Primer probe group and kit for early detection of high-grade cervical lesions and cervical cancer

Abstract

The invention provides a primer probe group and a kit for early detection of high-grade cervical lesions and cervical cancer. The nucleotide sequence of the primer probe group is as shown in any one of SEQ ID NO: 1-69; or a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more basic groups to the nucleotide sequence as shown in any one of SEQ ID NO: 1-69, or a nucleotide sequence with similar functions to the nucleotide sequence as shown in any one of SEQ ID NO: 1-69. The primer probe group supports household sampling of cervical specimens, is combined with high-risk HPV and polygene methylation for joint detection, has high sensitivity and specificity, can quickly screen cervical cancer and high-grade lesions, reduces the omission ratio and improves the detection accuracy. Especially, cervical cancer and high-grade lesions caused by non-HPV infection and non-high-risk HPV infection which cannot be detected by other screening methods can be detected. Unnecessary referral treatment is reduced, the operation process is simplified, the time and economic cost of the examinee are reduced, and meanwhile the medical burden is relieved.

Description

Technical Field

[0001] The present application relates to the field of molecular biology, and specifically, to reagents and kits for early detection of high-grade cervical lesions and cervical cancer. More specifically, it relates to a primer probe set, the use of a primer probe set in the preparation of a reagent or kit for early detection of high-grade cervical lesions and cervical cancer, and reagents and kits. Background Art

[0002] Cervical cancer is an important public health issue facing the world, and it is also one of the common malignant tumors that seriously threatens the health of women around the world. Human papillomavirus (HPV) is considered to be the main cause of cervical cancer. According to research, there are more than 100 subtypes of HPV, which are currently divided into low-risk and high-risk types based on whether they are carcinogenic. Under normal circumstances, the vast majority of HPV infections are temporary, and more than 90% of infections will automatically clear up within 2-3 years. Early screening is an effective way to prevent cervical precancerous lesions and cervical cancer. Currently, the commonly used detection methods in clinical practice in my country mainly include liquid-based cytology testing and HPV testing, as well as methylation testing for colposcopy diversion for those who are negative for HPV16 and HPV18 but positive for other high-risk HPVs.

[0003] DNA methylation is a form of epigenetic modification that can affect gene expression and silencing in normal mammalian cells. At the same time, in human tumor studies, it was found that DNA methylation can often lead to changes in CpG islands in the promoter region of tumor suppressor genes. Hypermethylation or hypomethylation in the promoter region of tumor suppressor genes can lead to cell transformation, making DNA methylation status a potential marker for tumor detection. DNA methylation mainly occurs in the promoter region of genes and is usually closely related to the inactivation of tumor suppressor gene expression. Currently, cervical cell methylation testing has been used to guide colposcopy triage in patients infected with high-risk HPV types other than type 16 / 18.

[0004] However, the current cervical cancer screening methods still have some problems. For example, the sampling of cervical cells usually needs to be performed by a doctor in the hospital, which is not convenient for patients. For cervical cancer caused by non-HPV infection or non-high-risk HPV infection, the existing screening methods are difficult to achieve early detection, which affects the early diagnosis and intervention of the disease. For those infected with 12 high-risk HPV types other than types 16 and 18, if methylation testing is not performed and colposcopy is performed directly, some patients may receive unnecessary overtreatment and increase their anxiety, especially in young women. Even after methylation testing, diversion microscopy is performed based on the results, and patients still need to visit, sample and test multiple times, which not only increases the workload of doctors, but also prolongs the treatment cycle and brings additional time and economic costs.

[0005] In view of this, this application is hereby filed. Summary of the invention

[0006] The present application aims to solve at least one of the problems in the prior art. To this end, the present application provides a primer probe set for accurate detection of high-grade cervical lesions and early cervical cancer.

[0007] Specifically, this application provides the following technical solutions:

[0008] In a first aspect of the present application, the present application proposes a primer-probe set, the primer-probe set comprising: a primer or a combination thereof; and / or a probe or a combination thereof;

[0009] I. The aforementioned primers or a combination thereof, having any one of the following nucleotide sequences: 1) having a nucleotide sequence as shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44; or 2) having a nucleotide sequence as shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44 by modification, substitution, deletion or addition of one or more bases, or having a nucleotide sequence as shown in SEQ ID NOs: NO:1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44; or 3) a sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NO:1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44, or a sequence having SEQ ID NO:1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44 A nucleotide sequence having a similar function to any of the nucleotide sequences shown in NO: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44; or 4) a complementary sequence to any of the sequences shown in 1)-3);

[0010] II. The aforementioned probes or a combination thereof, having any one of the following nucleotide sequences: 5) having a nucleotide sequence as shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45; or 6) having a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to a nucleotide sequence as shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, or having a nucleotide sequence with similar function to a nucleotide sequence as shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45; or 7) having a nucleotide sequence as shown in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45. A sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45; or 8) a complementary sequence to any one of the sequences shown in 5)-7).

[0011] In some examples of the present application, the aforementioned primer probe set may also include at least one of the following additional technical features:

[0012] In some examples of the present application, the aforementioned primer probe set further comprises:

[0013] III. The aforementioned primers or a combination thereof, having any one of the following nucleotide sequences: 9) having a nucleotide sequence as shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65; or 10) having a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to a nucleotide sequence as shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65, or having a nucleotide sequence with similar functions to a nucleotide sequence as shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65; or 11) having a nucleotide sequence as shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65. A sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65, or a nucleotide sequence having a functionally similar nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65; or 12) a complementary sequence to the sequence shown in 9)-11);

[0014] IV. The aforementioned probes or a combination thereof have any one of the following nucleotide sequences: 13) a nucleotide sequence as shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66; or 14) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence as shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66, or a nucleotide sequence that is functionally similar to the nucleotide sequence as shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66; or 15) a sequence that has at least 80% homology to the nucleotide sequence as shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66, or a nucleotide sequence that is functionally similar to the nucleotide sequence as shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66; or 16) a complementary sequence to the sequence as shown in 13)-15).

