fiber membrane, and drying to prepare a gold-labeled pad; (5) Coating a second antibody on a nitrocellulose membrane by streaking with a certain concentration to form a detection line; (6) Coating goat anti-mouse IgG antibody on a nitrocellulose membrane in a streak mode with a certain concentration to form a quality control line; (7) And sequentially laminating and pasting the sample pad, the gold mark pad, the nitrocellulose membrane and the water absorbing pad on the PVC backboard, and cutting into test strips with proper widths.
9. Use of the test strip according to claim 6 or 7 for detecting rice flower honey.
10. A method for detecting rice flower honey by using the test strip according to claim 6 or 7, which is characterized in that the method comprises the steps of diluting a honey sample to be detected with water, inserting the test strip to enable the diluted honey sample to soak a sample pad, and reading a result after a period of time; The result is judged to be effective when the quality control line shows red lines, and is invalid when the quality control line does not show color when the detection line shows color, and the honey sample to be detected is judged to be the honey in the rice ball or the honey mixed in the rice ball.

Description

Colloidal gold immunochromatography detection test strip for rice flower honey and preparation method and application thereof Technical Field The invention relates to the technical field of biotechnology and food detection, in particular to a rice flower honey colloidal gold immunochromatography detection test strip and a preparation method and application thereof. Background The rice flower honey is a natural sweet substance which is obtained by fully brewing after Chinese bees collect rice flower honey and are mixed with self secretion. The color of the rice flower honey is very special and black, so the rice flower honey is also called as black honey. As an important characteristic, chinese bee honey, the honeyed rice flower is a plant of genus Lemongrass (Leucosceptrum) of Labiatae (Lamiaceae), and grows in the form of shrub or small arbor, and flowers 2-4 months per year. The rice flower honey has long production period and high maturity under the influence of climate conditions, and is a high-end product in honey. Currently, common methods for honey plant source identification include sensory evaluation, sporopollen analysis, and mass spectrometry techniques. Although the result is accurate and reliable, the detection equipment is expensive, the analysis flow is complex, professional operation is needed, the pretreatment of the sample is complicated, the single detection cost is high, the period is long, and the requirements of rapid screening and large quantity detection cannot be met at all. Therefore, development of a rice ball honey type identification method capable of achieving both laboratory level accuracy and on-site rapid detection convenience is needed. Disclosure of Invention The invention aims to provide a rice flower honey colloidal gold immunochromatography detection test strip and a preparation method and application thereof. To achieve the object of the present invention, in a first aspect, the present invention provides any one of the following uses of Ycf a protein or a truncate thereof: 1) The method is used for identifying the rice flower honey; 2) As a characteristic marker of the rice flower honey; 3) The fingerprint spectrum is used for constructing the rice flower honey; the Ycf protein is: (A) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2, or (B) A protein which is derived from (A) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2; preferably, the truncated body of Ycf protein is 255-698 amino acid sequences of Ycf protein; more preferably, the truncations of Ycf protein are: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1, or (B) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1. In a second aspect, the invention provides a method for identifying the authenticity of the rice ball honey, which comprises the steps of detecting components in the honey to be detected, and judging that the honey to be detected is the rice ball honey or the rice ball honey mixed with the rice ball honey if the components contain Ycf protein or a truncated body thereof. In a third aspect, the invention provides an anti-Ycf protein monoclonal antibody which is secreted by hybridoma cell line A11 with the preservation number of CGMCC No.46751 or hybridoma cell line C9 with the preservation number of CGMCC No. 46752. In a fourth aspect, the present invention provides a composition comprising a monoclonal antibody secreted by hybridoma cell line a11 having a accession number of CGMCC No.46751 and a monoclonal antibody secreted by hybridoma cell line C9 having a accession number of CGMCC No. 46752. In a fifth aspect, the invention provides any one of the following uses of the monoclonal antibody or the composition: (i) For detecting Ycf protein or a truncate thereof in a sample; (ii) Preparing a reagent, test paper or kit for detecting Ycf protein or truncated body thereof. In a sixth aspect, the invention provides a test strip for detecting colloidal gold immunochromatography of rice flower honey, which comprises a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are sequentially stacked; The gold-labeled pad is coated with a gold-labeled antibody formed by combining a first antibody with colloidal gold, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a second antibody, and the quality control line is coated with goat anti-mouse IgG; wherein the first antibody is a monoclonal antibody secreted by a hybridoma cell strain A11 with the preservation number of CGMCC No.46751, and the second antibody is a monoclonal antibody secreted by a hybridoma cell strain C9 with the preservation number of CGMCC No.46752, or The first