Cervical cancer screening method based on TCT detection

Abstract

The invention relates to a cervical cancer screening method based on TCT detection, which comprises the following steps: S1, taking cervical exfoliated cells, and storing the cervical exfoliated cells in a TCT liquid-based cell sampling tube; s2, putting the TCT liquid-based cell sampling tube into a centrifugal machine, oscillating and uniformly mixing to obtain a first sample; taking 500 [mu] l of the first sample, and carrying out centrifugal treatment to obtain a second sample; s4, adding 1ml of absolute ethyl alcohol into the second sample to obtain a third sample; s5, carrying out centrifugal treatment on the third sample, discarding the supernatant, and taking the precipitate to obtain a fourth sample; s6, adding 1mL of normal saline into the fourth sample to obtain a fifth sample; s7, carrying out centrifugal treatment on the fifth sample, discarding the supernatant, and taking the precipitate to obtain a sixth sample; s8, adding 120 [mu] l of a cell lysis solution into the sixth sample, and carrying out heating treatment to obtain a seventh sample; and S9, carrying out centrifugal treatment on the seventh sample, and taking supernatant to obtain a detection sample for PCR amplification. The screening method provided by the invention can achieve the purpose of one-tube double-detection, and reduces the pain of a patient in sampling at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical testing, and in particular relates to a cervical cancer screening method based on TCT detection. Background Art

[0002] In the 1970s, German Professor Hausen discovered that the occurrence of cervical cancer is directly related to HPV infection. Since then, the study of cervical cancer has begun to shift from cytology to molecular medicine. HPV is a spherical DNA virus that can cause genital warts and genital cancer. Currently, there are as many as 130 types of high-risk and low-risk HPV isolated. The main cause of cervical cancer is persistent infection with HPV. HPV can be detected in almost all precancerous lesions and cervical cancer tissue specimens. Different types of HPV have different carcinogenic abilities. In view of the differences in carcinogenic activity, it is clinically important to detect HPV infection and identify high-risk HPV types. Therefore, the use of HPV testing in cervical cancer screening helps to increase the screening rate.

[0003] At present, HPV-positive people need to undergo two cervical cell sampling to complete the initial cervical screening. The reason is that the main components of the HPV cell preservation fluid and TCT liquid-based cell preservation fluid used in the test are different, which makes the same sampling tube cannot be used in both HPV-DNA testing and TCT liquid-based cell testing. Moreover, the above method has the following problems: 1. The positive rate of TCT cervical cytology decreases: This is because the samples needed for cervical cancer screening mainly come from the exfoliated cells on the surface of the cervix, and the exfoliated cells mainly come from the squamous epithelium on the surface of the cervix. A sufficient amount of cervical exfoliated cells need to be collected for an HPV test, and the squamous epithelial cells of the cervical epidermis need at least 30 days to regenerate a sufficient amount of mature cells; if the interval between multiple sampling is short, there will be too few or no effective cells, and the result is a false negative, so the two samplings should be separated by more than 20 days; 2. Increase the workload of primary care doctors for repeated sampling and recall of positive people; 3. Repeated vaginal sampling in a short period of time increases the probability of vaginal damage in the tested population, resulting in a decrease in the cooperation of the tested population and a decrease in the recall rate; 4. It will take a longer time for HPV-positive people to complete the initial screening, which is easy to cause missed screening.

[0004] Therefore, the present invention provides a cervical cancer screening method based on TCT detection to solve the above technical problems. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a cervical cancer screening method based on TCT detection, which can achieve the purpose of double detection in one tube while reducing the pain of sampling for patients.

