Reagent composition for cervical cancer detection, kit and application

Abstract

The invention relates to the technical field of biological detection, and discloses a reagent composition for cervical cancer detection, a kit and application. The reagent composition comprises a reagent for detecting the methylation level function of cervical cancer marker genes or fragments thereof, and the marker genes comprise a PAUPAR gene, an NETO1 gene and an NTM gene. According to the reagent composition, the methylation level of a specific marker gene combination is detected, efficient non-invasive screening of high-grade cervical lesions and cervical cancer can be achieved, and high detection specificity and sensitivity are achieved.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a reagent composition, kit, and application for cervical cancer detection. Background Technology

[0002] Cervical cancer (CC) is the fourth most common malignant tumor among women worldwide and the only malignant tumor with a clearly defined cause. Its main causative factor is persistent infection with high-risk human papillomavirus (HPV). Currently, commonly used cervical cancer screening methods mainly include cytological examination (such as TCT) and HPV testing, but both methods have significant limitations. TCT testing has low sensitivity, and its results largely depend on the pathologist's subjective experience, making false negatives common. Furthermore, its high dependence on the physician's operational and analytical skills and experience significantly affects the accuracy of the test results. While HPV testing has high sensitivity, its positive predictive value is low, resulting in many women with only transient infections or low-risk lesions requiring colposcopy and biopsy for further diagnosis.

[0003] Therefore, there is an urgent need to develop more precise and efficient early screening and diagnostic technologies to overcome the limitations of existing methods and improve the grim situation of cervical cancer prevention and control. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned problems in the prior art and provide a reagent composition, kit, and application for cervical cancer detection. The composition provided by this invention detects the methylation level of specific gene combinations, exhibiting high detection specificity and sensitivity. Furthermore, this composition can achieve efficient screening for high-grade cervical lesions and cervical cancer simply by detecting cervical exfoliated cell samples, thereby improving patient compliance and increasing detection efficiency.

[0005] To achieve the above objectives, a first aspect of the present invention provides a reagent composition for detecting cervical cancer, the reagent composition comprising a reagent for detecting the methylation level of a cervical cancer marker gene or a fragment thereof, wherein the marker gene includes... PAUPAR Gene, NETO1 Genes and NTM gene .

[0006] A second aspect of the present invention provides a kit for detecting cervical cancer, the kit comprising the reagent composition described in the first aspect.

[0007] The third aspect of the present invention provides the use of the reagent composition described in the first aspect, or the kit described in the second aspect, in screening cervical cancer drugs.

[0008] The fourth aspect of the present invention provides joint detection PAUPAR Gene, NETO1 Genes and NTM Application of reagents for gene methylation levels in the preparation of cervical cancer detection products.

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects: (1) The present invention performs combined detection of methylation levels for a combination of specific cervical cancer characteristic genes, which has a good screening effect on high-grade cervical lesions and cervical cancer, and the detection specificity and sensitivity are both at a high level.

[0010] (2) The composition of the present invention can be used for non-invasive early screening and diagnosis of high-grade cervical lesions and cervical cancer, which is conducive to improving patient compliance, reducing the difficulty of detection, reducing the dependence on the professionalism of testing personnel and equipment, and has the potential for clinical application and promotion.

[0011] (3) When using the composition / kit of the present invention for screening for high-grade cervical lesions and cervical cancer, it has high detection sensitivity and specificity for cervical exfoliated cell samples, reducing the misdiagnosis rate and missed diagnosis rate. Attached Figure Description

[0012] Figure 1 This is a graph showing the results of quantitative real-time PCR detection of a positive sample with a methylation ratio of 0.5% and a reaction concentration of 10 ng / mL, using the kit from the preparation example in Example 1.

[0013] Figure 2 This is a graph showing the results of quantitative real-time PCR detection of a negative sample containing 100 ng / mL of reagent using the kit from the preparation example in Example 1.

[0014] Figure 3 This is the ROC curve of prediction model A with "CIN2+" as the outcome variable, plotted in Example 2.

