WO2022057608A1 - 用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒 - Google Patents

用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒 Download PDF

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WO2022057608A1
WO2022057608A1 PCT/CN2021/115485 CN2021115485W WO2022057608A1 WO 2022057608 A1 WO2022057608 A1 WO 2022057608A1 CN 2021115485 W CN2021115485 W CN 2021115485W WO 2022057608 A1 WO2022057608 A1 WO 2022057608A1
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gene
methylation
primer
cervical
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熊驷骏
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北京起源聚禾生物科技有限公司
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Definitions

  • the invention belongs to the technical field of nucleic acid in vitro diagnosis, and in particular relates to a composition and a kit for applying a specific gene methylation marker to the early detection of cervical high-grade lesions and cervical cancer.
  • cervical cancer is the fourth most common cancer among women in the world. According to statistics, in 2018, there were 570,000 new cases of cervical cancer and 311,000 deaths worldwide. Cervical cancer has become the main cause of cancer death among women in most low- and middle-income countries. The etiology of cervical cancer is clear, and persistent infection with high-risk human papillomavirus (HPV) is a necessary condition. The process from cervical precancerous lesions to invasive cervical cancer can take up to ten years. Therefore, early cervical cancer screening can help. Effectively block the progression of precancerous lesions. In recent years, the incidence of cervical cancer has become younger and younger, and the incidence of women under the age of 35 has increased significantly, accounting for about 30% of new cases.
  • HPV human papillomavirus
  • HPV Human papillomavirus
  • HPV-16 and HPV-18 are basically associated with more than 90% of cervical cancers.
  • HPV nucleotides Since persistent high-risk HPV infection is the primary cause of cervical lesions, the detection of HPV nucleotides has a very high sensitivity in the detection of cervical lesions (about 90% in literature data), but because only a small number of High-risk HPV-infected patients present with cervical lesions, and thus HPV testing exhibits very low specificity (approximately 60.7%), especially in young women.
  • DNA methylation is a modification of epigenetics. Studies have reported that DNA methylation can affect gene expression and silencing in normal mammalian cells; at the same time, it has been found in human tumor research that DNA methylation can often lead to tumors. CpG island changes in the promoter region of repressed genes. Either hypermethylation or hypomethylation in the promoter regions of tumor suppressor genes may lead to cellular transformation, making DNA methylation status a potential marker for tumor detection.
  • DNA methylation occurs mainly in the promoter region of genes (where DNA begins to be transcribed into RNA) and is usually closely associated with the inactivation of tumor suppressor gene expression.
  • Commonly used methods for methylation research include: methylation-specific PCR (Methylation-specific PCR, MSP), bisulfite sequencing (Bisulfite sequencing PCR, BSP) and high-resolution melting curve method (High Resolution Melting, HRM) et al.
  • Methylation-specific PCR mainly relies on the combination of primers and target templates to perform PCR amplification to detect methylation sites; bisulfite sequencing method relies on sequencing primers for PCR amplification, and on this basis, subsequent sequencing is performed to achieve Detection of methylation sites; high-resolution melting curve method is mainly used to distinguish methylation and non-methylation by the change of melting temperature caused by the change of CG content in the sample. Each method has its own characteristics.
  • the BSP method has high accuracy and is easy to interpret intuitively, but it has low sensitivity, and the operation is relatively cumbersome and costly; the HRM method is relatively low in sensitivity, and the result analysis is slightly complicated; The detection sensitivity is high, the sample requirements are relatively low, the detection time is short, the cost is low, and the results are easy to interpret.
  • Cervical exfoliated cells are currently the main source of samples for cervical cancer screening. Whether it is liquid-based cytology testing or HPV testing, collecting the secretions of cervical exfoliated cells is a necessary operation. The remaining cell preservation solution can be used in this kit for methylation research, which will not bring additional inconvenience to patients in terms of sample source.
  • a kit to detect gene methylation high sensitivity and high specificity are very important index conditions. On the one hand, the normal gene background is relatively high, and on the other hand, the experimental process is limited by the current transformation technology. resulting in high losses.
  • the purpose of the present invention is to provide a composition and kit for early detection of cervical high-grade lesions and cervical cancer, which solves the problem that false positive results are prone to occur in conventional detection.
  • the present invention provides a composition for the early detection of cervical high-grade lesions and cervical cancer, comprising FAM19A4 gene methylation primer, JAM3 gene methylation primer and PAX1 gene methylation primer and 1 pair Internal reference gene GAPDH primer, the primer sequence is as follows:
  • FAM19A4 gene methylation forward primer F ATTTCGGTTAAAACGGTGAAATTTC
  • FAM19A4 gene methylation reverse primer R TACAAACTCCGCCTCCCG;
  • JAM3 gene methylation forward primer F ATAGGGTAGCGGCGGTTG
  • JAM3 gene methylation reverse primer R ACGACAACGACGACGACAC;
  • PAX1 gene methylation forward primer F TCGTGTTCGGGATTTCG
  • PAX1 gene methylation reverse primer R AAACAAATAAACAACCGCCGT;
  • GAPDH forward primer F TTATTTTTTGGTATGTGGTTGG
  • GAPDH reverse primer R ACCACCCTATTACTATAACCAAATT.
  • the composition for early detection of cervical high-grade lesions and cervical cancer due to the diversity of cervical cancer and cervical lesions of different grades, the combined detection of multi-gene methylation regions complements each other in function, which is beneficial to improve high-grade lesions and cervical lesions.
  • the detection rate of cancer as an aid for the early diagnosis of high-grade lesions and cervical cancer, including FAM19A4 gene methylation-specific primers, JAM3 gene methylation-specific primers, PAX1 gene methylation-specific primers and a pair of internal reference gene primers .
  • methylation-specific primers mainly distributes multiple CpG islands on the forward and reverse primers, and it is optimal to distribute 2-3 CG sites per primer to improve the specificity and accuracy of the primers for the methylated region. identification, increasing detection sensitivity and specificity.
  • the Taqman probe corresponding to the methylation site of the FAM19A4 gene also includes the Taqman probe corresponding to the methylation site of the JAM3 gene, the Taqman probe corresponding to the methylation site of the PAX1 gene, and the internal reference gene GAPDH
  • the corresponding Taqman probe, the probe sequence is as follows:
  • FAM19A4 gene probe FAM-TTAGTCGGGCGTAGTGGCGCGCGTTT-BHQ1;
  • JAM3 gene probe ROX-ATTCGTGGAGACGCGTCGTCGTTA-BHQ2;
  • PAX1 gene probe CY5-TTGGCGTTCGTGGGCGATACGGGATT-BHQ2;
  • the detection system is to detect four genes in one tube, and the four genes need to be reflected in the form of Ct values through different fluorescence channels, so the amplification of the four genes in the system does not interfere with each other, and the four genes
  • the amplification efficiency is consistent with the single-plex amplification efficiency, it means that the multiplex reaction system is not affected by inhibition. Therefore, the above specific sequences are determined through the design and screening of a large number of primers.
  • the combined detection of three target genes FAM19A4, JAM3 and PAX1 and a reasonable threshold setting can increase the accuracy of early detection of cervical cancer.
  • the invention uses a special primer design method and a clinical sample with accurate pathological information to determine a reasonable positive judgment value through the ROC curve, improves the accuracy of the screening of the reaction system and the reliability of early detection of cervical high-grade lesions and cervical cancer, and maximizes the The occurrence of false positive and false negative results is avoided, and the detection performance of the entire kit is significantly improved.
  • the methylated region was further specifically identified by the design of the probe, that is, the probe could distinguish whether the FAM19A4, JAM3 and PAX1 genes were methylated.
  • the design points for the probe are as follows: specifically distinguish the methylated and unmethylated regions; the binding free energy ⁇ G between the probe and the methylated template and that between the probe and the unmethylated template is 20kcal mol -1 ; The sequence after sulfate conversion has only three bases A, T, and G (except for the CG site).
  • the composition for early detection of cervical high-grade lesions and cervical cancer also includes a Taqman probe corresponding to the methylation site of the FAM19A4 gene, a Taqman probe corresponding to the methylation site of the JAM3 gene, and the methylation site of the PAX1 gene.
