CN118243929A - Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit - Google Patents
Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit Download PDFInfo
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Abstract
The invention discloses a joint detection kit for detecting precancerous lesions and cancerations of cervical cancer, and a preparation method and application thereof, wherein the joint detection kit at least comprises a plurality of test strips for detecting the following indexes: e5 protein, E6 protein, E7 protein and hTERT; 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 invention discovers that the joint detection of the E5 protein, the E6 protein, the E7 protein and the hTERT (telomerase component) can effectively improve the sensitivity and the specificity of detection of precancerous lesions and cancerations of cervical cancer, for example, compared with the detection of the E7 protein only, the detection rate, the sensitivity and the specificity of the joint detection kit are obviously improved.
Description
Technical Field
The invention relates to the technical field of detection of precancerous lesions and cancerations of cervical cancer, in particular to a joint detection kit for detection of precancerous lesions and cancerations of cervical cancer, and a preparation method and application thereof.
Background
Cervical cancer is the fourth cancer next to breast cancer among women worldwide, locating the first place of female genital tract malignancy. The related statistics show that nearly 50 thousands of new cervical cancer cases occur annually worldwide, wherein 83% of cases occur in 13.15 thousands of new cervical cancer cases in China in developing countries, accounting for 28.8%, 29 tens of thousands of cervical cancer cases annually worldwide die, and 3 tens of thousands of cervical cancer cases in China. 57 ten thousand cervical cancer patients in 2018 and 31.1 ten thousand deaths. Since early symptoms of cervical cancer are not obvious, early screening, auxiliary diagnosis, prevention and intervention are particularly important. Currently screening for cervical cancer mainly involves methods of detecting tissue cell levels and molecular levels. Such as clinical thin layer liquid-based cell smear (TCT) and colposcopy, require special instruments and special technicians, and the result judgment also requires a lot of experience support, which is not suitable for early rapid screening of cervical cancer. In addition, thin-layer liquid-based cell smear (TCT) detection is limited to analysis of whether the cell morphology is normal, and the abnormal cells are mostly cervical inflammation, not equivalent to cervical cancer, or require biopsy to determine precancerous lesions or cancerations of cervical cancer.
With the development of medical experimental techniques and the deepening of knowledge of the relationship between Human Papillomavirus (HPV) and cervical lesions, the examination method for HPV infection has also been developed from the tissue cell level to the molecular level. However, HPV detection is limited to detection of HPV virus, HPV infection is transient, HPV infection is not equal to cervical cancer, or biopsy is required to determine precancerous lesions or cancerations of cervical cancer. Common detection methods for HPV include sequencing, hybridization capture and gene chip methods. But also requires expensive equipment and requires high demands on the operator. The hybridization capture method is complex to operate, takes a long time, takes about 5 hours at a time, and is easy to cause cross reaction. The gene chip method products (such as PCR-reverse dot hybridization method and liquid chip method) have the defects that real-time detection cannot be realized, analysis operation of amplified products is needed, more than 4 hours are consumed, the same operation is complex, PCR product pollution possibly exists, and the requirement of mass screening of clinical cervical cancer cannot be met.
Disclosure of Invention
The invention aims at providing a joint detection kit for detecting precancerous lesions and canceration of cervical cancer aiming at complex detection of precancerous lesions and canceration and time required for detecting results in the prior art
The invention also aims to provide a preparation method of the joint inspection kit.
It is another object of the present invention to provide the use of the kit for joint inspection.
The technical scheme adopted for realizing the purpose of the invention is as follows:
A joint detection kit for detection of precancerous lesions and cancerous lesions of cervical cancer, comprising at least a plurality of test strips having the following indicators: e5 protein, E6 protein, E7 protein and hTERT;
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 above technical scheme, the precancerous lesions of cervical cancer comprise low-grade precancerous lesions of cervical cancer and high-grade precancerous lesions of cervical cancer, the detection rate of the low-grade precancerous lesions of cervical cancer is greater than 96%, the sensitivity is greater than 90%, the specificity is greater than 96%, the detection rate of the high-grade precancerous lesions of cervical cancer is greater than 94%, the sensitivity is greater than 89%, the specificity is greater than 95%, and the detection rate of the cervical cancer combined detection kit is greater than 91%, the sensitivity is greater than 87% and the specificity is greater than 92%.