[0015] Figure 4 This is the ROC curve of prediction model B with "CIN3+" as the outcome variable, plotted in Example 2. Detailed Implementation

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] In this invention, "methylation level" and "methylation status" both refer to the methylation status of the target sequence in the marker gene (which may include whether the target sequence in the marker gene is methylated, the degree of methylation, etc.), have similar meanings, and can be used interchangeably.

[0018] Studies have shown that methylation of specific genes is closely related to the occurrence of cancer. Based on this, gene testing products for early cervical cancer screening have been developed in this field. However, some of these products detect methylation of a single gene, while others require detection of methylation levels of a large number of genes. Many single-gene testing products suffer from poor sensitivity and specificity, which can easily lead to misdiagnosis. As a result, the screening effect for cervical cancer is still not ideal, with a high incidence of missed screenings and false positives. Multiple other testing methods are still needed for diagnosis. On the other hand, large-scale gene methylation testing methods lead to a significant increase in the cost of testing reagents and complex result analysis. Moreover, most current methods use individual detection of each gene methylation level, which can easily lead to sample contamination and operational errors due to the large number of samples tested, thus adversely affecting the accuracy of the test.

[0019] Through long-term research, the inventors of this invention have ingeniously discovered that the methylation levels of several specific genes are highly correlated with cervical cancer and high-grade cervical lesions. Using these genes for cervical cancer screening has excellent sensitivity and specificity, thereby effectively improving the screening accuracy and the effective utilization of medical resources.

[0020] Based on this, a first aspect of the present invention provides a reagent composition for detecting cervical cancer, the reagent composition comprising a reagent for detecting the methylation level of cervical cancer marker genes or fragments thereof, wherein the marker genes include PAUPAR Gene, NETO1 Genes and NTM gene .

[0021] The gene names involved in this invention have a general meaning in the art, and their complete sequences can be obtained through conventional methods in the art, such as by querying public bioinformatics databases such as NCBI.

[0022] The composition provided by this invention can target the complete PAUPAR Gene, NETO1 Genes and NTMThe methylation level of the gene can be detected, or the methylation level of a specific segment (e.g., a single segment or multiple segments) of the gene can be detected. The inventors discovered that detecting and analyzing the methylation level of a specific region within the gene can achieve the goal of screening for cervical cancer and high-grade cervical lesions with high sensitivity and specificity. Compared to detecting the entire gene, detecting specific segments is simpler and easier; therefore, this invention preferably uses a specific region in the composition. PAUPAR Gene, NETO1 Genes and NTM gene A reagent for detecting the methylation level in a specific region of the medium.

[0023] According to some preferred embodiments of the present invention, the reagent used to detect the methylation status of cervical cancer characteristic genes or fragments thereof is a reagent used to detect the methylation status of CpG island regions or fragments thereof in cervical cancer characteristic genes. CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G), and a "CpG island" refers to a region on the genome rich in CpG dinucleotides.

[0024] The composition provided by this invention can detect the methylation level in the complete sequence of the CpG island region in the aforementioned genes, or it can detect the methylation level in a portion of the region (such as a single fragment or multiple fragments).

[0025] According to some particularly preferred embodiments of the present invention, the reagent composition comprises a reagent having the function of detecting the methylation level of gene fragments (also referred to as “target sequences” or “target marker sequences” in the present invention) such as SEQ ID NO:1-3 or SEQ ID NO:17-19.

[0026] The composition of the present invention can detect the methylation level of the target sequence described above, or it can detect the methylation level of a segment in a characteristic gene that has at least 80% identity with the target sequence (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100%, or any range consisting of any two of the above values, or any intermediate value within that range). For example, it can detect the methylation level of a gene fragment that contains additional nucleotides on top of the above sequence.

[0027] In this invention, "identity" refers to the percentage of identical sequences between two sequences. For example, 90% identity between sequence 1 and sequence 2 means that 90% of sequence 1 is identical to sequence 2, or 90% of sequence 2 is identical to sequence 1. Differences between the two sequences can be due to nucleotide deletions, additions, or substitutions. For example, sequence b is obtained by deleting 20 consecutive nucleotides from the 5' end of sequence a, which contains 100 nucleotides. Sequence a and sequence b have 80% identity, while sequence b and sequence a have 100% identity.