[0006] The present invention provides a cervical cancer screening method based on TCT detection, which comprises the following steps:

[0007] S1, extracting a sample to be tested, and placing the sample to be tested in a TCT liquid-based cell sampling tube containing a TCT liquid-based cell preservation solution for preservation; wherein the sample to be tested is cervical exfoliated cells, and the components of the TCT liquid-based cell preservation solution include an organic solvent, water, and a fixative;

[0008] S2, placing the TCT liquid-based cell sampling tube containing the cervical exfoliated cells in a centrifuge, and shaking and mixing at a speed of 2500 r / min for 5 to 10 minutes to obtain a first sample;

[0009] S3, extracting 500ul of the first sample and placing it in a centrifuge, and centrifuging it at a speed of 13000r / min-15000r / min for 5 minutes to 10 minutes to precipitate the cervical exfoliated cells to obtain a second sample;

[0010] S4, adding 1 ml of anhydrous ethanol to the second sample to remove the organic solvent in the second sample to obtain a third sample;

[0011] S5, placing the third sample in a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, discarding the supernatant and taking the precipitate to obtain a fourth sample;

[0012] S6, adding 1 mL of physiological saline to the fourth sample to purify the cervical exfoliated cells to obtain a fifth sample;

[0013] S7, placing the fifth sample in a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, discarding the supernatant and taking the precipitate to obtain a sixth sample;

[0014] S8, adding 120 ul of cell lysate to the sixth sample, placing the sixth sample with the cell lysate in a heating device, and heating at a temperature of 95 degrees Celsius to 100 degrees Celsius for 10 minutes to 15 minutes to obtain a seventh sample;

[0015] S9, placing the seventh sample into a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, taking the supernatant to obtain a test sample for PCR amplification.

[0016] Preferably, the organic solvent includes ethanol, methanol and isopropanol; the cell lysis solution includes potassium chloride, tris(hydroxymethyl)amino and polyethylene glycol octylphenyl ether.

[0017] Preferably, the heating device is a dry thermostat; the reagent used for PCR amplification in S9 is a human papillomavirus genotyping (type 23) amplification reagent.

[0018] Compared with the related art, the present invention provides a cervical cancer screening method based on TCT detection, wherein anhydrous ethanol is added in step S4 to dissolve the organic solvent in the TCT liquid-based cell preservation solution into anhydrous ethanol, and the organic solvent is dissolved by the principle of similar dissolution of organic solvents, and then the waste liquid is discarded by centrifugation and supernatant discarding in step S5 to preserve cervical exfoliated cells; physiological saline is added in step S6, and the property that anhydrous ethanol can be miscible with water in any proportion is utilized, and then the waste liquid is discarded by centrifugation and supernatant discarding in step S7 to further purify the cells; after two rounds of centrifugal purification, the residual organic solvent in the TCT liquid-based cell preservation solution has been basically removed, thereby not affecting the downstream PCR amplification. The present invention optimizes the process of HPV detection, so that the sampled specimens in the TCT liquid-based cell preservation solution can be used for both TCT liquid-based cell detection and HPV-DNA detection, achieving the purpose of double detection in one tube, and it does not need to perform sampling twice, thereby shortening the screening cycle of cervical cancer, reducing the probability of missed diagnosis and sampling cost, and reducing the pain of sampling for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of a process of a cervical cancer screening method based on TCT detection according to the present invention;

[0020] Figure 2 A schematic diagram of the detection steps of a cervical cancer screening method based on TCT detection according to the present invention;

[0021] Figure 3 It is a schematic diagram of the detection results of a cervical cancer screening method based on TCT detection using the present invention. DETAILED DESCRIPTION

[0022] The present invention provides a cervical cancer screening method based on TCT detection, aiming to solve the problem in the existing cervical cancer screening method that the same sampling tube cannot be used for TCT liquid-based cell detection and HPV-DNA detection at the same time.