[0015] In some examples of the present application, the aforementioned primer probe set further comprises:

[0016] V. The aforementioned primers or a combination thereof, having any one of the following nucleotide sequences: 17) having a nucleotide sequence as shown in any one of SEQ ID NOs: 67-68; or 18) having a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence as shown in any one of SEQ ID NOs: 67-68, or having a nucleotide sequence with similar functions to the nucleotide sequence as shown in any one of SEQ ID NOs: 67-68; or 19) having at least 80% homology to the nucleotide sequence as shown in any one of SEQ ID NOs: 67-68, or having a nucleotide sequence with similar functions to the nucleotide sequence as shown in any one of SEQ ID NOs: 67-68; or 20) a complementary sequence to the sequence as shown in 17)-19);

[0017] VI, the aforementioned probes or a combination thereof, having any one of the nucleotide sequences shown below: 21) having the nucleotide sequence shown in SEQ ID NO: 69; or 22) having a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 69, or having a nucleotide sequence that is functionally similar to the nucleotide sequence shown in SEQ ID NO: 69; or 23) a sequence that has at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 69, or having a nucleotide sequence that is functionally similar to the nucleotide sequence shown in SEQ ID NO: 69; or 24) a complementary sequence to the sequence shown in 21)-23).

[0018] In the second aspect of the present application, the present application proposes the use of any primer probe set of the first aspect in the preparation of a reagent or kit for early detection of high-grade cervical lesions and cervical cancer.

[0019] In some examples of the present application, the above application may further include at least one of the following additional technical features:

[0020] In some examples of the present application, the aforementioned high-grade cervical lesions and early cervical cancer are caused by one or more infections of high-risk HPV, non-HPV, or non-high-risk HPV.

[0021] In some examples of the present application, high-grade cervical lesions and cervical cancer are caused by high-risk HPV infection. The high-grade cervical lesions and cervical cancer caused by high-risk HPV infection can be detected by high-risk HPV testing, and further colposcopy diversion examination can be completed through methylation testing.

[0022] The aforementioned high-risk HPV types include: at least one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 or HPV68.

[0023] In some examples of the present application, the aforementioned high-grade cervical lesions and cervical cancer are caused by non-HPV and / or non-high-risk HPV infection, and the high-grade cervical lesions and cervical cancer caused by the aforementioned non-HPV and / or non-high-risk HPV infection can be detected by detecting methylation markers.

[0024] The aforementioned methylation markers include: at least one of: PAX1 gene methylation, JAM3 gene methylation or ZNF671 gene methylation.

[0025] The above-mentioned high-grade cervical lesions and early cervical cancer caused by high-risk HPV infection can be detected by existing screening methods. However, for high-grade cervical lesions and early cervical cancer caused by non-HPV and / or non-high-risk HPV infection, it is difficult to achieve early detection based on existing screening methods, which affects the early diagnosis and intervention of the disease. To this end, the present application has developed a reagent or kit for high-grade cervical lesions and early cervical cancer caused by non-HPV and / or non-high-risk HPV infection. The detection sensitivity is high, and cervical cancer screening can be completed with only one sampling and one test, thereby improving screening efficiency, reducing the number of patient visits, and improving the patient's medical experience.

[0026] In the third aspect of the present application, the present application proposes a reagent. According to an embodiment of the present application, the aforementioned reagent includes: any example of the primer probe set in the first aspect and an acceptable auxiliary agent.

[0027] In some examples of the present application, the aforementioned reagent may also include at least one of the following additional technical features:

[0028] In some examples of the present application, when the aforementioned reagent is used for one test, the amount of primer added at 10 μmol / L is 0.10 μL-0.50 μL, optionally, 0.10 μL, 0.20 μL, 0.30 μL, 0.40 μL or 0.50 μL. The aforementioned primers include HPV type amplification primers and gene methylation amplification primers.

[0029] Those skilled in the art know that, when performing the detection, the aforementioned reagents also include internal reference gene amplification primers.

[0030] HPV types include: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68.

[0031] Gene methylation includes: PAX1 gene methylation, JAM3 gene methylation, ZNF582 gene methylation, PCDGB7 gene methylation, SOX11 gene methylation or ZNF671 gene methylation.

[0032] For example, as shown in Table 1, SEO ID NO: 1-2 are HPV 16 type amplification forward primers and reverse primers, respectively. When a reagent containing the primers is used for one test, the amount of 10 μmol / L forward primer added is 0.10 μL-0.50 μL, and the amount of 10 μmol / L reverse primer added is 0.10 μL-0.50 μL. It is understandable that the amount of other type amplification forward primers, reverse primers, and internal reference primers added in one test is the same as above, and due to space limitations, they will not be repeated here.

[0033] Exemplarily, as shown in Table 1, SEO ID NO: 46-47 are the forward primer and reverse primer for PAX1 gene methylation amplification, respectively. When a reagent containing the primer is used for one test, the amount of 10 μmol / L forward primer added is 0.10 μL-0.50 μL, and the amount of 10 μmol / L reverse primer added is 0.10 μL-0.50 μL. It is understandable that the amount of other gene methylation amplification forward primers, reverse primers, and internal reference gene primers added in one test is the same as above, and due to space limitations, they will not be repeated here.

[0034] In some examples of the present application, when the aforementioned reagent is used for one test, the amount of 10 μmol / L probe added is 0.05 μL-0.50 μL, optionally, 0.05 μL, 0.10 μL, 0.20 μL, 0.30 μL, 0.40 μL or 0.50 μL. The aforementioned probes include HPV type and gene methylation probes.

[0035] Those skilled in the art will appreciate that, when performing the detection, the aforementioned reagents also include an internal reference gene detection probe.

[0036] The descriptions about HPV types and gene methylation are the same as above and will not be repeated here.

[0037] For example, as shown in Table 1, SEO ID NO: 3 is a HPV 16 type detection probe. When a reagent containing the probe is used for one test, the amount of 10 μmol / L probe added is 0.05 μL-0.50 μL. It is understandable that the amount of other type detection probes and internal reference gene detection probes added in one test is the same as above, and due to space limitations, it will not be repeated here.

[0038] In the fourth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the aforementioned kit includes: any example of the primer probe set in the first aspect or any example of the reagent in the third aspect.

[0039] In some examples of the present application, the aforementioned kit may also include at least one of the following additional technical features:

[0040] In some examples of the present application, the aforementioned kit further includes: at least one of dNTPs, PCR reaction solution or methylation PCR reaction solution.

[0041] In some examples of the present application, the aforementioned PCR reaction solution includes DNA polymerase, UNG enzyme and DNA polymerase buffer.