  • the point of screening is to observe the Whether the amplification efficiency of the four genes in the standard samples with different methylation degrees is the best, 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.
  • probe sequences are composed of FAM, ROX, CY5 and VIC four-channel fluorescence.
  • the design technique that the binding free energy ⁇ G of the probe and the methylated template and the probe and the unmethylated template differs by 20kcal mol-1, the binding efficiency of the probe and the methylated template is significantly improved, which is more conducive to methyl
  • the identification of chemical sites further improves the specificity and sensitivity of the detection.
  • FAM19A4 gene methylation blocking primer JAM3 gene methylation blocking primer and PAX1 gene methylation blocking primer, and the blocking primer sequence is as follows:
  • FAM19A4 gene methylation blocking primer 1 GCCGGGCGTAGTGGCGCGCG-C3spacer;
  • FAM19A4 gene methylation blocking primer 2 AGTGGGTGTAGTGGTGTGTGT-C3spacer;
  • JAM3 gene methylation blocking primer 1 CTCGTGGAGACGCGCCGCC-C3spacer;
  • JAM3 gene methylation blocking primer 2 TTGTGGAGATGTGTTGTTGTT-C3spacer;
  • PAX1 gene methylation blocking primer 1 GCGCCCGTGGGCGACACGGG-C3spacer.
  • PAX1 gene methylation blocking primer 2 TGGTGTTTGTGGGTGATATGG-C3spacer.
  • Genomic DNA has a variety of template sequences in the transformation product, such as untransformed genomic DNA, methylated genomic DNA in the region containing methylation sites after transformation, and no methylation in the region containing methylation sites after transformation.
  • Sponsored genomic DNA, etc. Therefore, the composition of the conversion product after bisulfite conversion is relatively complex, and only relying on specific primers and probes is easily interfered by other sequence templates, and cannot well identify the methylated region of the corresponding gene.
  • special blocking primer design skills are introduced.
  • blocking primers can block Untransformed sequence templates; blocking primers can block unmethylated sequence templates in methylated regions after conversion. Such a design method simplifies the original complex transformed products, and blocks various sequences that interfere with the target sequence template, thereby exposing more methylated genomic DNA in the region containing methylation sites after transformation. , the added specific primers and probes can better bind to the target template, greatly improving the sensitivity and specificity of methylated region detection.
  • the composition for early detection of cervical high-grade lesions and cervical cancer further includes untransformed blocking primers corresponding to each gene and unmethylated blocking primers in the methylated regions after the transformation.
  • the amount of 100 ⁇ mol/L FAM19A4 gene forward primer F is 0.10-0.50 ⁇ L
  • 100 ⁇ mol/L FAM19A4 gene reverse primer R is 0.10-0.50 ⁇ L
  • 100 ⁇ mol/L JAM3 gene is added in an amount of 0.10-0.50 ⁇ L.
  • the addition amount of forward primer F is 0.10-0.50 ⁇ L
  • the addition amount of 100 ⁇ mol/L JAM3 gene reverse primer R is 0.10-0.50 ⁇ L
  • the addition amount of 100 ⁇ mol/L PAX1 gene forward primer F is 0.10-0.50 ⁇ L
  • 100 ⁇ mol/ The amount of the PAX1 gene reverse primer R added in L is 0.10-0.50 ⁇ L
  • the addition amount of the 100 ⁇ mol/L internal reference gene GAPDH forward primer F is 0.01-0.50 ⁇ L
  • the 100 ⁇ mol/L internal control gene GAPDH reverse primer R is added in an amount of 0.01 -0.50 ⁇ L.
  • the amount of 100 ⁇ mol/L FAM19A4 gene probe added is 0.05-0.50 ⁇ L
  • the amount of 100 ⁇ mol/L JAM3 gene probe added is 0.05-0.50 ⁇ L
  • the amount of 100 ⁇ mol/L PAX1 gene probe added It is 0.05-0.50 ⁇ L
  • the addition amount of 100 ⁇ mol/L internal reference gene GAPDH probe is 0.01-0.10 ⁇ L.
  • the amount of 100 ⁇ mol/L FAM19A4 gene blocking primer 1 is 0.50-1.00 ⁇ L
  • 100 ⁇ mol/L FAM19A4 gene blocking primer 2 is 0.50-1.00 ⁇ L
  • 100 ⁇ mol/L JAM3 gene blocking primer is added in an amount of 0.50-1.00 ⁇ L 1
  • the addition amount is 0.50-1.00 ⁇ L
  • 100 ⁇ mol/L JAM3 gene blocking primer 2 The addition amount is 0.50-1.00 ⁇ L, 100 ⁇ mol/L PAX1 gene blocking primer 1
  • the addition amount is 0.50-1.00 ⁇ L, 100 ⁇ mol/L PAX1 gene blocking Primer 2 was added in an amount of 0.50-1.00 ⁇ L.
  • the present invention also provides a kit for early detection of cervical high-grade lesions and cervical cancer, comprising the above-mentioned composition for early detection of cervical high-grade lesions and cervical cancer.
  • positive quality control substances and negative quality control substances are also included.
  • PCR reaction solution includes Taq DNA polymerase, dNTPs, Mg 2+ and 10 ⁇ DNA polymerase buffer.
  • the addition amount of 1U/ ⁇ L Taq DNA polymerase is 0.3-0.8 ⁇ L
  • the addition amount of 25mmol/L dNTPs is 2.0-4.0 ⁇ L
  • the addition amount of 1.5mmol/L Mg 2+ is 3.0- 5.0 ⁇ L
  • the amount of 10 ⁇ DNA polymerase buffer added is 4.0-6.0 ⁇ L.
  • the selection of Taq DNA polymerase and the ratio between dNTPs, Mg 2+ and 10 ⁇ DNA polymerase buffer are directly related to the amplification efficiency of the combination of primers and probes.
  • the present invention detects the methylation sites of FAM19A4, JAM3 and PAX1 genes including not only the promoter region of the gene, but also the coding region of the gene. Due to the diversity of cervical high-grade lesions and cervical cancer species and the diversity of HPV infection, the selection of The combined detection of methylated regions of multiple genes forms a complementation between functions, which significantly improves the sensitivity for cervical cancer and high-grade cervical lesions, but has high specificity for normal and low-grade cervical lesions.
  • the kit uses molecular epigenetic means and methylation detection technology to detect patients with high-grade cervical lesions and possible cervical cancer in advance, and carry out preventive treatment in advance.
  • the present invention adopts this technology to perform multiple multi-channel fluorescence multi-gene methylation detection, and the involved detection samples are easy to obtain, the detection method is simple to operate, intuitive to interpret, and the results are obtained within 8 hours.
  • the entire experimental process adopts a one-stop fully enclosed form to avoid the possibility of cross-contamination. Due to the high detection sensitivity of the present invention, it has better detection efficiency for low-concentration templates.
  • the combined ROC curve area of the three genes FAM19A4, JAM3 and PAX1 is 0.900.
  • the high sensitivity detected by this kit is suitable for the shunt of high-grade cervical lesions, with an overall specificity of 93.1% and an overall sensitivity of 83.9%.
  • CIN2 The detection rates of CIN3, cervical squamous cell carcinoma and cervical adenocarcinoma were 61.54%, 84.21%, 100% and 88.89%, respectively, and the negative detection rates of CIN1 and inflammation were 94.1% and 92.5%. From the above results, it can be seen that, This kit can better identify high-grade cervical lesions and cancer, realize shunting with low-grade lesions and inflammation, and reduce unnecessary colposcopy referrals to a certain extent.
  • FIG. 1 is a ROC curve diagram obtained by detecting 230 samples of a kit for early detection of cervical high-grade lesions and cervical cancer in the specific embodiment.
  • FIG. 2 is a graph showing the sigmoid amplification curve of FAM19A4, JAM3 and PAX1 genes and an internal reference gene (GAPDH) in the specific embodiment.
  • This patent relates to three cervical cancer-related markers:
  • FAM19A4 family with sequence similarity 19 member A4 gene is a member of the TAFA gene family; the TAFA gene family mainly encodes small molecule proteins, which are related to stress and inflammation.