[0028] According to a preferred embodiment of the present invention, the composition further includes a reagent for detecting an internal standard. An "internal standard" refers to a non-target gene (or fragment thereof) that is detected together with the target gene (or a fragment thereof). Adding an internal standard reagent during detection can further improve detection accuracy. Typically, a known conserved gene from the target analyte (e.g., the sample used for detection) can be selected as the internal standard.

[0029] Preferably, the internal label is at least one of the housekeeping genes. "Housekeeping genes," also known as "family-managing genes" or "housekeeping genes," are a class of genes that are stably expressed in all cells.

[0030] According to a particularly preferred embodiment of the present invention, the internal standard is... ACTB The gene (whose complete sequence can be found in GenBank accession number: NC_000007.14) or a fragment thereof. ACTB The gene is the gene that encodes human cytoskeleton actin (β-Actin).

[0031] Preferably, the target sequence of the internal standard is as shown in SEQ ID NO:4.

[0032] In this invention, the reagents used to detect the methylation level of the characteristic gene and (optionally) the internal standard can be conventional reagents used in methods capable of performing the corresponding detections in the art. For example, detection can be performed using amplification-sequencing, biochips, quantitative PCR for methylation, etc. Correspondingly, the reagents commonly used in the above-mentioned methods can be used in the composition of this invention.

[0033] For example, when using methylation PCR for detection, according to a preferred embodiment of the present invention, the composition includes nucleic acid primers and an optional probe. "Optional probe" means that the probe is not a necessary component of the composition and can be selected for inclusion or exclusion based on actual needs. For example, when using methylation PCR-electrophoresis or methylation PCR-sequencing for detection, the composition may not contain the probe; conversely, when using methylation quantitative real-time PCR for detection, the composition may contain the probe. "Methylation quantitative real-time PCR" refers to a method that involves digesting the region to be detected with a methylation-sensitive restriction endonuclease, followed by quantitative real-time PCR detection using primers and probes specifically designed for the detection target, thereby determining the methylation level of the region to be detected.

[0034] Any nucleic acid primer and (optional) probe capable of detecting the aforementioned characteristic gene and (optionally) internal standard are applicable to this invention. "Primer" refers to an oligonucleotide that, when placed under conditions that induce the synthesis of primer extension products complementary to the nucleic acid strand, i.e., in the presence of nucleotides and an inducer such as DNA or RNA polymerase and at suitable temperature and pH, can serve as a synthesis initiation site. Primers typically contain at least about 9, 10, 15, 20, or 25 or more nucleotides. "Probe" refers to a nucleic acid sequence that hybridizes to a target sequence under specified conditions and can be used to detect the presence of that target sequence. The probe is a fragment that hybridizes to at least 15 nucleotides in the target sequence of the target gene under stringent conditions. For better detection results, according to a particularly preferred embodiment of the invention, the composition comprises a combination of the following primers and probes: (1) Detection PAUPAR Primers and optional probes for gene methylation levels: Primer pairs with nucleotide sequences as shown in SEQ ID NO:5-6, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:7; Alternatively, primer pairs with nucleotide sequences as shown in SEQ ID NO:20-21, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:22; (2) Detection NETO1 Primers and probes for gene methylation levels: Primer pairs with nucleotide sequences as shown in SEQ ID NO:8-9, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:10; Alternatively, primer pairs with nucleotide sequences as shown in SEQ ID NO:23-24, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:25; (3) Detection NTMPrimers and probes for gene methylation levels: Primer pairs with nucleotide sequences as shown in SEQ ID NO:11-12, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:13; Alternatively, primer pairs with nucleotide sequences as shown in SEQ ID NO:26-27, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:28.

[0035] In some preferred embodiments, the composition further includes: (4) Detection of internal standard ( ACTB Primers and probes for the gene: primers with nucleotide sequences as shown in SEQ ID NO:14-15, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:16.

[0036] According to a preferred embodiment of the present invention, the probe is modified with a reporter group (usually located at the 5' end), preferably a fluorescent reporter group. Any fluorescent reporter group commonly used in the art can be used in the present invention, such as ATTO 425, HEX, FAM, ROX, CY5, Quasar705, AF405, etc.