[0042] In some examples of the present application, the aforementioned methylation PCR reaction solution includes DNA polymerase and DNA polymerase buffer.

[0043] In some examples of the present application, the aforementioned kit further includes: at least one of: an HPV test positive quality control product, an HPV test negative quality control product, a gene methylation test positive quality control product, or a gene methylation test negative quality control product.

[0044] The aforementioned primer probe set, reagent and kit for early detection of high-grade cervical lesions and cervical cancer have the following beneficial effects:

[0045] 1) The specimens suitable for the above-mentioned high-grade cervical lesions and early detection of cervical cancer are cervical cell specimens or cervical specimens collected at home. Self-collection of cervical specimens can not only save the examinee from the trouble of going to the hospital, reduce time and economic costs, but also greatly reduce the workload of doctors, which is suitable for screening in the Internet era;

[0046] 2) The aforementioned primer probe sets, reagents or kits are used for combined detection and screening of high-risk HPV and gene methylation, and cervical cancer screening can be completed with one sampling and one test, including colposcopy diversion. The number of visits to the hospital for patients who need colposcopy is reduced from more than 3 times to 1 time, which not only reduces the time cost, economic cost and mental burden of the patients, but also greatly reduces the workload of doctors, which will help to achieve the goal of 70% cervical cancer screening rate in my country by 2030;

[0047] The sensitivity of detecting cervical cancer based on the above-mentioned primer probe set, reagent or kit is the superposition of the sensitivity of HPV16, 18 and methylation detection, which helps to further improve the sensitivity of cervical cancer screening.

[0048] 3) The primer probe set, reagent or kit proposed in this application solves the problem of missed detection of cervical cancer caused by non-HPV infection, and can detect cervical lesions of such patients at an early stage. Early treatment can improve the cure rate and survival time of patients with cervical lesions;

[0049] 4) The primer probe set, reagent or kit proposed in this application solves the problem of missed detection of cervical cancer caused by non-high-risk HPV infection, and can detect cervical lesions of such patients at an early stage. Early treatment can improve the cure rate and / or survival time of cervical lesions of such patients;

[0050] 5) Based on the primer probe set, reagent or kit proposed in this application, 58.3-66.7% of cervical lesions caused by non-HPV and non-high-risk HPV infection that cannot be detected by existing cervical cancer screening methods can be detected;

[0051] 6) The primer probe set, reagent or kit proposed in this application can simultaneously detect multiple methylation sites of JAM3 and PAX1 genes, including not only the promoter region of the gene, but also the coding region of the gene, which can significantly improve the detection sensitivity of cervical cancer and high-grade cervical lesions, and has high specificity for normal and low-grade cervical lesions.

[0052] 7) The primer probe set, reagent or kit proposed in this application can simultaneously detect the methylation sites of PAX1, JAM3 and ZNF671 genes. Due to the diversity of high-grade cervical lesions and cervical cancer types and the diversity of HPV infection, multiple gene methylation regions are selected for joint detection to form functional complementarity, significantly improve the sensitivity to cervical cancer and high-grade cervical lesions, and have high specificity for normal and low-grade cervical lesions;

[0053] 8) The primer probe set, reagent or kit proposed in this application uses viral molecular biology and molecular epigenetic methods, and utilizes DNA fluorescence quantitative PCR detection technology and methylation detection technology to detect high-grade cervical lesions and possible cervical cancer patients early, maximize detection, and carry out preventive treatment early;

[0054] 9) The primer probe set, reagent or kit proposed in this application uses DNA fluorescence quantitative PCR detection technology and methylation detection technology to perform multiple multi-channel fluorescence multi-gene, multi-site methylation detection and high-risk HPV typing detection. The test samples involved can be obtained at home, the detection method is simple to operate, the interpretation is intuitive, and the results can be obtained within 3 hours. General fluorescence quantitative PCR instruments can meet the detection needs. The entire experimental process adopts a one-stop fully enclosed form to avoid the possibility of cross contamination. Because the detection of this application is highly sensitive, it has better detection efficiency for low-concentration templates;

[0055] 10) The high sensitivity of the primer probe set, reagent or kit proposed in the present application is suitable for the initial screening of high-grade cervical lesions. For cervical self-sampling, the overall specificity is 89.47%, and the overall sensitivity is 92.00%, among which the detection rates of CIN2+, CIN3+, and cervical cancer are 92.00%, 95.18%, and 100%, respectively; the specificity for CIN1 and inflammation and non-cervical epithelial lesion samples is 89.47%; for cervical exfoliated cells, the overall specificity is 87.97%, and the overall sensitivity is 92.80%, among which the detection rates of CIN2+, CIN3+, and cervical cancer are 92.80%, 96.39%, and 100%, respectively; the specificity for CIN1 and inflammation and non-cervical epithelial lesion samples is 87.97%. From the above results, it can be seen that this kit can better identify high-grade cervical lesions and cancer, achieve diversion from low-grade lesions and inflammation, and to a certain extent reduce unnecessary colposcopy referrals for patients;

[0056] 11) The primer probe set, reagent or kit proposed in this application is suitable for completing the screening of high-grade cervical lesions and cervical cancer with one self-sampling and one DNA extraction test, and can detect high-grade cervical lesions and cervical cancer caused by non-HPV infection and non-14 high-risk HPV infections that cannot be detected by conventional screening. It can provide technical support for the improvement of cervical cancer screening methods in my country, and at the same time can reduce the time cost, economic cost and mental burden of the examinees, and can also greatly reduce the workload of doctors, with significant economic and social benefits.