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Please see attached Figure 1As shown, the present invention provides a cervical cancer screening method based on TCT detection, which specifically includes the following steps:

[0025] S1, extracting a sample to be tested, and placing the sample to be tested in a TCT liquid-based cell sampling tube containing a TCT liquid-based cell preservation solution for preservation; wherein the sample to be tested is cervical exfoliated cells, and the components of the TCT liquid-based cell preservation solution include an organic solvent, water, and a fixative;

[0026] S2, placing the TCT liquid-based cell sampling tube containing the cervical exfoliated cells in a centrifuge, and shaking and mixing at a speed of 2500 r / min for 5 to 10 minutes to obtain a first sample;

[0027] S3, extracting 500ul of the first sample and placing it in a centrifuge, and centrifuging it at a speed of 13000r / min-15000r / min for 5 minutes to 10 minutes to precipitate the cervical exfoliated cells to obtain a second sample;

[0028] S4, adding 1 ml of anhydrous ethanol to the second sample to remove the organic solvent in the second sample to obtain a third sample;

[0029] S5, placing the third sample in a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, discarding the supernatant and taking the precipitate to obtain a fourth sample;

[0030] S6, adding 1 mL of physiological saline to the fourth sample to purify the cervical exfoliated cells to obtain a fifth sample;

[0031] S7, placing the fifth sample in a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, discarding the supernatant and taking the precipitate to obtain a sixth sample;

[0032] S8, adding 120 ul of cell lysate to the sixth sample, placing the sixth sample with the cell lysate in a heating device, and heating at a temperature of 95 degrees Celsius to 100 degrees Celsius for 10 minutes to 15 minutes to obtain a seventh sample;

[0033] S9, placing the seventh sample into a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, taking the supernatant to obtain a test sample for PCR amplification.

[0034] It should be noted that, since some alcoholic organic solvents are often present in the TCT liquid-based cell preservation solution, if such solvents are not removed cleanly, the subsequent PCR amplification reaction will be affected. Therefore, in this embodiment, anhydrous ethanol is added in step S4 to dissolve the organic solvent in the TCT liquid-based cell preservation solution into the anhydrous ethanol, and the organic solvent is dissolved by the principle of like dissolving like of organic solvents, and then the waste liquid is discarded by centrifugation and supernatant discarding in step S5 to preserve the cervical exfoliated cells; in addition, physiological saline is added in step S6, and the property that anhydrous ethanol can be miscible with water in any proportion is utilized, and then the waste liquid is discarded by centrifugation and supernatant discarding in step S7 to further purify the cells; after two rounds of centrifugation and purification, the residual organic solvent in the TCT liquid-based cell preservation solution has been basically removed, so as not to affect the downstream PCR amplification.

[0035] In this embodiment, the organic solvent includes ethanol, methanol and isopropanol; the cell lysis solution includes potassium chloride, tris(hydroxymethyl)amino and polyethylene glycol octylphenyl ether; the heating device is a dry thermostat; the reagent used for PCR amplification in S9 is a human papillomavirus genotyping (type 23) amplification reagent.

[0036] It is worth noting that Figure 2 As shown, the specific steps of the cervical cancer screening method based on TCT detection provided by the present invention are: using TCT liquid-based cell sampling tubes to preserve the patient's sample specimens, performing HPV testing on the sample specimens, and completing the screening if the HPV test is negative; if the HPV test is positive, the inspection personnel initiate the TCT additional process, directly transfer the sample specimens to the pathology center, and enter the TCT testing process. The detection steps are completed by the laboratory staff from the completion of sampling to entering the laboratory. The front-end doctors can understand the test results and circulation of the samples through the system. The entire initial screening process reduces the workload of the front-end doctors for two samplings, TCT sampling work, and HPV positive sample follow-up.

[0037] Please refer to Figure 3 As shown, in order to further verify the cervical cancer screening method based on TCT detection provided by the present invention, samples were taken for practical testing, and HPV detection was performed on TCT liquid-based cells, all of which met the standards for issuing reports; then HPV-positive specimens were directly transported to the pathology center for testing, and the samples also met the standards for issuing pathology reports. The entire positive screening time was shortened from the previous 20 days to 1 month to within a week; it greatly reduced the workload of grassroots gynecologists, relieved the pressure of screening work, and reduced the probability of missed diagnosis.