  • FAM19A4 is a ligand for formyl peptide receptor 1, which promotes phagocytosis and increases the release of reactive oxygen species from macrophages. Usually upregulated in lipopolysaccharide-stimulated monocytes and macrophages.
  • FAM19A4 is closely related to cervical cancer and is an important cervical cancer marker. It is also an important shunting method for patients with high-risk HPV infection in cervical cancer screening.
  • JAM3 junctional adhesion molecule 3
  • JAM3 is a member of the JAM gene family, which can directly affect the tight junction between epithelial cells and endothelial cells.
  • JAM3 also commonly referred to as JAMC
  • JAM3 plays an important regulatory role in tumor development, such as its methylation in colorectal cancer and cervical cancer.
  • PAX1 (paired box gene 1) gene is a member of the PAX gene family located on chromosome 20, which 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.
  • Cervical exfoliated cells are currently the main source of samples for cervical cancer screening. Whether it is liquid-based cytology testing or HPV testing, collecting the secretions of cervical exfoliated cells is a necessary operation. The remaining cell preservation solution can be used in this kit for methylation research, which will not bring additional inconvenience to patients in terms of sample source.
  • a kit to detect gene methylation high sensitivity and high specificity are very important index conditions. On the one hand, the normal gene background is relatively high, and on the other hand, the experimental process is limited by the current transformation technology. resulting in high losses.
  • Composition and kit for early detection of cervical high-grade lesions and cervical cancer detecting methylation sites in gene regions closely related to cervical cancer development in exfoliated cells of cervical secretions, including FAM19A4 gene, JAM3 gene and PAX1 gene .
  • the methylation sites that are closely related to cervical cancer in the above three genes were detected, and the hypermethylated CpG island regions in the FAM19A4, JAM3 and PAX1 genes were selected to realize the detection of multi-gene polymethylated regions in one tube reaction. detection.
  • the invention utilizes the technology of introducing blocking primers in the methylation detection, so that the methylated sequence is fully released, which not only improves the detection sensitivity, but also improves the detection specificity.
  • the composition for early detection of cervical high-grade lesions and cervical cancer due to the diversity of cervical cancer and cervical lesions of different grades, the combined detection of multi-gene methylation regions complements each other in function, which is beneficial to improve high-grade lesions and cervical lesions.
  • the detection rate of cancer as an aid for the early diagnosis of high-grade lesions and cervical cancer, including FAM19A4 gene methylation-specific primers, JAM3 gene methylation-specific primers, PAX1 gene methylation-specific primers and a pair of internal reference gene primers , the specific primer sequences are as follows:
  • FAM19A4 gene forward primer F ATTTCGGTTAAAACGGTGAAATTTC
  • FAM19A4 gene reverse primer R TACAAACTCCGCCTCCCG
  • JAM3 gene forward primer F ATAGGGTAGCGGCGGTTG
  • JAM3 gene reverse primer R ACGACAACGACGACGACAC
  • PAX1 gene forward primer F TCGTGTTCGGGATTTCG
  • PAX1 gene reverse primer R AAACAATAAACAACCGCCGT.
  • GPDH Internal reference gene
  • GPDH Internal reference gene reverse primer R: ACCACCCTATTACTATAACCAAATT.
  • the design of methylation-specific primers mainly distributes multiple CpG islands on the forward and reverse primers, and it is optimal to distribute 2-3 CG sites per primer to improve the specificity and accuracy of the primers for the methylated region. identification, increasing detection sensitivity and specificity.
  • the detection system is to detect four genes in one tube, and the four genes need to be reflected in the form of Ct values through different fluorescence channels, so the amplification of the four genes in the system does not interfere with each other, and the four genes When the amplification efficiency is consistent with the single-plex amplification efficiency, it means that the multiplex reaction system is not affected by inhibition. Therefore, the above specific sequences are determined through the design and screening of a large number of primers.
  • the combined detection of three target genes FAM19A4, JAM3 and PAX1 and a reasonable threshold setting can increase the accuracy of early detection of cervical cancer.
  • the invention uses a special primer design method and a clinical sample with accurate pathological information to determine a reasonable positive judgment value through the ROC curve, improves the accuracy of the screening of the reaction system and the reliability of early detection of cervical high-grade lesions and cervical cancer, and maximizes the The occurrence of false positive and false negative results is avoided, and the detection performance of the entire kit is significantly improved.
  • the methylated region was further specifically identified by the design of the probe, that is, the probe could distinguish whether the FAM19A4, JAM3 and PAX1 genes were methylated.
  • the design points for the probe are as follows: specifically distinguish the methylated and unmethylated regions; the binding free energy ⁇ G between the probe and the methylated template and that between the probe and the unmethylated template is 20kcal mol -1 ; The sequence after sulfate conversion has only three bases A, T, and G (except for the CG site).
  • the composition for early detection of cervical high-grade lesions and cervical cancer also includes a Taqman probe corresponding to the methylation site of the FAM19A4 gene, a Taqman probe corresponding to the methylation site of the JAM3 gene, and the methylation site of the PAX1 gene.
  • the point of screening is to observe the Whether the amplification efficiency of the four genes in the standard samples with different methylation degrees is the best, 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.
  • the specific nucleotide sequences of the four probes were determined as follows:
  • FAM19A4 gene probe FAM-TTAGTCGGGCGTAGTGGCGCGCGTTT-BHQ1;
  • JAM3 gene probe ROX-ATTCGTGGAGACGCGTCGTCGTTA-BHQ2;
  • PAX1 gene probe CY5-TTGGCGTTCGTGGGCGATACGGGATT-BHQ2;
  • GPDH Internal reference gene
  • probe sequences are composed of FAM, ROX, CY5 and VIC four-channel fluorescence.
  • the design technique that the binding free energy ⁇ G of the probe and the methylated template and the probe and the unmethylated template differs by 20kcal mol-1, the binding efficiency of the probe and the methylated template is significantly improved, which is more conducive to methyl
  • the identification of chemical sites further improves the specificity and sensitivity of the detection.
  • Genomic DNA has a variety of template sequences in the transformation product, such as untransformed genomic DNA, methylated genomic DNA in the region containing methylation sites after transformation, and no methylation in the region containing methylation sites after transformation.
  • Sponsored genomic DNA, etc. Therefore, the composition of the conversion product after bisulfite conversion is relatively complex, and only relying on specific primers and probes is easily interfered by other sequence templates, and cannot well identify the methylated region of the corresponding gene.
  • the present invention introduces special blocking primer design skills.
  • blocking primers It can block the untransformed sequence template; the blocking primer can block the unmethylated sequence template in the methylated region after transformation.
  • Such a design method simplifies the original complex transformed products, and blocks various sequences that interfere with the target sequence template, thereby exposing more methylated genomic DNA in the region containing methylation sites after transformation.
  • the added specific primers and probes can better bind to the target template, greatly improving the sensitivity and specificity of methylated region detection.
  • the composition for early detection of cervical high-grade lesions and cervical cancer further includes untransformed blocking primers corresponding to each gene and unmethylated blocking primers in the methylated regions after the transformation.
  • FAM19A4 gene blocking primer 1 GCCGGGCGTAGTGGCGCGCG-C3spacer
  • FAM19A4 gene blocking primer 2 AGTTGGGTGTAGTGGTGTGTGT-C3spacer
  • JAM3 gene blocking primer 1 CTCGTGGAGACGCGCCGCC-C3spacer
  • JAM3 gene blocking primer 2 TTGTGGAGATGTGTTGTTGTT-C3spacer
  • PAX1 gene blocking primer 1 GCGCCCGTGGGCGACACGGG-C3spacer.
  • PAX1 gene blocking primer 2 TGGTGTTTGTGGGTGATATGG-C3spacer.
  • PCR fluorescent probe method To use PCR fluorescent probe method to detect gene methylation, one of the most important points is to enrich and capture the fragments where the target methylation site is located as much as possible.
  • the extracted sample contains both the target methylated genome and a large number of unmethylated genomes at the same site; this leads to non-specific amplification (false positive results) in subsequent PCR amplification an important factor.
  • using the most mainstream bisulfite conversion technology to transform the extracted samples is limited by the current bisulfite conversion technology, except that about 80% of the target genome may be lost In addition, there will be a certain probability of untransformed templates; this is another possible cause of false positive results in PCR amplification.