[0057] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0059] Figure 1 A schematic diagram of the S-type amplification curve of HPV16, HPV18 and the internal reference gene (GAPDH) in cervical exfoliated cells provided in the examples of the present application;

[0060] Figure 2 Schematic diagram of the S-type amplification curves of HPV31, HPV39, HPV45 and HPV58 in cervical exfoliated cells provided in the examples of the present application;

[0061] Figure 3 Schematic diagram of the S-type amplification curves of HPV33, HPV56, HPV59 and HPV68 in cervical exfoliated cells provided in the examples of the present application;

[0062] Figure 4 Schematic diagram of the S-type amplification curves of HPV35, HPV51, HPV52 and HPV66 in cervical exfoliated cells provided in the examples of the present application;

[0063] Figure 5 Schematic diagram of the S-type amplification curves of PAX1, JAM3 and ZNF671 genes and the internal reference gene (GAPDH) in cervical exfoliated cells provided in the examples of the present application;

[0064] Figure 6 Schematic diagram of the S-type amplification curve of HPV16, HPV18 and the internal reference gene (GAPDH) in the cervical self-sampling provided in the examples of the present application;

[0065] Figure 7 A schematic diagram of the S-type amplification curves of HPV31, HPV39, HPV45 and HPV58 in cervical self-sampling provided in the embodiments of the present application;

[0066] Figure 8 A schematic diagram of the S-type amplification curves of HPV33, HPV56, HPV59 and HPV68 in cervical self-sampling provided in the embodiments of the present application;

[0067] Fig. 9 A schematic diagram of the S-type amplification curves of HPV35, HPV51, HPV52 and HPV66 in cervical self-sampling provided in the embodiments of the present application;

[0068] Fig.10 Schematic diagram of the S-type amplification curves of PAX1, JAM3 and ZNF671 genes and the internal reference gene (GAPDH) in cervical self-sampling provided in the examples of the present application;

[0069] Fig.11A A schematic diagram of a conventional HPV screening process (hospital examination) provided in an embodiment of the present application;

[0070] Fig. 11B Schematic diagram of the screening process of the kit of the present application provided in the embodiment of the present application (home sampling). DETAILED DESCRIPTION

[0071] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0072] In this article, unless otherwise specified, the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described here can be implemented in a sequence other than those illustrated or described here. In the present application, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or server comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. In the description of the present application, unless otherwise specified, "multiple" refers to two or more than two.

[0073] In this article, unless otherwise specified, under the premise of not substantially affecting the function of the nucleotide sequence (retaining at least 95% of the activity), those skilled in the art can replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6 or 7 or more) nucleotides of the sequence of the present application to obtain variants of primers or probes. They are all deemed to be included in the scope of protection of the present application. The variant sequence described in the present application can have at least 80% identity (or homology) with the reference sequence (sequence shown in Table 1), which means at least 80% with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity. The sequence identity described in the present application can be measured using sequence analysis software. For example, the computer program BLAST using default parameters. The sequences mentioned in this application are all shown from 5' to 3' end.

[0074] The sequences in this application are shown in Table 1.

[0075] Table 1

[0076]

[0077]

[0078] Note: F: indicates forward primer sequence; R indicates reverse primer sequence; P indicates fluorescent probe sequence; PAX1-1 indicates PAX1 gene region 1; PAX1-2 indicates PAX1 gene region 2.

[0079] Application of primer probe sets, reagents or kits in early detection of high-grade cervical lesions and cervical cancer

[0080] Applying the primer probe set, reagent or kit of the present application to the early detection of high-grade cervical lesions and cervical cancer includes: obtaining a sample to be tested; using the primer probe set, reagent or kit to perform nucleic acid amplification and fluorescence quantitative detection on the aforementioned sample to be tested; based on the detection results, classifying the HPV type and / or gene methylation type of the aforementioned sample to be tested.

[0081] In some examples of the present application, the aforementioned samples to be tested are selected from cervical cell specimens, which are collected by professional doctors; they can also be selected from cervical self-collected specimens, which are collected by the examinee at home, to avoid the need for routine screening to go to the hospital for consultation and sampling repeatedly.

[0082] Some exemplary methods of sample collection in this application are as follows:

[0083] Cervical exfoliated cell collection: Use the speculum to open the vagina to expose the cervix, place the sampling brush in the cervical canal close to the cervical opening, and slowly rotate the sampling brush in the same direction for more than 5 circles to avoid reverse rotation; after the sampling is completed, take out the sampler and place it in the cervical exfoliated cell sample preservation solution, bend the middle of the sampler to break it and leave the front part in the preservation solution, and shake the sampling bottle appropriately to ensure that the sample and the preservation solution are fully mixed. If there is a lot of cervical secretions during the sampling process, the cervical opening and its surrounding secretions should be gently wiped with a cotton swab before sampling.

[0084] Self-sampling of cervical exfoliated cells: Sampling is performed in a semi-sitting position. Hold the bottom of the handle and slowly insert the swab into the vagina until the safety guard touches the vaginal opening. (Note: the sampling head does not rotate out here) Rotate the handle clockwise 6-7 times to rotate the sampling head out of the vagina for sampling. Hold the handle and twist it 4-5 times in the same direction. (Note: During the twisting process, keep the safety guard touching the vaginal opening) Gently pull out the sampling swab from the vaginal opening, and continue to rotate the handle until it can no longer rotate, so that the connection of the sampling head is completely exposed. Bend the middle of the sampler to break it and leave the front part in the preservation solution, and shake the sampling bottle appropriately to ensure that the sample and the preservation solution are fully mixed.

[0085] The inventors performed HPV type detection and gene methylation detection on the above two types of collected specimens in 258 clinical samples, and the results are shown in Table 2. It can be seen that the HPV type detection result conformity rate is 98.84%, and the gene methylation detection result conformity rate is 94.57%. This shows that the primer probe set, reagent or kit of the present application is highly reliable in the detection of high-grade cervical lesions and early cervical cancer using self-sampled specimens.

[0086] Table 2 Comparison of detection performance and compliance rate of two samples

[0087] HPV type testing Gene methylation detection total 258 258 Type mismatch 3 / Yin and Yang don't match 0 14 Results compliance rate 98.84％ 94.57％

[0088] Persistent infection with high-risk HPV is the main cause of cervical cancer. In 2021, the WHO's Guidelines for Cervical Cancer Screening and Treatment of Precancerous Lesions (Second Edition) and the recommended methods for cervical cancer screening programs of the National Health Commission of my country recommend nucleic acid testing for 14 high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68). After persistent high-risk HPV infection, the risk of progression to cervical cancer and precancerous lesions is closely related to the HPV genotype. HPV16 / 18-positive people have a high risk of cervical cancer and precancerous lesions. In high-grade cervical lesions (HSIL), the HPV16 / 18 infection rate is about 52%; in cervical squamous cell carcinoma, HPV16 / 18 is the most common, accounting for 84.5%. HPV 18 can cause 50% of cervical adenocarcinoma in situ (AIS) and invasive carcinoma.

[0089] In addition, the 2023 "Guidelines for Cervical Cancer Screening in China" also records the nucleic acid detection of 14 high-risk HPV types. To this end, the inventors specifically designed specific primers, internal reference gene primers and corresponding detection probes for HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68.

