CN118067993A - Combined detection kit for intestinal polyp detection, and preparation method, detection method and application thereof - Google Patents
Combined detection kit for intestinal polyp detection, and preparation method, detection method and application thereof Download PDFInfo
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- 108090000235 Myeloperoxidases Proteins 0.000 claims abstract description 9
- 102100038246 Retinol-binding protein 4 Human genes 0.000 claims abstract description 9
- 101710137011 Retinol-binding protein 4 Proteins 0.000 claims abstract description 9
- 239000008280 blood Substances 0.000 claims abstract description 9
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- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 claims description 3
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a joint detection kit for intestinal polyp detection, a preparation method, a detection method and application thereof, wherein the joint detection kit at least comprises a plurality of test strips for detecting the following indexes: m2-pyruvate kinase, matrix metalloproteinase 9, myeloperoxidase, glutathione S-transferase Pi, cytidine deaminase, retinol binding protein 4, serine protease inhibitor F2, calprotectin, and fecal occult blood; the connection pad of each reagent strip is coated with a detection antibody-colloidal gold conjugate, the detection line of each reagent strip is coated with a specific capture antibody, and the specific capture antibodies on each test strip are different. The joint detection of the joint detection kit can effectively improve the sensitivity and specificity of the detection of intestinal polyps, the detection time is only 15min, and the detection rate is higher than 91%. And further improves the accuracy of detection and diagnosis of colorectal cancer caused by intestinal polyps.
Description
Technical Field
The invention relates to the technical field of joint inspection kits, in particular to a joint inspection kit for detecting intestinal polyp factors, a preparation method, a detection method and application thereof.
Background
Colorectal cancer is a cancer that originates in the epithelium of the large intestine, a common malignancy. Early symptoms are mostly changed into defecation habit and fecal character and are easily ignored; symptoms such as abdominal pain, abdominal mass, progressive wasting can occur in advanced stages. Early discovery, early diagnosis and early treatment are key to treatment, and mainly used for enteroscopy and surgical excision treatment.
90% Of colorectal cancers develop from colorectal polyps, a precancerous lesion, and within 4-5 years after the appearance of a polyp, the polyp becomes progressively malignant. Therefore, the polyp is comprehensively known and excised in advance, and the occurrence of colorectal cancer can be effectively reduced. Polyps are unwanted swelling that develops on the surface of human tissue, and modern medicine generally refers to neoplasms that develop on the surface of human mucosa as polyps. The polyp of the large intestine refers to local swelling lesions higher than intestinal mucosa, which are frequently distributed in multiple ways, are hidden to grow at ordinary times, and become colon cancer after atypical hyperplasia. The evolution process of polyp-atypical hyperplasia-cancer is mainly carried out for 10-15 years, and the precious time window provides opportunities for prevention and early diagnosis and early treatment of colorectal cancer. Currently, enteroscopy is the only method for detecting intestinal polyps, but needs to go to a hospital and a specialized person to operate a specialized instrument, so that the detection time is long.
Disclosure of Invention
The invention aims to provide a joint detection kit for detecting intestinal polyp factors, aiming at the problems of complex detection and time required by detection results in the prior art.
The invention further provides a preparation method of the joint inspection kit.
In another aspect of the invention, a method for detecting non-diagnostic objects based on the joint test kit is provided.
In another aspect of the invention, there is provided the use of the kit.
The technical scheme adopted for realizing the purpose of the invention is as follows:
a joint inspection kit for intestinal polyp detection, comprising at least nine test strips with the following indicators: m2-pyruvate kinase (M2-PK), matrix metalloproteinase 9 (MMP-9), myeloperoxidase (MPO), glutathione S-transferase Pi (GST-Pi), cytidine Deaminase (CDA), retinol binding protein 4 (RBP 4), serine protease inhibitor F2 (SERPIN-F2), calprotectin (CAL), and occult blood of stool (FOBT);
the connection pad of each reagent strip is coated with a detection antibody-colloidal gold conjugate, the detection line of each reagent strip is coated with a specific capture antibody, and the specific capture antibodies on each test strip are different.