[0037] According to a preferred embodiment of the present invention, the probe is further modified with a fluorescence quenching group (typically located at the 3' end). Any quenching group commonly used in the art for use with a fluorescent reporter group is applicable to the present invention. For example, it can be BHQ-0, BHQ-1, BHQ-2, BHQ-3, SQ1, SQ2, etc. Those skilled in the art are familiar with the pairing between fluorescent groups and their corresponding fluorescence quenching groups, and will not be elaborated further here.

[0038] According to some preferred embodiments of the present invention, the composition further includes a negative control reagent and / or a positive control reagent.

[0039] A negative control reagent refers to a reagent in which the target detection gene (such as the aforementioned characteristic gene or its fragment) is unmethylated. It may contain no nucleic acid or may contain a nucleic acid fragment or plasmid with a methylation level of 0 for the target detection gene. In the composition provided by this invention, the negative control may contain only one verified unmethylated characteristic gene or its fragment, or it may contain multiple verified unmethylated characteristic genes or their fragments.

[0040] A positive control reagent refers to a reagent containing a target gene known to be methylated (such as the aforementioned characteristic gene or its fragment). In the compositions provided by this invention, the positive control may contain only one verified methylated characteristic gene or its fragment, or it may contain multiple verified methylated characteristic genes or their fragments.

[0041] In the composition provided by the present invention, both the negative control and the positive control can be obtained by conventional means, such as by artificial synthesis.

[0042] A second aspect of the present invention provides a kit for detecting cervical cancer, the kit comprising the composition described in the first aspect.

[0043] The kit provided by this invention may contain only the core reagents for detecting cervical cancer (such as primers and optional probes used in the aforementioned characteristic gene detection), or it may include some convenient reagents for detection (such as negative and positive controls used in the aforementioned characteristic gene assays), or it may further include other conventional reagents required for the detection process (such as buffer systems, enzymes, nucleotides, and other reagents required for PCR detection; or reagents required for sample pretreatment steps such as nucleic acid extraction, purification, and methylation detection). Any reagent commonly used in the field for characteristic gene detection can be applied to this invention, and those skilled in the art can select and adjust it according to the actual detection technology used.

[0044] According to some preferred embodiments of the present invention, the kit further includes at least one of an enzyme, a buffer, a magnesium source, and deoxyribonucleoside triphosphates (dNTPs).

[0045] Preferably, the enzyme comprises a methylation-sensitive restriction endonuclease and / or a DNA polymerase. Any enzyme suitable for use in methylation-based quantitative PCR is applicable to this invention. Preferably, the methylation-sensitive restriction endonuclease comprises at least one of HpaII, HinP1I, and HhaI. Any DNA polymerase available in the art for methylation-based quantitative PCR can be used in this invention; for example, conventional DNA polymerases or hot-start DNA polymerases (such as H-Taq polymerase) can be used.

[0046] Preferably, the magnesium source comprises a water-soluble inorganic magnesium salt. Typically, the magnesium source can be provided in aqueous solution form, such as Mg... 2+ Magnesium chloride, magnesium sulfate, magnesium nitrate, etc., with concentrations of 1-6 mM.

[0047] More preferably, the kit also includes reagents for nucleic acid extraction and / or purification.

[0048] The reagents used for nucleic acid extraction and / or purification are primarily used to extract nucleic acids from samples, and the extracted nucleic acids are further detected using the reagents contained in the kit of this invention for detecting the methylation level of characteristic genes. Any reagents in the art that can be used to extract / purify nucleic acids from biological samples are applicable to this invention; they can be purchased commercially or prepared according to existing technology. Biological samples can be test samples collected from subjects in need, such as at least one of histological sections, tissue biopsies / paraffin-embedded tissues, cells, cervical smear samples, etc.

[0049] In this invention, there are no particular restrictions on the concentration of the various reagents contained in the kit, and they can be adjusted according to actual testing needs.