  • the target methylated template after conversion can be enriched and exposed to the matching primer environment to a certain extent. It was successfully captured, and the excitation and collection of fluorescence was realized. Therefore, in the development process of this kit, by adding blocking primers for the different functions of these three genes, to achieve the blocking of other sequences that may cause non-specific amplification, enrich the target sequence, and complete the kit Detection of methylation status of these three genes.
  • the kit used for the early detection of cervical high-grade lesions and cervical cancer requires a PCR reaction solution corresponding to methylation specificity in addition to the combination of primers and probes.
  • the selection of PCR reaction solution is particularly important.
  • Each gene primer probe in the system is particularly important.
  • the amplification efficiency of the system should be similar to that of the corresponding single-plex amplification, to ensure that the primers or probes in the system do not interfere with each other, and to give full play to the amplification effect of each set of primers and probes.
  • the PCR reaction solution includes DNA Taq polymerase, dNTPs and Mg 2+ , 10 ⁇ DNA polymerase buffer, etc.
  • the selection of DNA polymerase and the ratio with dNTPs, Mg 2+ , and 10 ⁇ DNA polymerase buffer are directly related to the amplification efficiency of the combination of primers and probes.
  • the kit components are shown in Table 1.
  • PAX1 Gene-R (100 ⁇ M) 0.2 PAX1 gene-FP (100 ⁇ M) 0.15 FAM19A4 Gene Blocking Primer 1 (100 ⁇ M) 0.85 FAM19A4 Gene Blocking Primer 2 (100 ⁇ M) 0.8 JAM3 Gene Blocking Primer 1 (100 ⁇ M) 0.75 JAM3 Gene Blocking Primer 2 (100 ⁇ M) 0.6 PAX1 Gene Blocking Primer 1 (100 ⁇ M) 0.6 PAX1 Gene Blocking Primer 2 (100 ⁇ M) 0.7 Internal reference gene-F (100 ⁇ M) 0.05 Internal reference gene-R (100 ⁇ M) 0.05 Internal reference gene-FP (100 ⁇ M) 0.05 purified water Make up to 20 ⁇ L
  • the detection test of the kit for the early detection of cervical high-grade lesions and cervical cancer using the composition and the components of the kit for the early detection of cervical high-grade lesions and cervical cancer, includes the following steps:
  • Sample source Select 230 cervical exfoliated cell samples from women aged not less than 21 years old with known clear pathological information: 41 cases were identified as cervical adenocarcinoma and squamous cell carcinoma samples; 34 cases were low-grade intraepithelial lesions (CIN1 ) samples; 102 samples from high-grade intraepithelial lesions (CIN2 and CIN3); 53 samples from inflammation.
  • OD260/280 was between 1.8 and 2.0.
  • step (3) bisulfite conversion is carried out from the cellular genomic DNA obtained in step (2), and the total conversion amount is 100ng-1000ng, and even genomic DNA templates as low as 50ng total amount can be detected, so that no methyl group occurs in the DNA.
  • the methylated 5' cytosine (C) is converted into uracil (U), while the methylated 5' cytosine (C) does not change, and finally the converted Bis-DNA is obtained.
  • the DNA bisulfite conversion kit (DP215) of Root Biochemical Technology (Beijing) Co., Ltd. should ensure the conversion efficiency of DNA and the final conversion yield of Bis-DNA during the conversion process.
  • the PCR mixture includes 100ng-1000ng and 100-300nM primers before transformation, 100-300nM probes, 400-1200nM blocking primers, 1U/ ⁇ L Taq DNA polymerase, 1-5mM MgCl 2 , 20-30mM dNTPs, 10 ⁇ DNA polymerase buffer.
  • the kit reaction system for methylation detection is determined by ROC curve to determine the positive judgment value, including cervical cancer (adenocarcinoma, squamous cell carcinoma, etc.) samples, low-grade cervical lesion samples (CIN1), Cervical high-grade lesions samples (CIN2 and CIN3) and inflammatory samples, that is, methylation of at least one gene in the detected FAM19A4, JAM3 and PAX1 genes, that is, high risk of cervical cancer.