[0090] Since cervical cancer and different grades of cervical lesions are diverse, the joint detection of multi-gene methylation regions is complementary in function, which is conducive to improving the detection rate of high-grade lesions and cervical cancer. In some preferred examples of this application, this application also provides methylation markers related to the early detection of high-grade cervical lesions and cervical cancer, including PAX1 gene, JAM3 gene and ZNF671 gene.

[0091] The PAX1 (paired box gene 1) gene is a member of the PAX gene family located on chromosome 20 and plays a key role in fetal development and cell proliferation. Methylation of the PAX1 gene promoter plays an important epigenetic regulatory role in the occurrence and development of tumors. Studies have shown that PAX1 is methylated and silenced in cervical cancer and ovarian cancer, so PAX1 is also regarded as a tumor suppressor gene.

[0092] JAM3 (junctional adhesion molecule 3) is a member of the JAM gene family, which can directly affect the tight junctions between epithelial cells and endothelial cells. A large number of literature reports that JAM3, also commonly known as JAMC, is a junction regulator. In recent years, many literature reports that JAM3 plays an important regulatory role in tumor development, such as its methylation in colorectal cancer and cervical cancer.

[0093] Zinc Finger Protein 671 (ZNF671), hypermethylation of ZNF671 occurs in many cancers, such as clear renal carcinoma, urothelial carcinoma, cervical cancer, etc. In 2019, Shoko Mase et al. found that ZNF671 has a tumor suppressor effect, and the silencing of gene expression caused by its DNA methylation is associated with the progression of cancer.

[0094] In order to further improve the detection sensitivity and specificity, the aforementioned methylation markers further include: ZNF582 gene, PCDGB7 gene and SOX11 gene.

[0095] In order to further improve the detection sensitivity and specificity, the inventors of the present application designed another pair of PAX1 gene methylation primers. The detection sensitivity can be improved by using two pairs of PAX1 methylation primers for amplification. The specific sequences are shown in Table 1.

[0096] Among them, the specific primers for the above methylation markers, the internal reference gene primers and the detection probe sequences are shown in Table 1.

[0097] After obtaining the above-mentioned specific primers and probes, the optimal PCR reaction solution system is further determined for the HPV type primers. For the detection of HPV DNA, relevant DNA polymerases are required to screen for polymerases with better amplification efficiency. In this reaction system containing multiple primers and probes, the selection of PCR reaction solution is particularly important. The amplification efficiency of each gene primer probe in the system must be similar to that of its corresponding single-plex amplification to ensure that the primers or probes in the system do not interfere with each other and give full play to the amplification effect of each set of primers and probes. The PCR reaction solution includes 2×DNA polymerase buffer (+UNG) and probe primers. Screening of DNA polymerases and their interaction with dNTPs, Mg 2+ The ratio of 10× DNA polymerase buffer and 10× DNA polymerase buffer is directly related to the amplification efficiency of the combination of primers and probes. The components of the kit are shown in Table 3.

[0098] Table 3 Kit components

[0099]

[0100] After obtaining the above-mentioned specific primers and probes, the optimal PCR reaction solution system is further determined for the gene methylation primers. For the detection of methylation sites, special DNA polymerases related to methylation detection are required to screen for polymerases with better amplification efficiency. In this reaction system containing multiple primer probes, the selection of PCR reaction solution is particularly important. The amplification efficiency of each gene primer probe in the system must be similar to that of its corresponding single-plex amplification to ensure that the primers or probes in the system do not interfere with each other and give full play to the amplification effect of each set of primer probes. The PCR reaction solution includes 2×DNA polymerase buffer and probe primers. Screening of DNA polymerases and their interaction with dNTPs, Mg 2+ The ratio between 10× DNA polymerase buffer and 10× DNA polymerase buffer is directly related to the amplification efficiency of the combination of primers and probes. The components of the kit are shown in Table 4.

[0101] Table 4 Kit components

[0102]

[0103] After determining the kit components, the process further includes performing nucleic acid amplification on the sample. The exemplary steps of sample processing and DNA extraction and conversion are as follows:

[0104] After receiving the samples, the laboratory will perform pre-test processing on the samples according to the following process: a. Record the sample number to complete the sample type verification; b. Extract DNA from the samples under sterile operation; c. Divide the extracted DNA specimens into two equal parts, one group for HPV detection, and the other group for gene methylation detection after DNA conversion and purification.

[0105] DNA extraction: DNA from cervical exfoliated cells and cervical self-sampled specimens was extracted using the universal nucleic acid extraction kit (magnetic bead method) produced by Cuizhen Biotechnology (Zhengzhou) Co., Ltd.

[0106] DNA conversion and purification: Use the sulfite conversion and purification kit of Cuizhen Biotechnology (Zhengzhou) Co., Ltd.

[0107] PCR amplification:

[0108] The components of the PCR reaction system (20 μL / person) are shown in Table 5

[0109] Table 5 PCR reaction system

[0110] Components Amount per person (μL) PCR reaction solution 1 / 2 / 3 / 4 / II 16 DNA 4 Purified water Top up to 20

[0111] The PCR reaction program is shown in Table 6.

[0112] Table 6 PCR reaction program

[0113]

[0114] The fluorescence channels of each detection probe are shown in Table 7.

[0115] Table 7 Correspondence between probes and fluorescence channels

[0116] Probe Name Fluorescence channels HPV 16-P ROX HPV 18-P HEX HPV 31-P FAM HPV 33-P FAM HPV 35-P FAM HPV 39-P CY5 HPV 45-P ROX HPV 51-P CY5 HPV 52-P HEX HPV 56-P HEX HPV 58-P HEX HPV 59-P ROX HPV 66-P ROX HPV 68-P CY5 PAX1-P1 ROX PAX1-P2 ROX JAM3-P FAM ZNF671-P HEX GAPDH-P CY5

[0117] Interpretation of results:

[0118] 1) Interpretation of HPV results:

[0119] Positive quality control: Ct values ​​of FAM, ROX, VIC / HEX, and CY5 channels are ≤34, and the amplification curve has an obvious exponential growth period.

[0120] Negative quality control: There is no amplification curve in the FAM, ROX, VIC / HEX, and CY5 channels, or the amplification curve is a straight line or a slightly oblique line, with no obvious exponential growth period and no Ct value.