In the above technical scheme, the detection limit of the E5 protein is 35ng/mL, the detection limit of the E6 protein is 25ng/mL, the detection limit of the E7 protein is 20ng/mL, and the detection limit of the hTERT is 30ng/mL.
In the above technical scheme, the quality control line 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 sample processing reagent further comprises a sample processing reagent, wherein the sample processing reagent comprises Tris, naCI, tritonX-100 and BSA, and the sample processing 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 inspection line, wherein the coating amount of each specific capture antibody on the quality inspection line is 1.5mg/mL.
In another aspect of the present invention, a method for detecting a non-diagnostic target based on the joint inspection kit is provided, 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 the result.
In another aspect, the invention provides the application of the joint inspection kit in products for detecting premalignant lesions and canceration of cervical cancer.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention discovers that the joint detection of the E5 protein, the E6 protein, the E7 protein and the hTERT (telomerase component) can effectively improve the sensitivity and the specificity of detection of precancerous lesions and cancerations of cervical cancer, for example, compared with the detection of the E7 protein only, the detection rate, the sensitivity and the specificity of the joint detection kit are obviously improved.
2. The joint inspection kit greatly reduces the false negative of cervical cancer screening through four joint inspection of E5 protein, E6 protein, E7 protein and hTERT, reduces the false positive caused by HPV infection one-way property, and can be used for cervical cancer screening at different stages, especially early screening, and greatly improves the detection accuracy of precancerous lesions and canceration of cervical cancer.
3. The detection time of the joint detection kit provided by the invention is only 15min, compared with the detection time required by the prior art, the sample sampling is more convenient, the detection rate of the lesion before cervical cancer is higher than 91%, and the cervical cancer change detection rate is higher than 89%. The detection and diagnosis precision and efficiency of cervical cancer pre-lesion and canceration are improved.
Drawings
Fig. 1 is a schematic structural diagram of a test paper of the joint inspection kit of the present invention.
FIG. 2 shows immunohistochemical verification of the expression levels of biomarker panels consisting of related proteins in pre-cervical cancer lesions and cancerous tissue and normal cervical epithelial cell tissue in accordance with an embodiment of the present invention.
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 detection of precancerous lesions and cancerous lesions of cervical cancer, comprising four test strips as shown in fig. 1 with the following indices detected separately: e5 protein, E6 protein, E7 protein, hTERT (telomerase moiety);
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 pad 1, 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 also comprises a sample treatment reagent, wherein the sample treatment reagent comprises Tris, naCI, tritonX-100 and BSA, 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 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 and dissolves detection antibody-colloidal gold conjugate on a polyester film along a test strip, if E5 protein, E6 protein, E7 protein and hTERT exist in the sample, the E5 protein, the E6 protein, the E7 protein and the hTERT are combined with the detection antibody-colloidal gold conjugate to form Ag-Ab-colloidal gold conjugate, the conjugate continues to crawl onto a nitrocellulose film to form Ab-Ag-Ab-colloidal gold together with a specific capture antibody pre-coated on a detection line 3, and a marked reddish strip, namely a T line, is formed by accumulation and color development, and represents a positive result. If the sample does not contain E5 protein, E6 protein, E7 protein and hTERT, no obvious red band is 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 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-treatment:
Step S1.1, taking out a sample releasing agent from the kit;
step S1.2, before collecting cervical exfoliated cells, operating according to the use requirement of a sampling cervical brush;
Step S1.3, the test tube with the sample releasing agent is gently turned upside down three times and placed on the table top. Then holding the test tube with one hand and gently unscrewing the test tube cover with the other hand to ensure that the test tube opening and the cover opening are both upward;
s1.4, inserting a cervical sampling brush into a test tube port, lightly pressing the cervical sampling brush with force, and placing the cervical sampling brush in place;
s1.5, fixing the sampling brush by one hand, slightly pushing the sampling brush by the other hand, immersing the brush head in liquid, and rotating for 10 circles after the brush head contacts the bottom of the test tube to fully scratch the rib positions at the bottom of the reagent bottle;
and S1.6, pulling the sampling brush out of the test tube by one hand, lightly screwing a test tube cover, and standing the test tube vertically at room temperature for 20-30 minutes.