[0050] To achieve better detection results (such as improved sensitivity, specificity, and accuracy), according to some preferred embodiments of the present invention, the reagents included in the kit contain Mg. 2+ The final concentration of the primer can be 1-6 mM; the final concentration of the dNTPs can be 1-80 mM; the final concentration of the methylation-sensitive restriction endonuclease can be 0.01-30 U / μL; the final concentration of the primer can be 0.1-40 μM; and the final concentration of the probe can be 0.1-20 μM. The final concentrations of primers and probes refer to the final concentration of one primer / probe.

[0051] The present invention further provides a method for detecting cervical cancer, the method comprising detecting a sample using the composition described in the first aspect or the kit described in the second aspect.

[0052] The present invention further provides the use of the reagent composition described in the first aspect and / or the kit described in the second aspect in screening cervical cancer samples.

[0053] This invention further provides for SORCS3 Gene, DMRTA2 Genes and SIX6 Application of combined detection of gene methylation levels in cervical cancer detection.

[0054] The methods and applications provided by this invention can be diagnostic or non-diagnostic. For example, diagnostic aspects may include using the compositions or kits provided by this invention to test samples from subjects in need, determining whether they have a risk of cervical cancer or whether they have cervical cancer, thereby determining subsequent treatment plans (e.g., whether to conduct further diagnosis and testing, whether to conduct further treatment, etc.). As another example, non-diagnostic aspects may include using the compositions or kits provided by this invention to test samples in research or non-diagnostic testing, such as using the compositions or kits provided by this invention to test experimental samples in cervical cancer mechanism research, drug development, etc.

[0055] Based on this, the third aspect of the present invention provides the use of the reagent composition described in the first aspect, or the kit described in the second aspect, in screening cervical cancer drugs.

[0056] For example, in the drug screening process, the reagent composition or kit of the present invention can be used to detect the methylation level of the aforementioned cervical cancer characteristic genes in cell samples or experimental animals after drug administration, thereby determining whether the efficacy of the drug meets the criteria for proceeding to the next step of the experiment.

[0057] The fourth aspect of the present invention provides joint detection PAUPAR Gene, NETO1 Genes and NTM Application of reagents for gene methylation levels in the preparation of cervical cancer detection products.

[0058] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0059] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0060] Preparation Example 1. Identify the target gene and prepare the target sequence and primers and probes for detecting the target sequence. by PAUPAR Gene, NETO1 Genes and NTM The combination of genes is used as a characteristic gene, and its target sequences are shown in SEQ ID NO:1-3 as follows:

[0061]

[0062]

[0063] To further improve detection accuracy, the ACTB gene was used as an internal standard. The target sequence of the internal standard is shown below:

[0064] The primers and probes used for detection are shown in Table 1, and all were synthesized by Hunan Kangde Biotechnology Co., Ltd. In Table 1, primers marked with "F" are upstream primers, primers marked with "R" are downstream primers, and probes are marked with "P".

[0065] Table 1

[0066] 2. Prepare the unit reaction reagent kit for PCR reaction. Prepare the corresponding reagents according to the required amount of reagents for the unit reaction reagent kits (i.e., reagent kits used to test one sample in one PCR reaction) in Table 2.

[0067] Table 2

[0068] *In Table 2, the PCR amplification buffer was purchased from Hunan Kangde Biotechnology Co., Ltd., catalog number PCR buffer (S10); H-Taq enzyme was purchased from Feipeng Biotechnology Co., Ltd.; the methylation-sensitive restriction endonuclease mix was purchased from Hunan Kangde Biotechnology Co., Ltd., catalog number MRE S01, catalog number ME012-01; "upstream primer", "downstream primer" and "probe" are the amounts used for one sequence; the reagents are packaged separately according to the "PCR reaction solution" and "enzyme" in the table, that is, the PCR reaction solution is a mixture of the listed components, and the enzyme is a mixture of the listed enzymes.

[0069] 3. Prepare samples Negative sample: Human genomic DNA with no target gene methylation verified by sequencing, at a concentration of 10 ng / µL.