In the technical scheme, the detection rate of the joint inspection kit is more than 91%, the sensitivity is more than 84%, and the specificity is more than 93%.
In the above technical scheme, the detection limit of M2-pyruvate kinase is 5ng/mL, the detection limit of matrix metalloproteinase 9 is 20ng/mL, the detection limit of myeloperoxidase is 10ng/mL, the detection limit of glutathione S-transferase Pi is 30ng/mL, the detection limit of cytidine deaminase is 20ng/mL, the detection limit of retinol binding protein 4 is 1ng/mL, the detection limit of serine proteinase inhibitor F2 is 30ng/mL, the detection limit of calprotectin is 20ng/mL, and the detection limit of fecal occult blood is 100ng/mL.
In the above technical scheme, the quality control region on the test strip is pre-coated with goat anti-mouse IgG antibody.
In the above technical scheme, the test strip comprises a sample pad, a connecting pad, a detection pad and an absorption pad which are sequentially overlapped.
In the above technical scheme, the joint inspection kit further comprises a sample treatment reagent, wherein the sample treatment reagent comprises Tris, citric acid monohydrate, calcium chloride, BSA and Proclin300, and the sample treatment method comprises the following steps: placing the sample into a test tube containing a sample processing reagent, and then sealing the test tube, shaking and uniformly mixing.
In another aspect of the present invention, a method for preparing the joint inspection kit is provided, comprising the steps of:
step 1, coating a detection line: firstly preparing a specific capture antibody coating liquid, and then respectively coating the specific capture antibody coating liquid on detection lines of detection pads of the reagent strips;
And 2, coating detection antibody-colloidal gold.
In the above technical solution, the preparation method further includes: and step 3, coating a quality control line, wherein the coating amount of each specific capture antibody on the quality control line is 3mg/mL.
In another aspect of the present invention, there is provided a method for detecting a non-diagnostic purpose of the joint inspection kit, comprising the steps of:
Step S1, sample collection and post-treatment;
step S2, dripping the sample processed in the step 1 into a sample adding hole of the joint inspection kit, and waiting for 15min;
and S3, observing and recording results, wherein at least two positives in the nine indexes are intestinal polyp positives.
In another aspect of the invention, there is provided the use of the kit in a product for intestinal polyp detection.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention discovers that the joint detection of M2-pyruvate kinase, matrix metalloproteinase 9, myeloperoxidase, glutathione S-transferase Pi, cytidine deaminase, retinol binding protein 4, serine proteinase inhibitor, calprotectin and fecal occult blood can effectively improve the sensitivity and specificity of the detection of intestinal polyps, for example, compared with the detection of fecal occult blood only, the detection rate, sensitivity and specificity of the joint detection adopting the invention are all obviously improved.
2. The detection time of the joint inspection kit provided by the invention is only 15min, and compared with the detection time required by the prior art, the joint inspection kit is shorter, the sample sampling is more convenient, and the detection rate is higher than 91%. And further improves the accuracy of detection and diagnosis of colorectal cancer caused by intestinal polyps.
Drawings
Fig. 1 is a schematic structural diagram of a test paper of the joint inspection kit of the present invention.
FIG. 2 is a schematic diagram showing steps of sample collection and post-treatment.
FIG. 3 shows the immunohistochemistry of an embodiment of the present invention to verify the expression level of a biomarker panel consisting of related proteins in intestinal polyp tissue and normal intestinal tissue.
