Joint detection kit for detecting intestinal polyps, and preparation method therefor, detection method therefor and use thereof

Abstract

A joint detection kit for detecting intestinal polyp factors, and a preparation method therefor, a detection method therefor and the use thereof. The joint detection kit at least comprises a plurality of test strips for detecting 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. Conjugate pads (2) of each reagent strip are coated with detection antibody-colloidal gold conjugates. Detection lines (3) of the reagent strip are coated with specific capture antibodies, and the specific capture antibodies on each test strip are different. The joint detection performed by the joint detection kit can effectively improve the sensitivity and specificity of the detection on intestinal polyps. The detection time is merely 15 min, with the detection rate of more than 91%. Therefore, the accuracy on the detection and diagnosis of colorectal cancer caused by intestinal polyps is improved.

Description

A combined detection kit for intestinal polyp detection and a preparation method, detection method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of combined detection kits, in particular to a combined detection kit for intestinal polyp factor detection and a preparation method, detection method and application thereof. BACKGROUND

[0002] Colorectal cancer is a cancer originating from the epithelium of the large intestine, and is a common malignant tumor. Early symptoms are mainly changes in bowel habits and stool appearance, which are easily ignored. In the later stage, symptoms such as abdominal pain, abdominal mass and progressive weight loss may occur. Early detection, early diagnosis and early treatment are the key to treatment, mainly through colonoscopy and surgical resection. Colorectal cancer is one of the common malignant tumors in China, and the incidence of rectal cancer is higher than that of colorectal cancer, accounting for 9.9% of all malignant tumors, and the mortality rate accounts for 8.0% of the mortality rate of all malignant tumors. The incidence of the disease in men is higher than that in women, the incidence in cities is higher than that in rural areas, and the incidence in eastern regions is higher than that in central and eastern regions and western regions. The incidence of colorectal cancer in China begins to rise after the age of 25, and peaks at the age of 41-65.

[0003] 90% of colorectal cancer is caused by colorectal polyps, which is a precancerous lesion. Within 4-5 years after the appearance of polyps, polyps will gradually become cancer. Therefore, a comprehensive understanding of polyps and early removal can effectively reduce the occurrence of colorectal cancer. Polyps refer to the excess lumps on the surface of human tissue, and modern medicine generally refers to the neoplasm growing on the surface of human mucosa as polyps. Colonic polyps refer to local raised lesions of the colonic mucosa, which are often distributed in multiple ways, and are hidden and grow at ordinary times, and then become colon cancer after atypical hyperplasia. The evolution process of "polyp-atypical hyperplasia-cancer" takes about 10-15 years, which provides an opportunity for the prevention and early diagnosis and treatment of colorectal cancer. At present, colonoscopy is the only method for detecting intestinal polyps, but it needs to go to the hospital and requires professional personnel to operate professional instruments, and the result detection time is long. SUMMARY

[0004] The purpose of the present application is to provide a combined detection kit for intestinal polyp factor detection in view of the complicated intestinal polyp detection and long time required for detection results in the prior art.

[0005] Another purpose of the present application is to provide a preparation method of the combined detection kit.

[0006] Another aspect of the present application provides a detection method based on the combined detection kit for non-diagnostic purposes.

[0007] Another aspect of the present application provides an application of the combined detection kit.

[0008] The technical scheme adopted to achieve the object of the present application is:

[0009] A combined detection kit for intestinal polyp detection, comprising at least nine test strips for detecting the following indexes: 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 (RBP4), serine protease inhibitor F2 (SERPIN-F2), calprotectin (CAL), and fecal occult blood (FOBT).

[0010] The connecting pad of each reagent strip is coated with a detection antibody-colloidal gold conjugate, and the detection line of the reagent strip is coated with a specific capture antibody.

[0011] In the above technical scheme, the combined detection kit has a detection rate of greater than 91%, a sensitivity of greater than 84%, and a specificity of greater than 93%.

[0012] In the above technical scheme, the detection limit of M2-pyruvate kinase is 5 ng / mL, the detection limit of matrix metalloproteinase 9 is 20 ng / mL, the detection limit of myeloperoxidase is 10 ng / mL, the detection limit of glutathione S-transferase Pi is 30 ng / mL, the detection limit of cytidine deaminase is 20 ng / mL, the detection limit of retinol binding protein 4 is 1 ng / mL, the detection limit of serine protease inhibitor F2 is 30 ng / mL, the detection limit of calprotectin is 20 ng / mL, and the detection limit of fecal occult blood is 100 ng / mL.

[0013] In the above technical scheme, the quality control area on the test strip is pre-coated with a goat anti-mouse IgG antibody.

[0014] In the above technical scheme, the test strip comprises a sample pad, a connecting pad, a detection pad, and an absorption pad that are sequentially overlapped.

[0015] In the above technical scheme, the combined detection kit further comprises a sample processing reagent, which comprises Tris, monohydrate citric acid, calcium chloride, BSA, and Proclin 300, and the sample processing method is as follows: after the sample is placed in a test tube containing the sample processing reagent, the test tube is sealed and shaken to mix uniformly.