  • cervical cancer adenocarcinoma, squamous cell carcinoma, etc.
  • CIN1 low-grade cervical lesion samples
  • CIN2 and CIN3 Cervical high-grade lesions samples
  • inflammatory samples that is, methylation of at least one gene in the detected FAM19A4, JAM3 and PAX1 genes, that is, high risk of cervical cancer.
  • the detection rates of CIN2, CIN3, cervical squamous cell carcinoma and cervical adenocarcinoma were 61.54%, 84.21%, 100% and 88.89%, respectively; the negative detection rates of CIN1 and inflammation were 94.1% and 92.5%. From the above results, it can be seen that this kit can better identify high-grade cervical lesions and cervical cancer, achieve shunting with low-grade lesions and inflammation, and reduce unnecessary colposcopy referrals to a certain extent.
  • the internal standard channel has a S-shaped amplification curve and the Ct value is less than or equal to 33, and the PAX1, FAM19A4 and JAM3 gene channels have no S-shaped amplification curve or the ⁇ Ct value of the PAX1 gene is greater than 7.08 and the ⁇ Ct value of the FAM19A4 gene is greater than 8.94 and the ⁇ Ct value of JAM3 gene>11.62, the result is negative;
  • the internal standard channel has a S-shaped amplification curve, and the Ct value is ⁇ 33, the ⁇ Ct value of the PAX1 gene detection is ⁇ 7.08, or the ⁇ Ct value of the FAM19A4 gene detection is ⁇ 8.94, or the ⁇ Ct value of the JAM3 gene is ⁇ 11.62, and the three genes are at least If the detection of one channel meets the requirements of the corresponding ⁇ Ct value, the judgment result is positive;

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Abstract

本发明公开了用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒，用于宫颈高级别病变和宫颈癌早期检测的组合物，包括FAM19A4基因甲基化引物、甲基化位点对应的探针和甲基化封闭引物，JAM3基因甲基化引物、甲基化位点对应的探针和甲基化封闭引物，PAX1基因甲基化引物、甲基化位点对应的探针和甲基化封闭引物，内参基因GAPDH的1对引物和甲基化位点对应的探针。

Description

用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒 技术领域
本发明属于核酸体外诊断的技术领域，具体涉及一种将特定的基因甲基化标志物应用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒。
背景技术
目前，宫颈癌是世界女性第四大易发癌种，据统计，2018年全球宫颈癌新发病例570,000例，死亡311,000例，宫颈癌成为多数中低收入国家女性癌症死亡的主要原因。宫颈癌病因明确，高危型人乳头瘤病毒(human papillomavirus，HPV)持续感染是必要条件，由宫颈癌前病变发展至浸润性宫颈癌的过程可长达十年，因而早期进行宫颈癌筛查可以有效阻断癌前病变的进展。近几年宫颈癌的发病趋势愈发年轻化，小于35岁女性发病率明显上升，约占新发病例的30％，另有数据显示，宫颈癌的发病率存在地理差异，农村居民的发病率高于城市居民，我国中部地区较东、西部发病率高，这些差异可能部分归因于缺乏对宫颈癌的有效筛查及防治措施。近年来，随着基因检测和基因编辑技术的发展、预防性HPV疫苗的推广以及治疗性疫苗的开发研究，为宫颈癌早期筛查及预防指明了新的方向。
人乳头瘤病毒(HPV，全称为human papillomavirus)被认为是宫颈癌发生最主要原因，其和宫颈癌的关系最先于在上世纪70年代的时候由德国的科学家Harald zur Hausen提出的，而后得到了验证。据研究，HPV有多达100多种亚型，目前通常根据它的致癌性与否分为低危型和高危型：低危型的HPV通常不会导致恶性病变，但是高危型的HPV感染可能会最终导致宫颈癌前病变和宫颈癌，例如，最主要的两种高危型HPV病毒HPV-16和HPV-18基本跟90％以上的宫颈癌相关。在通常情况下，绝大多数的HPV感染都是暂时的，约超过90％以上的感染会在2～3年里清除掉，而这被认为要归因于人类的细胞调节免疫系统。但值得注意的是，在感染后对HPV-16病毒的抗体似乎只能起到相对普通的效果，没能够自发清除掉的患者在随后的数年或数10年中有发展成为宫颈上皮内瘤病变CIN(cervical intraepithelial neoplasia)甚至宫颈癌的危险。
早期筛查是预防宫颈癌癌前病变及宫颈癌的一种有效方法，目前临床上常用的检测手段主要包括液基细胞学检测和HPV检测。相较于传统的巴氏涂片法(灵敏度在30～87％之间，有时灵敏度甚至低于20％)，液基细胞学检测具有一定的提升，但仍有一定比例的假阴性结果(有研究表明，假阴性概率约为50％)。由于持续性的高危型HPV感染是宫颈病变的首要原因，对HPV核苷酸的检测在对宫颈病变的检测上具有极高的灵敏度(文献数据约为90％)，但由于仅有小部分的高危HPV感染患者会表现出宫颈病变，因而HPV检测表现出极低的特异性(约为60.7％)，特别是在年轻女性中。
DNA甲基化是表观遗传学的一种修饰方式，研究报告，DNA甲基化能影响正常哺乳细胞的基因表达和沉默；同时，在人类肿瘤研究中发现，DNA甲基化通常能导致肿瘤抑制基因启动子区域的CpG岛变化。肿瘤抑制基因启动子区域的高甲基化或者低甲基化都可能导致细 胞转化，而使DNA甲基化状态成为肿瘤检测的潜在标志物。
DNA甲基化主要发生在基因的启动子区域(DNA开始转录为RNA的位置)，通常与抑癌基因的表达失活紧密相关。常见的应用于甲基化研究的方法有：甲基化特异性PCR(Methylation-specific PCR，MSP)、亚硫酸氢盐测序法(Bisulfite sequencing PCR，BSP)和高分辨率熔解曲线法(High Resolution Melting，HRM)等。甲基化特异性PCR主要依靠引物与目标模板的结合以进行PCR扩增来检测甲基化位点；亚硫酸氢盐测序法依靠测序引物进行PCR扩增，再此基础上进行后续测序以实现对甲基化位点的检测；高分辨率溶解曲线法主要通常样本中的CG含量的变化导致的溶解温度的变化以此对甲基化与非甲基化情况进行区分。每种方法都有各自的特点，BSP法结果准确性较高，易于直观进行判读，但灵敏度较低，操作相对更为繁琐成本高；HRM法灵敏度相对较低，同时结果分析略复杂；PCR法检测灵敏度高，对样本的要求相对较低，同时检测时长较短、成本较低，结果易于判读。
宫颈脱落细胞是目前宫颈癌筛查中最主要的样本来源，无论是液基细胞学的检测还是HPV的检测，采集宫颈脱落细胞的分泌物都是必要的操作。而其剩余的细胞保存液，即可应用于本试剂盒做甲基化的情况研究，在样本来源上，不会给患者带来额外的不便。而对于一款对基因甲基化情况进行检测的试剂盒，高灵敏度和高特异性是非常重要的指标条件，一方面在于正常的基因背景较高，一方面在于实验过程中限制于目前转化技术而造成的高损失。