In the figure: 1-sample pad, 2-connection pad, 3-detection line, 4-quality detection line, 5-detection pad, 6-absorption pad, 7-backing, 8-treated sample.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A joint inspection kit for intestinal polyp detection, comprising nine test strips as shown in fig. 1 with the following indices detected separately: m2-pyruvate kinase (M2-PK), matrix metalloproteinase 9 (MMP-9), myeloperoxidase (MPO), glutathione S-transferase Pi (GST-Pi), cytidine Deaminase (CDA), retinol binding protein 4 (RBP 4), serine protease inhibitor F2 (SERPIN-F2), calprotectin (CAL), and occult blood of stool (FOBT);
The connection pad 2 of each reagent strip is coated with a detection antibody-colloidal gold conjugate, the detection line 3 of the reagent strip is coated with a specific capture antibody, and the specific capture antibodies on each test strip are different.
Specifically, the test strip includes a sample pad1, a connection pad 2 (polyester material is used in this embodiment), a detection pad 5 (nitrocellulose membrane is used in this embodiment), and an absorption pad 6, which are sequentially overlapped on a backing 7. The detection line 3 (T line) and the quality control line 4 (C line) are arranged on the detection pad 5, and the quality control line 4 is pre-coated with goat anti-mouse IgG antibody.
The joint inspection kit further comprises a sample treatment reagent, wherein the sample treatment reagent comprises Tris, citric acid monohydrate, calcium chloride, BSA and Proclin300, and the sample treatment method comprises the following steps: placing the sample into a test tube containing a sample processing reagent, and then sealing the test tube, shaking and uniformly mixing. The test tube adopts the excrement quantitative sampler disclosed in patent 2020201791735
The test paper upper coating of the joint inspection kit has the detection limit shown in table 1.
TABLE 1 coating amount and detection limit of specific capture antibody
The joint inspection kit is developed by utilizing the principles of a colloidal gold immunochromatography technology and a sandwich immunoassay. The detection line 3 on the nitrocellulose membrane of the test paper is coated with a specific capture antibody, the quality control line 4 is coated with a goat anti-mouse IgG antibody, and the polyester membrane is pre-coated with a specific detection antibody marked by colloidal gold. During detection, a sample upwards crawls along a test strip to dissolve a detection antibody-colloidal gold conjugate on a polyester film, if intestinal polyp factors exist in the sample, the intestinal polyp factors are combined with the detection antibody-colloidal gold conjugate to form an Ag-Ab-colloidal gold conjugate, the conjugate continues to crawl onto a nitrocellulose film to form a specific collection of a specific capture antibody pre-coated with a detection line 3, ab-Ag-Ab-colloidal gold is formed, and a strip with obvious reddish color is formed by stacking and developing, namely a T line, which represents a positive result. If the sample does not contain an intestinal polyp factor, no distinct red band is formed at the detection line 3 position, which represents a negative result. The free detection antibody-colloidal gold continues to climb to the upper part of the test paper to enable the quality testing line 4 to be combined with the pre-coated goat anti-mouse IgG antibody, and the combination is piled up for color development to form a reddish strip, which represents the quality testing line 4, namely a C line.
Example 2
The detection method based on the joint inspection kit comprises the following steps of:
Step S1, sample collection and post-processing, as shown in fig. 2:
s1.1, taking out a sample lysate from the kit;
Step S1.2, before collecting the excrement, correctly installing a sampling pad provided by the kit on a toilet according to the use requirement of the sampling pad;
Step S1.3, excreting the feces on the sampling pad;
S1.4, unscrewing a cover of the sample lysate tube, and taking out a sampling rod;
and S1.5, directly penetrating the sampling rod into excrement, rotating and then extracting. Repeating the above operation at 5 different positions in the sample until the recess is filled with the sample;
And S1.6, inserting a sampling rod into the sample lysate tube, screwing the cover, holding the sample tube upside down and shaking for 10 times, so that the excrement is dissolved in the liquid, and waiting for detection.
And S2, respectively dripping the samples 8 after the post-treatment in the step 1 into sample adding holes on sample pads 1 of nine test strips of the joint inspection kit, and waiting for 15min. Waiting for the results of nine indexes;
and S3, observing and recording results, wherein at least two positives in the nine indexes are intestinal polyp positives.