[0016] In another aspect of the present application, a preparation method of the combined detection kit is provided, comprising the following steps:

[0017] Step 1, detection line coating: first prepare a specific capture antibody coating solution, and then coat the specific capture antibody coating solution on the detection line of the detection pad of the reagent strip, respectively;

[0018] Step 2, detection antibody-gold colloid coating.

[0019] In the above technical solution, the preparation method further comprises: step 3, quality control line coating, and the coating amount of each specific capture antibody on the quality control line is 3 mg / mL.

[0020] In another aspect of the present application, a non-diagnostic purpose detection method of the combined detection kit is provided, comprising the following steps:

[0021] Step S1, sample collection and post-processing;

[0022] Step S2, drop the sample after post-processing in step 1 into the sample addition hole of the combined detection kit, and wait for 15 min;

[0023] Step S3, observe and record the results, and at least two positive results in the nine indicators are positive for intestinal polyps.

[0024] In another aspect of the present application, the combined detection kit is applied to a product for intestinal polyp detection.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] 1. The present application finds that the combined detection of M2-pyruvate kinase, matrix metalloproteinase 9, myeloperoxidase, glutathione S-transferase Pi, cytidine deaminase, retinol binding protein 4, serine protease inhibitor, calprotectin and fecal occult blood can effectively improve the sensitivity and specificity of intestinal polyp detection, such as the detection rate, sensitivity and specificity of the combined detection of the present application are significantly improved compared with only detecting fecal occult blood.

[0027] 2. The detection time of the combined detection kit of the present application is only 15 min, which is shorter than the time required by the prior art, the sample sampling is more convenient, and the detection rate is higher than 91%. Further, the detection and diagnosis of colorectal cancer caused by intestinal polyps are improved in accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0028] Fig. 1 shows the structure of the test paper of the combined detection kit of the present application.

[0029] Fig. 2 shows a step diagram of sample collection and post-processing.

[0030] Fig. 3 shows the expression level of the biomarker group of related proteins in intestinal polyp tissue and normal intestinal tissue verified by immunohistochemistry of the embodiment of the present application.

[0031] In the figure: 1-sample pad, 2-connecting pad, 3-detection line, 4-quality control line, 5-detection pad, 6-absorbent pad, 7-backing, 8-treated sample. DETAILED DESCRIPTION

[0032] The application will be further described in connection with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the application and should not be used to limit the application.

[0033] Example 1

[0034] A combined detection kit for intestinal polyp detection comprises nine test strips as shown in Figure 1 for detecting the following indicators respectively: 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 (RBP4), serine protease inhibitor F2 (SERPIN-F2), calprotectin (CAL) and fecal occult blood (FOBT).

[0035] The connecting pad 2 of each reagent strip is coated with a detection antibody-colloidal gold conjugate, and the detection line 3 of the reagent strip is coated with a specific capture antibody. The specific capture antibodies on each test strip are not the same.

[0036] Specifically, the test strip comprises a sample pad 1, a connecting pad 2 (polyester material is used in this embodiment), a detection pad 5 (nitrocellulose membrane is used in this embodiment) and an absorbent pad 6 which are sequentially overlapped on the backing 7. The detection pad 5 is provided with a detection line 3 (T line) and a quality control line 4 (C line), and the quality control line 4 is pre-coated with a goat anti-mouse IgG antibody.

[0037] The combined detection kit further comprises a sample treatment reagent, which comprises Tris, citric acid monohydrate, calcium chloride, BSA and Proclin 300. The sample treatment method is to place the sample in a test tube containing the sample treatment reagent, then seal the test tube and shake to mix. The test tube uses the fecal quantitative sampler disclosed in patent 2020201791735

[0038] The coating on the test paper of the combined detection kit and its detection limit are shown in Table 1.

[0039] Table 1 Specific capture antibody coating amount and detection limit

[0040]

[0041] The combined detection kit of the application is developed by using the principle of colloidal gold immunochromatography technology and sandwich immunoassay. The detection line 3 on the nitrocellulose membrane of the test paper is coated with specific capture antibody, the quality control line 4 is coated with goat anti-mouse IgG antibody, and the polyester film is pre-coated with colloidal gold-labeled specific detection antibody. During detection, the sample climbs along the test strip and dissolves the detection antibody-colloidal gold conjugate on the polyester film. If the sample contains enteromucosal factor, the enteromucosal factor and the detection antibody-colloidal gold conjugate combine to form Ag-Ab-colloidal gold conjugate, which continues to climb to the nitrocellulose membrane and specifically collects with the specific capture antibody pre-coated on the detection line 3 to form Ab-Ag-Ab-colloidal gold, and accumulates to form a distinct micro-red band, which represents a positive result. If the sample does not contain enteromucosal factor, no obvious red band will be formed at the position of the detection line 3, which represents a negative result. The free detection antibody-colloidal gold continues to climb to the quality control line 4 and combines with the pre-coated goat anti-mouse IgG antibody to form a micro-red band, which represents the quality control line 4, i.e. the C line.