目前PCR扩增中出现非特异性扩增(产生假阳性结果)的现象较多，目前最主流的重亚硫酸盐转化技术对提取后的样本进行转化处理，受限于目前重亚硫酸盐转化技术的限制，除了会有约80％左右的目的基因组可能发生损耗外，还会有一定概率的未转化完成的模板，是存在可能造成假阳性结果的原因。
有鉴于此，特提出本发明。
发明内容
本发明的目的是提供用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒，解决了常规检测易出现假阳性结果的问题。
为了实现上述目的，本发明提供的一种用于宫颈高级别病变和宫颈癌早期检测的组合物，包括FAM19A4基因甲基化引物、JAM3基因甲基化引物和PAX1基因甲基化引物和1对内参基因GAPDH引物，引物序列如下：
FAM19A4基因甲基化正向引物F：ATTTCGGTTAAAACGGTGAAATTTC，
FAM19A4基因甲基化反向引物R：TACAAACTCCGCCTCCCG；
JAM3基因甲基化正向引物F：ATAGGGTAGCGGCGGTTG，
JAM3基因甲基化反向引物R：ACGACAACGACGACGACAC；
PAX1基因甲基化正向引物F：TCGTGTTCGGGATTTCG，
PAX1基因甲基化反向引物R：AAACAAATAAACAACCGCCGT；
内参基因GAPDH正向引物F：TTATTTTTTGGTATGTGGTTGG，
内参基因GAPDH反向引物R：ACCACCCTATTACTATAACCAAATT。
用于宫颈高级别病变和宫颈癌早期检测的组合物，由于宫颈癌以及不同级别宫颈病变具有多样性，多基因甲基化区域的联合检测在功能上相互互补，有利于提高高级别病变以及宫颈癌的检测率，作为辅助高级别病变和宫颈癌的早期诊断，包括FAM19A4基因甲基化特异性引物、JAM3基因甲基化特异性引物和PAX1基因甲基化特异性引物及1对内参基因引物。
针对甲基化特异性引物的设计主要在正向和反向引物上分布多个CpG岛，每个引物分布2-3个CG位点为最佳，提高引物对甲基化区域的特异性精确识别，增加检测灵敏度和特异性。
进一步地，还包括FAM19A4基因的甲基化位点对应的Taqman探针、JAM3基因的甲基化位点对应的Taqman探针、PAX1基因的甲基化位点对应的Taqman探针和内参基因GAPDH对应的Taqman探针，探针序列如下：
FAM19A4基因探针：FAM-TTAGTCGGGCGTAGTGGCGCGCGTTT-BHQ1；
JAM3基因探针：ROX-ATTCGTGGAGACGCGTCGTCGTTA-BHQ2；
PAX1基因探针：CY5-TTGGCGTTCGTGGGCGATACGGGATT-BHQ2；
内参基因GAPDH探针：
VIC-TTTGGTGGTTGGTTTAGAAAAAGGGTTTTGA-BHQ1。
检测体系为一管对四个基因进行检测，并且需要将四个基因通过不同的荧光通道以Ct值的形式体现出来，因此在体系中四个基因之间的扩增相互没有干扰，四个基因的扩增效率与其单重扩增效率一致时，说明在多重反应体系没有受到抑制影响。因此通过大量引物的设计筛选，确定以上具体序列。通过三种目的基因FAM19A4、JAM3和PAX1的组合检测以及合理的阈值设定来增加宫颈癌早期检测的准确性。本发明通过特殊的引物设计方法和准确病理信息的临床样本经过ROC曲线确定合理的阳性判断值，提高了反应体系筛选的准确性和宫颈高级别病变及宫颈癌早期检测的可靠性，最大限度的避免了假阳性和假阴性结果的出现，使得整个试剂盒的检测性能得到明显提升。
后续通过探针的设计对甲基化区域进一步特异性识别，即探针能够区分FAM19A4、JAM3和PAX1基因是否发生甲基化。对于探针的设计要点为：特异区分甲基化和非甲基化区域；探针与甲基化模板和探针与非甲基化模板的结合自由能ΔG相差20kcal mol -1；因重亚硫酸盐转化之后的序列只有A、T、G三种碱基(除CG位点外)，引物或者探针之间非常容易产生干扰，相互之间存在扩增影响，设计时需要避免；用于宫颈高级别病变和宫颈癌早期检测的组合物中还包括FAM19A4基因的甲基化位点对应的Taqman探针、JAM3基因的甲基化位点对应的Taqman探针和PAX1基因的甲基化位点对应的Taqman探针以及内参基因对应的Taqman探针，除了4对特异性甲基化扩增引物之外，在这个反应体系中增加4个不同荧光通道标记的探针，筛选要点就是观察在不同甲基化程度的标准样本中4个基因的扩增效率是否为最佳，荧光曲线是否为标准的S型扩增曲线，与各基因单重扩增相比荧光曲线是否保持一致的趋势。
四种探针序列，分别由FAM、ROX、CY5和VIC四通道荧光组成。采用探针与甲基化模板和探针与非甲基化模板的结合自由能ΔG相差20kcal mol-1的设计技巧，使探针与甲基化模板的结合效率明显提升，更有利于甲基化位点的识别，进一步提高检测的特异性和灵敏 度。
进一步地，还包括FAM19A4基因甲基化封闭引物、JAM3基因甲基化封闭引物和PAX1基因甲基化封闭引物，封闭引物序列如下：
FAM19A4基因甲基化封闭引物1：GCCGGGCGTAGTGGCGCGCG-C3spacer；
FAM19A4基因甲基化封闭引物2：AGTTGGGTGTAGTGGTGTGTGT-C3spacer；
JAM3基因甲基化封闭引物1：CTCGTGGAGACGCGCCGCC-C3spacer；
JAM3基因甲基化封闭引物2：TTGTGGAGATGTGTTGTTGTT-C3spacer；
PAX1基因甲基化封闭引物1：GCGCCCGTGGGCGACACGGG-C3spacer。
PAX1基因甲基化封闭引物2：TGGTGTTTGTGGGTGATATGG-C3spacer。
基因组DNA在转化产物中存在多种模板序列，比如有未被转化的基因组DNA、转化后含甲基化位点区域发生甲基化的基因组DNA、转化后含甲基化位点区域未发生甲基化的基因组DNA等等。因此经过重亚硫酸转化之后的转化产物组成比较复杂，仅依靠特异性引物和探针容易受到其他序列模板的干扰，不能很好的识别对应基因的甲基化区域。为了充分将转化后产物中的发生甲基化区域序列模板释放出来，以便特异性的引物和探针能够更好的识别结合，引入特殊的封闭引物设计技巧，封闭引物设计要点：封闭引物能够封闭未发生转化的序列模板；封闭引物能够封闭转化后甲基化区域未发生甲基化的序列模板。这样的设计方式使原本复杂的转化后产物简单化，多种与目标序列模板相互干扰的序列被封闭，进而使转化后含甲基化位点区域发生甲基化的基因组DNA更多的暴露出来，加入的特异性引物和探针能更好的目标模板结合，大大提高甲基化区域检测的灵敏度和特异性。用于宫颈高级别病变和宫颈癌早期检测的组合物还包括各基因对应的未发生转化的封闭引物和转化后甲基化区域未发生甲基化的封闭引物。除了4对特异性甲基化引物和4个多通道荧光标记的探针，在这个反应体系中增加6个特异性的封闭引物。筛选要点是：封闭引物与引物探针之间不产生影响，不干扰扩增效率；封闭引物的加入增强检测的灵敏度和特异性。
优选地，进行一次检测时，100μmol/L的FAM19A4基因正向引物F加入量为0.10-0.50μL，100μmol/L的FAM19A4基因反向引物R加入量为0.10-0.50μL，100μmol/L的JAM3基因正向引物F加入量为0.10-0.50μL，100μmol/L的JAM3基因反向引物R加入量为0.10-0.50μL，100μmol/L的PAX1基因正向引物F加入量为0.10-0.50μL，100μmol/L的PAX1基因反向引物R加入量为0.10-0.50μL，100μmol/L的内参基因GAPDH正向引物F加入量为0.01-0.50μL，100μmol/L的内参基因GAPDH反向引物R加入量为0.01-0.50μL。
优选地，进行一次检测时，100μmol/L的FAM19A4基因探针加入量为0.05-0.50μL，100μmol/L的JAM3基因探针加入量为0.05-0.50μL，100μmol/L的PAX1基因探针加入量为0.05-0.50μL，100μmol/L的内参基因GAPDH探针加入量为0.01-0.10μL。
优选地，进行一次检测时，100μmol/L的FAM19A4基因封闭引物1加入量为0.50-1.00μL，100μmol/L的FAM19A4基因封闭引物2加入量为0.50-1.00μL，100μmol/L的JAM3基因封闭引物1加入量为0.50-1.00μL，100μmol/L的JAM3基因封闭引物2加入量为0.50-1.00μL，100μmol/L的PAX1基因封闭引物1加入量为0.50-1.00μL，100μmol/L的PAX1基因封闭引 物2加入量为0.50-1.00μL。
本发明还提供一种用于宫颈高级别病变和宫颈癌早期检测的试剂盒，包括上述的用于宫颈高级别病变和宫颈癌早期检测的组合物。
进一步地，还包括阳性质控品和阴性质控品。
进一步地，还包括PCR反应液，所述PCR反应液包括Taq DNA聚合酶、dNTPs、Mg 2+和10×DNA聚合酶buffer。
针对甲基化位点的检测，需要特殊的甲基化检测相关的DNA聚合酶，筛选具有更优扩增效率的聚合酶，在这个包含多重引物探针的反应体系中，PCR反应液的选择尤其重要，体系中每一个基因引物探针的扩增效率需与其对应的单重扩增时类似，确保体系中的引物或者探针之间不相互干扰，充分发挥每一组引物探针的扩增效果。