[0070] Positive samples: prepared by mixing fully methylated human genomic DNA and unmethylated normal human cervical exfoliated cell genomic DNA. These include: concentrations of 1 ng / μL containing 10% fully methylated human genomic DNA (1 ng / μL unmethylated normal human cervical exfoliated cell genomic DNA: 1 ng / μL fully methylated human genomic DNA = 9:1); concentrations of 1 ng / μL containing 1% fully methylated human genomic DNA (1 ng / μL unmethylated normal human cervical exfoliated cell genomic DNA: 1 ng / μL fully methylated human genomic DNA = 99:1); and concentrations of 1 ng / μL containing 0.5% fully methylated human genomic DNA (1 ng / μL unmethylated normal human cervical exfoliated cell genomic DNA: 1 ng / μL unmethylated fully methylated human genomic DNA = 199:1).

[0071] 4. Prepare the reagent kit According to the target detection capacity of a single kit, combine and package the reagents prepared in steps 1-3.

[0072] Example 1 Using the kit prepared in the example, negative and positive samples were used as test samples to detect gene methylation levels, in order to determine its sensitivity and specificity.

[0073] Specific testing methods include: Take a unit reaction reagent kit, mix the reagents in the PCR tube, add 10 μL of the sample to be tested, mix well, and then place the PCR tube in the Hongshi Real-Time PCR Analyzer and perform the PCR reaction according to the reaction conditions in Table 3.

[0074] Table 3

[0075] Select the corresponding channel pair based on the fluorescent labeling group on the probe. PAUPAR Gene, NETO1 Gene, NTM Genes and internal standards ACTB Genetic testing was performed.

[0076] After the reaction is complete, the instrument automatically saves the results, which can be automatically analyzed using the instrument's built-in software. The intersection of the amplification curve and the threshold line is recorded as the Ct value (Ct is an abbreviation for cycle threshold, which refers to the number of cycles that the fluorescence signal in the PCR reaction tube takes to reach the set threshold).

[0077] Figure 1 and Figure 2The detection results for a positive sample with a methylation ratio of 0.5% (10 ng / reaction) and a negative sample with a methylation ratio of 100 ng / reaction are shown. As can be seen from the figure, all target genes showed obvious amplification curves in the positive sample, while only the internal standard showed an amplification curve in the negative sample. This indicates that the kit of the present invention has excellent specificity, and the fact that it can detect methylation of target sequences even at such a low final concentration in the positive sample demonstrates the kit's excellent sensitivity.

[0078] Example 2 The kit prepared in the example was used to test actual samples to verify its effectiveness in detecting cervical cancer.

[0079] The samples used in this embodiment included 474 clinically collected cervical exfoliated cell samples, of which 23 were cervical cancer samples, 229 were cervical intraepithelial neoplasia grade 1 (CIN1) samples, 49 were cervical intraepithelial neoplasia grade 2 (CIN2) samples, 35 were cervical intraepithelial neoplasia grade 3 (CIN3) samples, and 138 were normal samples. Informed consent was obtained from relevant personnel for the research content of this embodiment.

[0080] Specific testing and verification methods include: (1) Sample processing Nucleic acid was extracted from each sample using Sansure Biotech's S10025 nucleic acid extraction reagent and the Natch48 nucleic acid extractor.

[0081] (2) Sample testing and result analysis Following the method described in Example 1, methylation fluorescence PCR was performed on the extracted nucleic acids from each sample. Based on the amplification results, the ΔCt value (Ct(target gene) - Ct(internal reference gene)) was calculated, and logistic regression fitting and ROC curve analysis were performed to obtain the prediction model. Model A - with "CIN2+" as the outcome variable: P=EXP(3.7708-0.2334ΔCt(PAUPAR)+0.0223ΔCt(NETO1)-0.1129ΔCt(NTM)) / (1+EXP(3.7708-0.2334ΔCt(PAUPAR)+0.0223ΔCt(NETO1)-0.1129ΔCt(NTM))) When the p-value calculated by the prediction model is greater than or equal to 0.244, it is judged as positive; when it is less than 0.244, it is judged as negative.

[0082] Model B - with "CIN3+" as the outcome variable: P=EXP(2.7186-0.2164ΔCt(PAUPAR)+0.1245ΔCt(NETO1)-0.2599ΔCt(NTM)) / (1+EXP(2.7186-0.2164ΔCt(PAUPAR)+0.1245ΔCt(NETO1)-0.2599ΔCt(NTM))) When the P-value calculated by the prediction model is greater than or equal to 0.199, it is judged as positive; when it is less than 0.199, it is judged as negative.