[0121] The above two conditions must be met at the same time, otherwise the test is invalid and should be retested.

[0122] Internal standard gene: In the sample test, the Ct value of the 1Cy5 channel of the reaction solution is ≤32, and the amplification curve has an obvious exponential growth period; if the Ct value is >32, it is likely that there are inhibitors in the sample DNA or the amount of sample DNA is insufficient. It is recommended to re-sample or re-extract DNA and re-test.

[0123] Target gene: In the sample test, if the Ct value of the target gene channel is ≤37 and the amplification curve has an obvious exponential growth period, it can be judged as positive according to the specific type. If the Ct value is greater than 37 or the target gene amplification curve is not S-shaped, it can be judged as negative according to the specific type.

[0124] 2) Interpretation of methylation results:

[0125] Positive quality control: Ct values ​​of FAM, ROX, VIC / HEX, and CY5 channels are ≤35, and the amplification curve has an obvious exponential growth period.

[0126] Negative quality control: There is no amplification curve in the FAM, ROX, VIC / HEX, and CY5 channels, or the amplification curve is a straight line or a slightly oblique line, with no obvious exponential growth period and no Ct value.

[0127] The above two conditions must be met in the same experiment, otherwise the results of this experiment will be invalid.

[0128] Internal standard gene: In the sample test, the Ct value of the internal standard gene channel is ≤35, and the amplification curve has an obvious exponential growth period; if the Ct value is >35, it is likely that there are inhibitors in the sample DNA or the amount of sample DNA is insufficient. It is recommended to re-sample or re-extract the converted DNA and re-test it.

[0129] Target gene: If the Ct value of the target gene channel in the sample test is ≤38 and the amplification curve has an obvious exponential growth period, the gene can be judged as positive. If the Ct value is greater than 38 or the target gene amplification curve is not S-shaped, the gene can be judged as negative. If any target gene is positive, the sample is judged as positive.

[0130] This application uses a special primer design method and clinical samples with accurate pathological information to determine a reasonable positive judgment value through the ROC curve, thereby improving the accuracy of reaction system screening and the reliability of early detection of high-grade cervical lesions and cervical cancer, avoiding the occurrence of false positive and false negative results to the greatest extent, and significantly improving the detection performance of the entire kit.

[0131] The methylated regions were further specifically identified through the design of probes, that is, the probes were able to distinguish whether the PAX1, JAM3 and ZNF671 genes were methylated. The key points for the probe design are: specific distinction between methylated and non-methylated regions; because the sequence after bisulfite conversion only has three bases, A, T, and G (except for the CG site), it is very easy for primers or probes to interfere with each other and have amplification effects on each other, which needs to be avoided during design.

[0132] The composition for early detection of high-grade cervical lesions and cervical cancer also includes Taqman probes corresponding to the methylation sites of the PAX1 gene, Taqman probes corresponding to the methylation sites of the JAM3 gene, Taqman probes corresponding to the methylation sites of the ZNF671 gene, and Taqman probes corresponding to the internal reference gene. In addition to 4 pairs of specific methylation amplification primers, 4 probes labeled with different fluorescent channels are added to this reaction system. The key point of screening is to observe whether the amplification efficiency of the 4 genes in standard samples with different methylation degrees is optimal, whether the fluorescence curve is a standard S-shaped amplification curve, and whether the fluorescence curve maintains a consistent trend compared with the single-plex amplification of each gene.

[0133] The above-mentioned composition and kit for early detection of high-grade cervical lesions and cervical cancer were used to perform clinical sample testing. The test results of some clinical samples are as follows: Figure 1-10 As shown. Among them, the HPV typing detection system is divided into four tubes, the first tube detects HPV16, 18 and internal reference genes, the second tube detects HPV31, 38, 45, 58, the third tube detects HPV33, 56, 59, 68, and the fourth tube detects HPV35, 51, 52, 66. And the four genes need to be reflected in the form of Ct values ​​through different fluorescence channels, so that the amplification between the four HPVs in the system does not interfere with each other. The 14 HPV typing detection performances of this application are consistent with those of the marketed test kits.

[0134] The gene methylation detection system detects four genes in one tube, and the four genes need to be displayed in the form of Ct values ​​through different fluorescence channels. The two pairs of primers for the PAX1 gene use the same fluorescence channel. No matter which pair of primers has amplification, it will be displayed. When more than one pair of primers has amplification, the fluorescence signal will be enhanced.

[0135] In the system, the amplification of the four genes does not interfere with each other. When the amplification efficiency of the four genes is consistent with their single-plex amplification efficiency, it means that there is no inhibition in the multiplex reaction system. Therefore, the above specific sequences are determined through the design and screening of a large number of primers. The accuracy of early detection of cervical cancer is increased through the combined detection of the three target genes PAX1, JAM3 and ZNF671 and the reasonable threshold setting.

[0136] Specifically, the steps include:

[0137] (1) Sample source: A total of 258 female patients aged ≥25 years and ≤64 years were enrolled. Cervical exfoliated cell samples and cervical cell self-sampling samples were collected for high-risk HPV and PAX1, JAM3, and ZNF671 gene methylation testing. The patients were also scheduled for colposcopy and pathological examination.

[0138] The colposcopy and pathology results were as follows: 26 samples were identified as cervical adenocarcinoma and squamous cell carcinoma; 54 samples were low-grade intraepithelial lesions (CIN1); 42 samples were high-grade intraepithelial lesions (CIN2); 57 samples were high-grade intraepithelial lesions (CIN3); and 79 samples were non-cervical epithelial lesions.

[0139] (2) The genomic DNA of the cells was extracted from the above 258 cervical exfoliated cell samples and cervical self-sampling samples. The universal nucleic acid extraction kit (magnetic bead method) of Cuizhen Biotechnology (Zhengzhou) Co., Ltd. was used to extract the genomic DNA of the above samples.

[0140] The quality of DNA extraction must be guaranteed during the extraction process in order to conduct subsequent experiments. The quality standard of DNA after extraction is: OD260 / 280 between 1.6 and 2.0.

[0141] (3) The cell genomic DNA obtained in step (2) is subjected to bisulfite conversion, and the total conversion amount is 100ng-1000ng. Even a total amount of genomic DNA template as low as 50ng can be detected, so that the unmethylated 5' cytosine (C) in the DNA is converted into uracil (U), while the methylated 5' cytosine (C) does not change, and finally the converted BisDNA is obtained. In this embodiment, the sulfite conversion purification kit of Cuizhen Bio (Zhengzhou) Co., Ltd. is selected. During the conversion process, the conversion efficiency of DNA and the final conversion yield of Bis-DNA must be guaranteed.