优选地，进行一次检测时，1U/μL的Taq DNA聚合酶加入量为0.3-0.8μL，25mmol/L的dNTPs加入量为2.0-4.0μL，1.5mmol/L的Mg 2+加入量为3.0-5.0μL，10×DNA聚合酶buffer加入量为4.0-6.0μL。
Taq DNA聚合酶的筛选以及与dNTPs、Mg 2+、10×DNA聚合酶buffer之间的配比直接关系到引物和探针的组合的扩增效率。
本发明提供的用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒，具有如下有益效果：
1、本发明检测FAM19A4、JAM3和PAX1基因甲基化的位点不仅包括基因的启动子区域，还包括基因的编码区域，由于宫颈高级别病变及宫颈癌种类多样性以及HPV感染多样性，选择多基因甲基化区域联合检测，形成功能间的互补，显著提升对宫颈癌以及宫颈高级别病变的灵敏度，但对正常及宫颈低级别病变具有高特异性。该试剂盒通过分子表观遗传手段，利用甲基化检测技术对宫颈高级别病变及宫颈癌可能的患者提早检出，提早进行预防治疗。
2、主要采用多重多通道荧光和封闭技术手段，通过特异性引物探针与甲基化序列准确识别，加上多条封闭引物的作用将甲基化模板充分释放出来，以及优化的特殊的甲基化DNA聚合酶，对FAM19A4、JAM3和PAX1基因甲基化位点精确检测。对比于仅用PCR特异性引物进行检测的方法，加上特异性探针对甲基化位点可以进行双重识别，可以显著提高检测的灵敏度、准确性。并且引入多重封闭引物技术。本发明采用这种技术进行多重多通道荧光多基因的甲基化检测，涉及到的检测样本易获取、检测方法操作简单、判读直观、8个小时内出结果，通用的荧光定量PCR仪器均能满足检测需求，整套实验流程采用一站式全封闭形式，避免了交叉污染的可能。因本发明检测高灵敏性，对低浓度模板有更好的检测效率。FAM19A4、JAM3和PAX1三个基因联合ROC曲线面积为0.900，本试剂盒检测的高灵敏性适用于宫颈高级别病变的分流，整体特异性为93.1％，整体灵敏度为83.9％，其中，对CIN2、CIN3、宫颈鳞癌和宫颈腺癌的检出率分别为61.54％、84.21％、100％和88.89％，对CIN1和炎症的阴性检出率为94.1％和92.5％，从以上结果可以看出，本试剂盒能较好地对宫颈高级别病变以及癌进行鉴别，实现与低级别病变以及炎症的分流，在一定程度上能减少患者不必要的阴道镜转诊。
附图说明
图1为本具体实施方式中用于宫颈高级别病变和宫颈癌早期检测的试剂盒检测230例样本所得ROC曲线图。
图2为本具体实施方式中FAM19A4、JAM3和PAX1基因和内参基因(GAPDH)的S型扩增曲线图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面结合具体实施方式对本发明作进一步的详细说明。
本专利涉及三个宫颈癌相关的标志物：
FAM19A4(family with sequence similarity 19 member A4)基因，是TAFA基因家族中的一员；TAFA基因家族主要编码小分子蛋白，与压力和炎症相关。FAM19A4是甲酰肽受体1的配体，能促进吞噬作用以及增加巨噬细胞释放的活性氧。通常在脂多糖刺激的单核细胞和巨噬细胞中上调。近年，一些研究报告显示FAM19A4与宫颈癌具有紧密联系，是重要的宫颈癌标志物同时在宫颈癌筛查中对高危HPV感染的患者是重要的分流手段。
JAM3(junctional adhesion molecule 3)是JAM基因家族中的一员，JAM基因家族能直接对上皮细胞和内皮细胞的紧密连接作用产生影响。大量文献研究报告JAM3，通常也被称作JAMC，是连接调节因子。近年来，许多文献报告JAM3在肿瘤发展中对肿瘤有重要的调节作用，如其甲基化在结直肠癌和宫颈癌中均有表现。
PAX1(paired box gene 1)基因是位于20号染色体的PAX基因家族中的一员，对胎儿发育和细胞增殖有着关键作用。PAX1基因启动子的甲基化在肿瘤的发生和发展中起着重要的表观遗传学调控作用。有研究表明PAX1在宫颈癌和卵巢癌中发生甲基化而沉默，因而PAX1也被当作是肿瘤抑制基因。
宫颈脱落细胞是目前宫颈癌筛查中最主要的样本来源，无论是液基细胞学的检测还是HPV的检测，采集宫颈脱落细胞的分泌物都是必要的操作。而其剩余的细胞保存液，即可应用于本试剂盒做甲基化的情况研究，在样本来源上，不会给患者带来额外的不便。而对于一款对基因甲基化情况进行检测的试剂盒，高灵敏度和高特异性是非常重要的指标条件，一方面在于正常的基因背景较高，一方面在于实验过程中限制于目前转化技术而造成的高损失。
用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒，对宫颈分泌物脱落细胞中与宫颈癌发展密切相关基因区域甲基化位点进行检测，包括FAM19A4基因、JAM3基因和PAX1基因。针对上述三个基因中与宫颈癌发生密切的甲基化位点进行检测，选取FAM19A4、JAM3和PAX1基因中发生高度甲基化的CpG岛区域，实现一管反应检测多基因多甲基化区域的检测。本发明利用在甲基化检测中引入封闭引物的技术，使甲基化的序列充分释放出来，不仅提高检测的灵敏度，而且提高检测的特异性。
用于宫颈高级别病变和宫颈癌早期检测的组合物，由于宫颈癌以及不同级别宫颈病变具有多样性，多基因甲基化区域的联合检测在功能上相互互补，有利于提高高级别病变以及宫 颈癌的检测率，作为辅助高级别病变和宫颈癌的早期诊断，包括FAM19A4基因甲基化特异性引物、JAM3基因甲基化特异性引物和PAX1基因甲基化特异性引物及1对内参基因引物，具体引物序列如下：
FAM19A4基因正向引物F：ATTTCGGTTAAAACGGTGAAATTTC，
FAM19A4基因反向引物R：TACAAACTCCGCCTCCCG；
JAM3基因正向引物F：ATAGGGTAGCGGCGGTTG，
JAM3基因反向引物R：ACGACAACGACGACGACAC；
PAX1基因正向引物F：TCGTGTTCGGGATTTCG，
PAX1基因反向引物R：AAACAAATAAACAACCGCCGT。
内参基因(GAPDH)正向引物F：TTATTTTTTGGTATGTGGTTGG，
内参基因(GAPDH)反向引物R：ACCACCCTATTACTATAACCAAATT。
针对甲基化特异性引物的设计主要在正向和反向引物上分布多个CpG岛，每个引物分布2-3个CG位点为最佳，提高引物对甲基化区域的特异性精确识别，增加检测灵敏度和特异性。检测体系为一管对四个基因进行检测，并且需要将四个基因通过不同的荧光通道以Ct值的形式体现出来，因此在体系中四个基因之间的扩增相互没有干扰，四个基因的扩增效率与其单重扩增效率一致时，说明在多重反应体系没有受到抑制影响。因此通过大量引物的设计筛选，确定以上具体序列。通过三种目的基因FAM19A4、JAM3和PAX1的组合检测以及合理的阈值设定来增加宫颈癌早期检测的准确性。本发明通过特殊的引物设计方法和准确病理信息的临床样本经过ROC曲线确定合理的阳性判断值，提高了反应体系筛选的准确性和宫颈高级别病变及宫颈癌早期检测的可靠性，最大限度的避免了假阳性和假阴性结果的出现，使得整个试剂盒的检测性能得到明显提升。
后续通过探针的设计对甲基化区域进一步特异性识别，即探针能够区分FAM19A4、JAM3和PAX1基因是否发生甲基化。对于探针的设计要点为：特异区分甲基化和非甲基化区域；探针与甲基化模板和探针与非甲基化模板的结合自由能ΔG相差20kcal mol -1；因重亚硫酸盐转化之后的序列只有A、T、G三种碱基(除CG位点外)，引物或者探针之间非常容易产生干扰，相互之间存在扩增影响，设计时需要避免；用于宫颈高级别病变和宫颈癌早期检测的组合物中还包括FAM19A4基因的甲基化位点对应的Taqman探针、JAM3基因的甲基化位点对应的Taqman探针和PAX1基因的甲基化位点对应的Taqman探针以及内参基因对应的Taqman探针，除了4对特异性甲基化扩增引物之外，在这个反应体系中增加4个不同荧光通道标记的探针，筛选要点就是观察在不同甲基化程度的标准样本中4个基因的扩增效率是否为最佳，荧光曲线是否为标准的S型扩增曲线，与各基因单重扩增相比荧光曲线是否保持一致的趋势。经过大量设计筛选，4个探针具体核苷酸序列确定如下：
FAM19A4基因探针：FAM-TTAGTCGGGCGTAGTGGCGCGCGTTT-BHQ1；
JAM3基因探针：ROX-ATTCGTGGAGACGCGTCGTCGTTA-BHQ2；
PAX1基因探针：CY5-TTGGCGTTCGTGGGCGATACGGGATT-BHQ2；
内参基因(GAPDH)探针：VIC-TTTGGTGGTTGGTTTAGAAAAAGGGTTTTGA-BHQ1。
四种探针序列，分别由FAM、ROX、CY5和VIC四通道荧光组成。采用探针与甲基化模板和探针与非甲基化模板的结合自由能ΔG相差20kcal mol-1的设计技巧，使探针与甲基化模板的结合效率明显提升，更有利于甲基化位点的识别，进一步提高检测的特异性和灵敏度。