And S2, respectively dripping the samples 8 after the post-treatment in the step 1 into sample adding holes on sample pads 1 of four test strips of the joint detection kit, and waiting for 15min. Waiting for the results of the four indexes;
and S3, observing and recording the result, wherein at least one positive index of the four indexes is cervical cancer pre-lesion or cancerous positive.
Example 3
The human body sample was detected by the detection method of example 2, the detection results are shown in table 2, table 3 and table 4, 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 calculation of CIN1 (low cervical precancerous lesions) joint detection kit detection and E7 protein detection
TABLE 3 calculation of CIN2-3 (high cervical precancerous lesion) joint detection kit detection and E7 protein detection
TABLE 4 statistical calculation results of cervical cancer mutation detection kit detection and E7 protein detection
As can be seen from the data in tables 2, 3 and 4, the comparison of the results of the detection of the joint detection reagent and the detection of the E7 protein item list shows that the joint detection result of the invention improves the sensitivity and specificity of the detection of the precancerous lesions and the cancerous lesions, and further improves the accuracy of the detection and the diagnosis of the precancerous lesions and the cancerous lesions.
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 (10)
1. A joint inspection kit for detection of precancerous lesions and cancerous lesions of cervical cancer, comprising at least a plurality of test strips having the following indicators: e5 protein, E6 protein, E7 protein and hTERT;
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 the detection of precancerous lesions and canceration according to claim 1, wherein the precancerous lesions of cervical cancer comprise low-grade precancerous lesions and high-grade precancerous lesions of cervical cancer, the detection rate of the low-grade precancerous lesions of cervical cancer is greater than 96%, the sensitivity is greater than 90%, the specificity is greater than 96%, the detection rate of the high-grade precancerous lesions of cervical cancer is greater than 94%, the sensitivity is greater than 89%, the specificity is greater than 95%, the detection rate of the joint detection kit of cervical cancer is greater than 91%, the sensitivity is greater than 87%, and the specificity is greater than 92%.
3. The joint detection kit for cervical cancer precancerous lesions and canceration detection according to claim 1, wherein the detection limit of the E5 protein is 35ng/mL, the detection limit of the E6 protein is 25ng/mL, the detection limit of the E7 protein is 20ng/mL, and the detection limit of the hTERT is 30ng/mL.
4. The joint detection kit for the detection of precancerous lesions and cancerations of cervical cancer according to claim 1, wherein the quality control line on the test strip is pre-coated with goat anti-mouse IgG antibodies.
5. The joint detection kit for the detection of precancerous lesions and cancerations of cervical cancer according to claim 1, wherein the test strip comprises a sample pad, a connection pad, a detection pad and an absorption pad which are sequentially overlapped.
6. The joint detection kit for detection of precancerous lesions and carcinomas of cervical cancer according to claim 1, further comprising sample processing reagents comprising Tris, naCI, tritonX-100 and BSA, 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 inspection line, wherein the coating amount of each specific capture antibody on the quality inspection line is 1.5mg/mL.
9. A method for detecting a non-diagnostic object based on the joint inspection kit according to any one of claims 1 to 6, characterized by 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 the result.
10. Use of a joint test kit according to any one of claims 1-6 in a product for the detection of pre-cervical lesions and carcinomas.
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