[0083] The ROC curve analysis results for prediction model A and prediction model B are respectively referred to Figure 3 and Figure 4 The figure shows the AUC value of the overall model and indicates the optimal cutoff value for this population.

[0084] The optimal cutoff value was used to determine the methylation test results. The detection results of prediction model A and prediction model B for high-grade cervical lesions and cervical cancer are shown in Table 4. In the table, "CIN2-" represents CIN1 and normal samples; "CIN2+" represents CIN2, CIN3 and cervical cancer; "CIN3-" represents CIN2, CIN1 and normal samples; "CIN3+" represents CIN3 and cervical cancer; CIN1 and normal samples were both negative.

[0085] Sample detection sensitivity (%) = (Number of positive cases detected / Total number of positive cases) × 100% Sample detection specificity (%) = (Total number of negative detections / Total number of negative samples) × 100% Table 4

[0086] As can be seen from Table 4, the composition of the present invention has high detection sensitivity and specificity for high-grade cervical lesions and cervical cancer. The sensitivity and specificity for detecting high-grade cervical lesions CIN2 and CIN3 and cervical cancer (CIN2+) are 70.1% and 91.8%, respectively, and the sensitivity and specificity for detecting CIN3 and cervical cancer (CIN3+) are 84.5% and 91.3%, respectively.

[0087] Therefore, it can be seen that adopting PAUPAR , NETO1 , NTMThe three-target combination biomarker demonstrates high sensitivity in detecting cervical exfoliated cell samples of high-grade cervical lesions and cervical cancer, indicating that these three biomarkers are tissue-specific methylation gene combinations for high-grade cervical lesions and cervical cancer. Using this combination for screening high-grade cervical lesions and cervical cancer results in high accuracy and a low false negative rate. Furthermore, the specificity of the target combination biomarker used in this invention for detecting high-grade cervical lesions and cervical cancer samples exceeds 90%, demonstrating that this detection system possesses good specificity and a low false positive rate, providing an effective and accurate new method for screening and diagnosing high-grade cervical lesions and cervical cancer.

[0088] Example 3 Following the method in Example 2, the clinical samples used in Example 2 were tested, and the test results of each clinical sample were analyzed. The difference was that the primers and probes used in the kit to detect the marker gene target sequences were replaced with primers and probes targeting the following target sequences (the target sequence and primer / probe for the internal standard remained unchanged). The specific primer and probe sequences are shown in Table 5, where "F" indicates the upstream primer, "R" indicates the downstream primer, and "P" indicates the probe. The analysis and calculation results for the clinical samples are shown in Table 6.

[0089]

[0090]

[0091]

[0092] Table 5

[0093] Table 6

[0094] By comparing the test results of different target sequence combinations in Table 6, it can be seen that although the detection sensitivity and specificity fluctuate to some extent with the change of target sequence combination, overall, the detection sensitivity and specificity using the method selected in this invention are still effective. PAUPAR , NETO1 , NTM The three-target combination biomarker can achieve a high level of sensitivity in detecting cervical exfoliated cell samples of high-grade cervical lesions and cervical cancer, thereby achieving the goal of rapid and accurate screening for cervical cancer and advanced precancerous lesions.

[0095] Comparative Example 1 The clinical samples used in Example 2 were tested, and the testing and judgment methods were the same as in Example 2. The difference was that one of the target genes was replaced with... CADM1 The diagnostic performance of combined detection of different target genes for cervical cancer was compared.

[0096] PAUPAR Gene, NETO1 Gene, NTM Genes and internal standards ACTB The target sequences of the gene are shown in SEQ ID NO:1-4, and their primers and probes are detailed in Table 1. CADM1 The target sequence of the gene is shown in SEQ ID NO:29, and its primers and probes are shown in Table 7 below.

[0097]

[0098] Table 7

[0099] Note: CC- CADM1 -P is modified with a fluorescent reporter group at the 5' end and a corresponding fluorescent quencher group at the 3' end. The specific modified group depends on the fluorescent reporter group modified on the probes of other genes being detected together (so that the fluorescent reporter group modified on this probe is detected in a different channel than the fluorescent reporter group modified on the probes of other genes).