[0042] Example 2

[0043] A detection method based on the combined detection kit, comprising the following steps:

[0044] Step S1, sample collection and post-processing, as shown in Figure 2:

[0045] Step S1.1, take out the sample lysate from the kit;

[0046] Step S1.2, before collecting the feces, correctly install the sampling pad provided by the kit on the toilet according to the use requirements of the sampling pad;

[0047] Step S1.3, excrete the feces on the sampling pad;

[0048] Step S1.4, unscrew the cap of the sample lysate tube, and take out the sampling stick;

[0049] Step S1.5, directly stab the sampling stick into the excrement, rotate and then pull out. Repeat the above operation at 5 different positions in the sample until the grooves are filled with the sample;

[0050] Step S1.6, insert the sampling stick into the sample lysate tube, tighten the cap, and shake the sample tube up and down for 10 times, so that the feces are dissolved in the liquid, and the sample is ready for detection.

[0051] Step S2, add the sample 8 after step 1 post-processing to the sample pad 1 of the nine test strips of the combined detection kit respectively, and wait for 15 min. Wait for the results of the nine indicators;

[0052] Step S3, observing and recording the results, at least two positive indicators of nine indicators is positive for intestinal polyps.

[0053] Example 3

[0054] The detection method of Example 2 was used to detect human samples, the number of samples was 268, and the detection results are shown in Table 2. The method for calculating linear correlation is as follows: scatter plot is made for the detection results of two kinds of reagents, the detection value of the comparison reagent is taken as the X axis, and the detection value of the evaluation reagent is taken as the Y axis. Medcalc statistical software is used to perform linear correlation analysis on the detection values of the two kinds of reagents, and the regression equation y, the correlation coefficient r, and the 95% confidence interval are obtained.

[0055] Table 2 Statistical calculation results of the detection of the combined reagent kit of the application and the detection of fecal occult blood items

[0056]

[0057] From the data in Table 2, it can be seen that the comparison of the detection results of the combined reagent of the application and the single detection of FOB items shows that the combined detection results of the application improve the sensitivity and specificity of the detection of intestinal polyps, and further improve the accuracy of the detection and diagnosis of colorectal cancer caused by intestinal polyps.

[0058] The above is only the preferred embodiment of the application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the application, and these improvements and refinements should also be considered within the protection scope of the application.

Claims

1. A combined reagent kit for intestinal polyp detection, characterized by, at least nine test strips for detecting the following indicators: M2-pyruvate kinase, matrix metalloproteinase 9, myeloperoxidase, glutathione S-transferase Pi, cytosine deaminase, retinol binding protein 4, serine protease inhibitor F2, calprotectin and fecal occult blood; The connecting pad of each reagent strip is coated with a detection antibody-gold colloid conjugate, and the detection line of the reagent strip is coated with a specific capture antibody, and the specific capture antibodies on each test strip are different.

2. The combined test kit for the detection of intestinal polyp according to claim 1, wherein The detection rate of the combined detection kit is greater than 91%, the sensitivity is greater than 84%, and the specificity is greater than 93%.

3. The combined test kit for the detection of intestinal polyp according to claim 1, wherein The detection limit of M2-pyruvate kinase is 5 ng / mL, the detection limit of matrix metalloproteinase 9 is 20 ng / mL, the detection limit of myeloperoxidase is 10 ng / mL, the detection limit of glutathione S-transferase Pi is 30 ng / mL, the detection limit of cytosine deaminase is 20 ng / mL, the detection limit of retinol binding protein 4 is 1 ng / mL, the detection limit of serine protease inhibitor F2 is 30 ng / mL, the detection limit of calprotectin is 20 ng / mL, and the detection limit of fecal occult blood is 100 ng / mL.

4. The combined test kit for the detection of intestinal polyp according to claim 1, wherein The quality control area on the reagent strip is pre-coated with a goat anti-mouse IgG antibody.

5. The combined test kit for the detection of intestinal polyp according to claim 1, wherein The test strip comprises a sample pad, a connecting pad, a detection pad and an absorption pad connected in sequence.

6. The combined test kit for the detection of intestinal polyp according to claim 1, wherein The sample processing reagent comprises Tris, monohydrate citric acid, calcium chloride, BSA and Proclin 300, and the sample processing method is to place the sample in a test tube containing the sample processing reagent, then seal the test tube and shake to mix.

7. A method for preparing the combined test kit according to any one of claims 1 to 6, characterized in that, The method comprises the following steps: Step 1, detection line coating: first prepare a specific capture antibody coating solution, then coat the specific capture antibody coating solution on the detection line of the detection pad of the reagent strip respectively; Step 2, detection antibody-gold colloid coating.

8. The production method according to claim 7, wherein Further comprising: step 3, quality control line coating, the coating amount of each specific capture antibody on the quality control line is 3 mg / mL.

9. Use of the combined detection kit according to any one of claims 1-6 in a product for detecting intestinal polyps.

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