[0038] In addition, compared with the previous HPV+TCT two-time sampling, the use of TCT one-time sampling, in the entire cervical screening process, in addition to the advantages in workflow such as shortening the screening cycle, reducing missed diagnosis, and reducing sampling costs, the advantages of TCT one-time sampling are also reflected in statistical data: statistical analysis of 8701 cervical cancer screening samples, a total of 707 HPV high-risk positive cases were detected, with a positive rate of 8.13%; 520 cases of cervical cytology abnormalities were screened out among HPV high-risk positive cases, with an abnormality rate of 72.2%; compared with the previous HPV+TCT two-time sampling screening data, there was no significant difference in the HPV high-risk positive rate, and the cytology abnormality rate increased from 45.5% in the past to 72.2%, which greatly improved the abnormal detection rate.

[0039] Compared with the related art, the present invention provides a cervical cancer screening method based on TCT detection, wherein anhydrous ethanol is added in step S4 to dissolve the organic solvent in the TCT liquid-based cell preservation solution into anhydrous ethanol, and the organic solvent is dissolved by the principle of similar dissolution of organic solvents, and then the waste liquid is discarded by centrifugation and supernatant discarding in step S5 to preserve cervical exfoliated cells; physiological saline is added in step S6, and the property that anhydrous ethanol can be miscible with water in any proportion is utilized, and then the waste liquid is discarded by centrifugation and supernatant discarding in step S7 to further purify the cells; after two rounds of centrifugal purification, the residual organic solvent in the TCT liquid-based cell preservation solution has been basically removed, thereby not affecting the downstream PCR amplification. The present invention optimizes the process of HPV detection, so that the sampled specimens in the TCT liquid-based cell preservation solution can be used for both TCT liquid-based cell detection and HPV-DNA detection, achieving the purpose of double detection in one tube, and it does not need to perform sampling twice, thereby shortening the screening cycle of cervical cancer, reducing the probability of missed diagnosis and sampling cost, and reducing the pain of sampling for patients.

[0040] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A cervical cancer screening method based on TCT detection, characterized in that: The method comprises the following steps: S1, extracting a sample to be tested, and placing the sample to be tested in a TCT liquid-based cell sampling tube containing a TCT liquid-based cell preservation solution for preservation; wherein the sample to be tested is cervical exfoliated cells, and the components of the TCT liquid-based cell preservation solution include an organic solvent, water, and a fixative; S2, placing the TCT liquid-based cell sampling tube containing the cervical exfoliated cells in a centrifuge, and shaking and mixing at a speed of 2500 r / min for 5 to 10 minutes to obtain a first sample; S3, extracting 500ul of the first sample and placing it in a centrifuge, and centrifuging it at a speed of 13000r / min-15000r / min for 5 minutes to 10 minutes to precipitate the cervical exfoliated cells to obtain a second sample; S4, adding 1 ml of anhydrous ethanol to the second sample to remove the organic solvent in the second sample to obtain a third sample; S5, placing the third sample in a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, discarding the supernatant and taking the precipitate to obtain a fourth sample; S6, adding 1 mL of physiological saline to the fourth sample to purify the cervical exfoliated cells to obtain a fifth sample; S7, placing the fifth sample in a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, discarding the supernatant and taking the precipitate to obtain a sixth sample; S8, adding 120 ul of cell lysate to the sixth sample, placing the sixth sample with the cell lysate in a heating device, and heating at a temperature of 95 degrees Celsius to 100 degrees Celsius for 10 minutes to 15 minutes to obtain a seventh sample; S9, placing the seventh sample into a centrifuge, centrifuging at a speed of 13000 r / min-15000 r / min for 5 minutes to 10 minutes, taking the supernatant to obtain a test sample for PCR amplification.

2. A cervical cancer screening method based on TCT detection according to claim 1, characterized in that: The organic solvent includes ethanol, methanol and isopropanol; the cell lysis solution includes potassium chloride, tris(hydroxymethyl)amino and polyethylene glycol octylphenyl ether.

3. A cervical cancer screening method based on TCT detection according to claim 1, characterized in that: The heating device is a dry thermostat; the reagent used for PCR amplification in S9 is a human papillomavirus genotyping (type 23) amplification reagent.

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