[0100] The sensitivity and specificity of cervical exfoliated cell samples were calculated using the method described in Example 2 for combined detection of different target gene combinations. The results are detailed in Table 8.

[0101] Table 8

[0102] As can be seen from Table 8, the detection results of different target gene combinations in the above-mentioned target gene joint detection system are different. Among them, the target gene combination selected in this invention ( PAUPAR + NETO+NTM The detection sensitivity and specificity of this combination are the highest. That is, among these combined detection systems, the false negative rate and false positive rate are the lowest when using this combination for screening high-grade cervical lesions and cervical cancer. This indicates that the combined detection of this gene combination for cervical cancer screening is more suitable for promotion and application in clinical and research work.

[0103] The research related to this invention was funded by the Hunan Provincial Science and Technology Innovation Program, project number 2024RC9025.

[0104] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A reagent composition for detecting cervical cancer, characterized in that, The reagent composition includes reagents for detecting the methylation level of cervical cancer marker genes or fragments thereof, wherein the marker genes include PAUPAR Gene, NETO1 Genes and NTM gene .

2. The reagent composition according to claim 1, wherein, The reagent composition includes reagents that have the function of detecting the methylation level of gene fragments with nucleotide sequences such as SEQ ID NO:1-3 or SEQ ID NO:17-19.

3. The reagent composition according to claim 1 or 2, wherein, The reagent composition also includes a reagent for detecting an internal standard; Preferably, the internal standard is selected from at least one of the human housekeeping gene or a fragment thereof; More preferably, the internal standard is ACTB The gene or a fragment thereof, preferably the nucleotide sequence of the internal standard as shown in SEQ ID NO:

4.

4. The reagent composition according to any one of claims 1-3, wherein, The reagent composition includes nucleic acid primers and optional probes; Preferably, the reagent composition comprises a combination of the following primers and optional probes: (1) Detection PAUPAR Primers and optional probes for gene methylation levels: Primer pairs with nucleotide sequences as shown in SEQ ID NO:5-6, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:7; Alternatively, primer pairs with nucleotide sequences as shown in SEQ ID NO:20-21, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:22; (2) Detection NETO1 Primers and probes for gene methylation levels: Primer pairs with nucleotide sequences as shown in SEQ ID NO:8-9, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:10; Alternatively, primer pairs with nucleotide sequences as shown in SEQ ID NO:23-24, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:25; (3) Detection NTM Primers and probes for gene methylation levels: Primer pairs with nucleotide sequences as shown in SEQ ID NO:11-12, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:13; Alternatively, primer pairs with nucleotide sequences as shown in SEQ ID NO:26-27, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:28; More preferably, the reagent composition further includes: (4) Detection ACTB Primers and probes for the gene: primer pairs with nucleotide sequences as shown in SEQ ID NO:14-15, and optionally, probes with nucleotide sequences as shown in SEQ ID NO:

16.

5. The reagent composition according to claim 4, wherein, The probe is modified with a fluorescent reporter group and a fluorescent quencher group.

6. The reagent composition according to any one of claims 1-5, wherein, The reagent composition also includes a negative control reagent and / or a positive control reagent.

7. A reagent kit for detecting cervical cancer, characterized in that, The kit comprises the reagent composition according to any one of claims 1-6.

8. The kit according to claim 7, wherein, The kit also includes at least one of the following: enzyme, buffer, magnesium source, and deoxyribonucleoside triphosphate; Preferably, the enzyme comprises a methylation-sensitive restriction endonuclease and / or a DNA polymerase, and more preferably, the methylation-sensitive restriction endonuclease comprises at least one of HpaII, HinP1I, and HhaI; Preferably, the magnesium source comprises a water-soluble inorganic magnesium salt; More preferably, the kit also includes reagents for nucleic acid extraction and / or purification.

9. The use of the reagent composition of any one of claims 1-6, or the kit of claim 7 or 8, in screening cervical cancer drugs.

10. Joint detection PAUPAR Gene, NETO1 Genes and NTM Application of reagents for gene methylation levels in the preparation of cervical cancer detection products.

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