[0142] (4) Prepare the PCR reaction system according to Table 5.

[0143] (5) Adding samples: As shown in Table 5, 4 μL of the genomic DNA, positive control material I and negative control material extracted in step (2) were added to each of the mixed solution systems 1 / 2 / 3 / 4 prepared in step (4); 4 μL of the Bis-DNA clinical sample, positive control material II and negative control material converted in step (3) were added to the mixed solution system II prepared in step (4).

[0144] (6) PCR reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 15 s, 58°C annealing and extension for 45 s, 45 cycles.

[0145] (7) The test results are shown in Table 8.

[0146] Table 8 Gene methylation and 14 high-risk HPV test results of 258 clinical samples

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] Test result analysis:

[0155] A total of 258 clinical samples were tested using the kit of this application, and the clinical case results showed that among the 258 subjects, there were 26 cases of cervical cancer, 42 cases of high-grade intraepithelial lesions (CIN2), 57 cases of high-grade intraepithelial lesions (CIN3), 54 cases of low-grade intraepithelial lesions (CIN1), and 79 cases of non-cervical epithelial lesions.

[0156] The test results using the kit of this application are as follows: Cervical exfoliated cell specimens: For CIN2+ specimens, the methylation detection sensitivity is 89.60%, and the HPV16 / 18 combined methylation detection sensitivity is 92.80%; For CIN3+ specimens, the methylation detection sensitivity is 92.77%, and the HPV16 / 18 combined methylation detection sensitivity is 96.39%; For cervical cancer (including cervical adenocarcinoma) specimens, the methylation detection sensitivity is 100%; For CIN1 and non-cervical epithelial lesion samples, the specificity is 87.97%.

[0157] For cervical self-sampling specimens, the methylation detection sensitivity for CIN2+ specimens was 88.00%, and the sensitivity of HPV16 / 18 combined methylation detection was 92.00%; the methylation detection sensitivity for CIN3+ specimens was 91.57%, and the sensitivity of HPV16 / 18 combined methylation detection was 95.18%; for cervical cancer (including cervical adenocarcinoma) specimens, the methylation detection sensitivity was 100%; the specificity for CIN1 and non-cervical epithelial lesion samples was 89.47%.

[0158] From the above results, it can be seen that the test results of the cervical self-sampling specimens proposed by this kit are consistent with those of traditional cervical exfoliated cell specimens in terms of sensitivity and specificity; the sensitivity and specificity of the combined detection of the three genes PAX1, JAM3 and ZNF671 in this kit are comparable to those of the marketed kits. The sensitivity of this kit after using HPV combined with gene methylation detection is better than that of the marketed products, and it can increase the detection rate of CIN2+ and CIN3+ by more than 3% based on methylation detection. The results are shown in Tables 9 and 10.

[0159] Table 9 Comparison of sensitivity of two samples

[0160]

[0161] Table 10 Comparison of the specificity of the two specimens for CIN1+ and cases without cervical epithelial lesions

[0162] Cervical exfoliated cell specimen Self-collected cervical specimens Number of methylation-negative cases 117 / 133 119 / 133 Specificity% 87.97 89.47

[0163] The relationship between PAX1 gene methylation and cervical cancer was recognized the earliest, and it has the closest relationship with cervical cancer. When using PAX1 gene methylation to diagnose high-grade cervical lesions, this application uses two pairs of primers to jointly detect more PAX1 methylation sites to improve sensitivity. The detection rate of cervical self-sampling specimens using two pairs of primers can be increased by 8%. The results are shown in Table 11.

[0164] Table 11 PAX1 gene multi-locus detection rate

[0165]

[0166] The methylation detection of the kit of the present application adopts the combination of PAX1, ZNF671 and JAM3. Each of the three genes can increase the detection rate alone, and the sensitivity of the combined detection reaches the highest. The results are shown in Table 12.

[0167] Table 12 Performance of single gene and multi-gene methylation in diagnosing high-grade cervical lesions

[0168]

[0169] In the detection of high-grade cervical epithelial lesions and cervical cancer caused by non-HPV infection and non-high-risk HPV infection, 66 out of 258 patients had negative results for high-risk HPV. According to the existing cervical cancer screening program, these patients are classified as low-risk patients and are screened once every 5 years in the later stage. However, in the study of this kit, 2 patients with cervical adenocarcinoma and 10 patients with CIN2+ and above cervical lesions were found through microscopic examination and / or pathological examination. When the cervical exfoliated cells of these 12 patients were tested using the kit of this application, 8 methylation-positive patients were successfully detected, with a detection rate of 66.67%; 7 methylation-positive patients were successfully detected by cervical self-sampling specimens, with a detection rate of 58.33%. The results are analyzed as shown in Table 13.

[0170] Cervical cancer caused by non-HPV infection or non-high-risk HPV infection accounts for about 10-15% of all cervical cancers and high-grade cervical lesions. There are at least 50,000 new cases of such cervical cancer lesions worldwide each year. Up to now, there is no method in the world that can achieve early diagnosis through early screening. Such patients are often in the middle and late stages when clinically discovered. The kit of the present application can detect more than 50% of the above-mentioned cervical lesions, avoiding the missed detection of cervical cancer caused by non-HPV infection and non-high-risk HPV infection, which is of great significance for early screening, early diagnosis and early treatment of cervical cancer.

[0171] Table 13 Methylation detection rates of high-grade cervical lesions caused by non-HPV and non-high-risk HPV infection

[0172]

[0173]

[0174] Analysis of the economic and social benefits of this application

[0175] Early screening is an effective way to prevent cervical precancerous lesions and cervical cancer. Currently, the commonly used detection methods in clinical practice mainly include liquid-based cytology and HPV testing. Compared with the traditional Pap smear method (sensitivity is between 30-87%, and sometimes the sensitivity is even less than 20%), liquid-based cytology has a certain improvement, but there is still a high proportion of false negative results (studies have shown that the probability of false negatives is about 50%). Since persistent high-risk HPV infection is the primary cause of cervical lesions, the detection of HPV nucleotides has a high sensitivity in the detection of cervical lesions, but because only a small number of high-risk HPV-infected patients will show cervical lesions, HPV testing shows extremely low specificity (about 60.7%), especially in young women.