Example 3
The human body samples were detected by the detection method of example 2, the number of samples was 268, the detection results are shown in table 2, and the method for calculating the linear correlation is as follows: and (3) performing a scatter diagram on the measurement results of the two reagents, taking the measurement value of the comparative reagent as an X axis, the measurement value of the checking reagent as a Y axis, and performing linear correlation analysis on the measurement values of the two reagents by Medcalc statistical software to obtain a confidence interval with a regression equation of Y, a correlation coefficient r and 95%.
TABLE 2 statistical calculation results of the joint inspection kit detection and fecal occult blood item detection of the present invention
As can be seen from the data in Table 2, the comparison of the results of the joint inspection reagent detection and FOB item single detection of the invention shows that the joint inspection result of the invention improves the sensitivity and specificity of the detection of intestinal polyps, and further improves the accuracy of the detection and diagnosis of colorectal cancer caused by intestinal polyps.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A joint inspection kit for intestinal polyp detection, comprising at least nine test strips with the following indicators: m2-pyruvate kinase, matrix metalloproteinase 9, myeloperoxidase, glutathione S-transferase Pi, cytidine deaminase, retinol binding protein 4, serine protease inhibitor F2, calprotectin, and fecal occult blood;
the connection pad of each reagent strip is coated with a detection antibody-colloidal gold conjugate, the detection line of each reagent strip is coated with a specific capture antibody, and the specific capture antibodies on each test strip are different.
2. The joint detection kit for intestinal polyp detection according to claim 1, wherein the joint detection kit has a detection rate of greater than 91%, a sensitivity of greater than 84%, and a specificity of greater than 93%.
3. The joint test kit for intestinal polyp detection according to claim 1, wherein the limit of detection of M2-pyruvate kinase is 5ng/mL, the limit of detection of matrix metalloproteinase 9 is 20ng/mL, the limit of detection of myeloperoxidase is 10ng/mL, the limit of detection of glutathione S-transferase Pi is 30ng/mL, the limit of detection of cytidine deaminase is 20ng/mL, the limit of detection of retinol binding protein 4 is 1ng/mL, the limit of detection of serine proteinase inhibitor F2 is 30ng/mL, the limit of detection of calprotectin is 20ng/mL, and the limit of detection of fecal occult blood is 100ng/mL.
4. The joint detection kit for intestinal polyp detection of claim 1, wherein the quality control zone on the strip is pre-coated with goat anti-mouse IgG antibody.
5. The joint test kit for intestinal polyp detection of claim 1, wherein the test strip comprises a sample pad, a connection pad, a detection pad, and an absorption pad that overlap in sequence.
6. The joint test kit for intestinal polyp detection of claim 1, further comprising sample processing reagents including Tris, citric acid monohydrate, calcium chloride, BSA, and Proclin300, the sample processing method being: placing the sample into a test tube containing a sample processing reagent, and then sealing the test tube, shaking and uniformly mixing.
7. A method of preparing a joint test kit according to any one of claims 1 to 6, comprising the steps of:
step 1, coating a detection line: firstly preparing a specific capture antibody coating liquid, and then respectively coating the specific capture antibody coating liquid on detection lines of detection pads of the reagent strips;
And 2, coating detection antibody-colloidal gold.
8. The method of manufacturing of claim 7, further comprising: and step 3, coating a quality control line, wherein the coating amount of each specific capture antibody on the quality control line is 3mg/mL.
9. Use of a joint test kit according to any one of claims 1-6 in a product for the detection of intestinal polyps.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118243929A (en) * | 2024-05-30 | 2024-06-25 | 弗雷米德生物医药技术（天津）有限公司 | Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit |
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| CN118243929B (en) * | 2024-05-30 | 2024-08-13 | 弗雷米德生物医药技术（天津）有限公司 | Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit |
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