[0176] Routine HPV screening is generally ( Fig.11A ): Cytology → HPV test → methylation test diversion → colposcopy. Those who are HPV16 and 18 positive and those who are not HPV16 and 18 positive and methylation positive need to undergo colposcopy. Those who are not HPV16 and 18 positive need to complete the screening process. Three samplings and four visits to the hospital to determine whether there are high-grade cervical epithelial lesions or cervical cancer. Each examination takes about half a day. There are about 300 million women of appropriate age in China who need cervical cancer screening. The screening rate reaches 50%, which is about 150 million people. According to the current screening model, the workload of cervical cancer screening doctors will be staggering, and the time cost of the screener is also high.

[0177] Using the kit of this application for cervical cancer screening ( Fig. 11B), after placing an order for testing on the Internet, you only need to take samples at home for testing. If the test results are negative for HPV and methylation, you do not need to go to the hospital to complete the screening process; if HPV16, 18 and methylation are positive, you only need to go to the hospital once for a colposcopy. If there are lesions, they can be treated at the same time. On average, each person can save 2.5 days of medical treatment time, and it can greatly reduce the workload of the screening doctor. The simplification of the screening process will also effectively increase the screening rate, which will help achieve the national cervical cancer screening and early diagnosis and treatment goals.

[0178] The combined detection of HPV and gene methylation proposed in this application can improve the screening sensitivity of cervical cancer and high-grade lesions, reduce the number of times patients seek medical treatment, and detect cervical cancer and high-grade lesions caused by non-HPV and non-high-risk HPV infections. It not only refers to the HPV and gene methylation combined detection kit, but also includes the combined use of HPV detection kits and cervical cancer methylation detection kits that can be easily completed by ordinary technicians in this field under the inspiration of this application.

[0179] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0180] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in the field can change, modify, replace and modify the above embodiments within the scope of the present application without departing from the principles and purpose of the present application.

Claims

1. A primer probe set, characterized in that: include: Primers or combinations thereof; and / or probe or a combination thereof; 1. The primer or a combination thereof has any one of the following nucleotide sequences: 1) having a nucleotide sequence as shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44; or 2) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44; or 3) a sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, 40-41, 43-44; or 4) a complementary sequence to any of the sequences shown in 1) to 3); II. The probe or a combination thereof has any one of the following nucleotide sequences: 5) having a nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45; or 6) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45; or 7) a sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45; or 8) A complementary sequence to any of the sequences in 5) to 7).

2. The primer probe set according to claim 1, characterized in that: Further including: III. The primer or a combination thereof has any one of the following nucleotide sequences: 9) having a nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65; or 10) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65; or 11) a sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 46-47, 49-50, 52-53, 55-56, 58-59, 61-62, 64-65; or 12) a complementary sequence to the sequence shown in 9)-11); IV. The probe or a combination thereof has any one of the following nucleotide sequences: 13) having a nucleotide sequence shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, and 66; or 14) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66; or 15) a sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 48, 51, 54, 57, 60, 63, 66; or 16) A complementary sequence to the sequence shown in 13)-15).

3. The primer probe set according to claim 2, characterized in that: Further including: V. The primer or a combination thereof has any one of the following nucleotide sequences: 17) having a nucleotide sequence shown in any one of SEQ ID NOs: 67-68; or 18) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in any one of SEQ ID NOs: 67-68, or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 67-68; or 19) a sequence having at least 80% homology to the nucleotide sequence shown in any one of SEQ ID NOs: 67-68 or a nucleotide sequence having a similar function to the nucleotide sequence shown in any one of SEQ ID NOs: 67-68; or 20) A complementary sequence to the sequence shown in 17)-19); VI, the probe or a combination thereof, having any one of the nucleotide sequences shown below: 21) having the nucleotide sequence shown in SEQ ID NO: 69; or 22) a nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence shown in SEQ ID NO: 69, or a nucleotide sequence having a similar function to the nucleotide sequence shown in SEQ ID NO: 69; or 23) a sequence having at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 69 or a nucleotide sequence having a similar function to the nucleotide sequence shown in SEQ ID NO: 69; or 24) A complementary sequence to the sequence shown in 21)-23).

4. Use of the primer probe set according to any one of claims 1 to 3 in the preparation of a reagent or kit for early detection of high-grade cervical lesions and cervical cancer.

5. The use according to claim 4, characterized in that: The high-grade cervical lesions and cervical cancer are caused by high-risk HPV infection; Optionally, high-grade cervical lesions and cervical cancer caused by the high-risk HPV infection can be detected by high-risk HPV detection, and further colposcopy triage examination can be completed by methylation detection; Optionally, the high-risk HPV includes at least one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 or HPV68; The use according to claim 4 is characterized in that the high-grade cervical lesions and cervical cancer are caused by non-HPV and / or non-high-risk HPV infection; Preferably, high-grade cervical lesions and cervical cancer caused by non-HPV and / or non-high-risk HPV infection can be detected by detecting methylation markers; Preferably, the methylation marker comprises at least one of: PAX1 gene methylation, JAM3 gene methylation or ZNF671 gene methylation.

6. A reagent, characterized in that include: The primer probe set according to any one of claims 1 to 3 and an acceptable auxiliary agent.

7. The reagent according to claim 6, characterized in that When performing one test, the amount of 10 μmol / L primer added is 0.10 μL-0.50 μL, wherein the primers include HPV type amplification primers and gene methylation amplification primers; Optionally, when performing one detection, the amount of 10 μmol / L probe added is 0.05 μL-0.50 μL, wherein the probe includes an HPV type detection probe and a gene methylation detection probe.

8. A kit, characterized in that: include: The primer probe set according to any one of claims 1 to 3 or the reagent according to any one of claims 6 to 7.

9. The kit according to claim 8, characterized in that Further including: At least one of dNTPs, PCR reaction solution or methylation PCR reaction solution; Optionally, the PCR reaction solution includes DNA polymerase, UNG enzyme and DNA polymerase buffer; Optionally, the methylation PCR reaction solution includes DNA polymerase and DNA polymerase buffer.

10. The kit according to claim 8, characterized in that Further including: At least one of the HPV test positive quality control product, HPV test negative quality control product, gene methylation test positive quality control product or gene methylation test negative quality control product.

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