基因组DNA在转化产物中存在多种模板序列，比如有未被转化的基因组DNA、转化后含甲基化位点区域发生甲基化的基因组DNA、转化后含甲基化位点区域未发生甲基化的基因组DNA等等。因此经过重亚硫酸转化之后的转化产物组成比较复杂，仅依靠特异性引物和探针容易受到其他序列模板的干扰，不能很好的识别对应基因的甲基化区域。为了充分将转化后产物中的发生甲基化区域序列模板释放出来，以便特异性的引物和探针能够更好的识别结合，本发明引入特殊的封闭引物设计技巧，封闭引物设计要点：封闭引物能够封闭未发生转化的序列模板；封闭引物能够封闭转化后甲基化区域未发生甲基化的序列模板。这样的设计方式使原本复杂的转化后产物简单化，多种与目标序列模板相互干扰的序列被封闭，进而使转化后含甲基化位点区域发生甲基化的基因组DNA更多的暴露出来，加入的特异性引物和探针能更好的目标模板结合，大大提高甲基化区域检测的灵敏度和特异性。用于宫颈高级别病变和宫颈癌早期检测的组合物还包括各基因对应的未发生转化的封闭引物和转化后甲基化区域未发生甲基化的封闭引物。除了4对特异性甲基化引物和4个多通道荧光标记的探针，在这个反应体系中增加6个特异性的封闭引物。筛选要点是：封闭引物与引物探针之间不产生影响，不干扰扩增效率；封闭引物的加入增强检测的灵敏度和特异性；通过大量设计筛选，具体核苷酸序列如下：
FAM19A4基因封闭引物1：GCCGGGCGTAGTGGCGCGCG-C3spacer；
FAM19A4基因封闭引物2：AGTTGGGTGTAGTGGTGTGTGT-C3spacer；
JAM3基因封闭引物1：CTCGTGGAGACGCGCCGCC-C3spacer；
JAM3基因封闭引物2：TTGTGGAGATGTGTTGTTGTT-C3spacer；
PAX1基因封闭引物1：GCGCCCGTGGGCGACACGGG-C3spacer。
PAX1基因封闭引物2：TGGTGTTTGTGGGTGATATGG-C3spacer。
要应用PCR荧光探针法实现对基因甲基化的检测，其中最为重要的一个点在于要尽可能多地实现对目的甲基化位点所在片段的富集与捕捉。提取出的样本中既包含目标的甲基化后基因组，同时还含有大量相同位点非甲基化的基因组；而这是导致后续PCR扩增中出现非特异性扩增(产生假阳性结果)的一个重要因素。另一方面，采用目前最主流的重亚硫酸盐转化技术对提取后的样本进行转化处理，受限于目前重亚硫酸盐转化技术的限制，除了会有约80％左右的目的基因组可能发生损耗外，还会有一定概率的未转化完成的模板存在；而这是PCR扩增的另一可能造成假阳性结果的原因。因而，尽可能地将没有发生甲基化的模板以及没有转化的模板消耗掉或者阻隔掉，能一定程度上使目标的转化后甲基化模板实在富集而暴露在匹配的引物环境中，因而被成功捕捉到，实现荧光的激发与收集。因而，在本试剂盒的研发过程中，通过加入针对此三种基因不同作用的封闭引物，来实现对可能引起非特异性扩增的其它序列的阻断，使目标序列富集，而完成试剂盒对此三种基因甲基化状态的检测。
用于宫颈高级别病变和宫颈癌早期检测的试剂盒，除了引物和探针的组合，需要对应甲基化特异性的PCR反应液，因本发明是针对甲基化位点的检测，需要特殊的甲基化检测相关的DNA聚合酶，筛选具有更优扩增效率的聚合酶，在这个包含多重引物探针的反应体系中，PCR反应液的选择尤其重要，体系中每一个基因引物探针的扩增效率需与其对应的单重扩增时类似，确保体系中的引物或者探针之间不相互干扰，充分发挥每一组引物探针的扩增效果。PCR反应液包括DNA Taq聚合酶、dNTPs和Mg 2+、10×DNA聚合酶buffer等。DNA聚合酶的筛选以及与dNTPs、Mg 2+、10×DNA聚合酶buffer之间的配比直接关系到引物和探针的组合的扩增效率。试剂盒组分如表一。
表一 试剂盒组分表
Figure PCTCN2021115485-appb-000001
其中扩增反应体系中PCR反应液的配制见表二。
表二 PCR反应液(25ul/人份)组分表
组分 一人份加入量(μL)
Taq DNA聚合酶(1U/μL) 0.5
dNTPs(25mM) 3
Mg 2+(1.5mM) 4
10×DNA聚合酶buffer 5
纯化水 补足至25μL
其中引物探针混合液的配制见表三。
表三 引物探针混合液(20μL/人份)组分表
组分 一人份加入量(μL)
FAM19A4基因-F(100μM) 0.2
FAM19A4基因-R(100μM) 0.2
FAM19A4基因-FP(100μM) 0.1
JAM3基因-F(100μM) 0.25
JAM3基因-R(100μM) 0.25
JAM3基因-FP(100μM) 0.15
PAX1基因-F(100μM) 0.3
PAX1基因-R(100μM) 0.2
PAX1基因-FP(100μM) 0.15
FAM19A4基因封闭引物1(100μM) 0.85
FAM19A4基因封闭引物2(100μM) 0.8
JAM3基因封闭引物1(100μM) 0.75
JAM3基因封闭引物2(100μM) 0.6
PAX1基因封闭引物1(100μM) 0.6
PAX1基因封闭引物2(100μM) 0.7
内参基因-F(100μM) 0.05
内参基因-R(100μM) 0.05
内参基因-FP(100μM) 0.05
纯化水 补足至20μL
用于宫颈高级别病变和宫颈癌早期检测的试剂盒的检测试验，利用上述用于宫颈高级别病变和宫颈癌早期检测的组合物及试剂盒的成分，包括以下步骤：
(1)样本来源：选取已知明确病理信息结果的230例年龄不小于21岁女性的宫颈脱落细胞样本：41例鉴定为宫颈腺癌、鳞癌样本；34例为低级别上皮内病变(CIN1)样本；102例高级别上皮内病变(CIN2和CIN3)样本；53例炎症样本。
(2)对上述230例宫颈脱落细胞样本进行细胞基因组DNA的提取，采用血液/细胞/组织基因组DNA提取试剂盒对上述样本进行基因组DNA的提取，本实施例中选择采用天根生化科技(北京)有限公司的血液/细胞/组织基因组DNA提取试剂盒(DP304)进行提取。
提取过程中需保证DNA的提取质量，以进行后续试验。对提取之后的DNA质量监控，OD260/280在1.8～2.0之间。
(3)由步骤(2)中得到的细胞基因组DNA，进行重亚硫酸盐转化，转化总量为100ng-1000ng，甚至能够检测低至50ng总量的基因组DNA模板，使DNA中未发生甲基化的5’胞嘧啶(C)转化为尿嘧啶(U)，而发生甲基化的5’胞嘧啶(C)不发生改变，最后得到转化后的Bis-DNA，本实施例中选择采用天根生化科技(北京)有限公司的DNA重亚硫酸盐转化试剂盒(DP215)转化过程中要保证DNA的转化效率以及最后的Bis-DNA的转化得率。
(4)按照表二和表三配制PCR反应液和引物探针混合液，不同的配比和反应条件与最后的PCR扩增效率密切相关，需要进行不断筛选比较，筛选最优条件。具体优选如下，PCR混合物包括转化前模板总量为100ng-1000ng和100-300nM引物，100-300nM探针，400-1200nM封闭引物，1U/μL的Taq DNA聚合酶，1-5mM的MgCl 2，20-30mM的dNTPs，10×DNA聚合酶buffer。
(5)加样，如表一所示，向步骤(4)中配制的混合液体系中加入5μL阳性质控品和转化好的Bis-DNA临床样本作为阴性质控品。
(6)筛选最优PCR反应条件：96℃预变性5min；94℃变性15s，60℃退火延伸35s，45个循环；25℃保持10min。以步骤(3)中得到的Bis-DNA为模板，进行PCR扩增。进行信号收集，60℃收集FAM、VIC、ROX以及CY5信号。
(7)检测结果分析
通过选取病理信息明确的临床样本对甲基化检测的试剂盒反应体系通过ROC曲线对阳性判断值进行确定，包含宫颈癌(腺癌、鳞癌等)样本、低级别宫颈病变样本(CIN1)、宫颈高级别病变样本(CIN2和CIN3)以及炎症样本，即所检测FAM19A4、JAM3和PAX1基因中至少一个基因发生甲基化，即宫颈癌的发生为高风险。
利用上述试剂盒反应体系检测共230例样本，其中包括宫颈癌样本41例，低级别上皮内病变(CIN1)样本34例，102例高级别上皮内病变(CIN2和CIN3)样本，53例炎症样本，检测结果如表四。对比临床病理结果，使用本甲基化检测试剂盒得到的三个基因联合ROC曲线面积为0.900(如图1所示)，整体特异性为93.1％，整体灵敏度为83.9％。其中，对CIN2、CIN3、宫颈鳞癌和宫颈腺癌的检出率分别为61.54％、84.21％、100％和88.89％；对CIN1和炎症的阴性检出率为94.1％和92.5％。从以上结果可以看出，本试剂盒能较好地对宫颈高级别病变以及宫颈癌进行鉴别，实现与低级别病变以及炎症的分流，在一定程度上能减少患者不必要的阴道镜转诊。
表四 试剂盒反应体系检测共230例样本的检测结果
Figure PCTCN2021115485-appb-000002
Figure PCTCN2021115485-appb-000003
Figure PCTCN2021115485-appb-000004
Figure PCTCN2021115485-appb-000005
(8)结果判读分析
a.满足内标通道有S型扩增曲线，且Ct值≤33，PAX1、FAM19A4和JAM3基因通道均无S型扩增曲线或PAX1基因的△Ct值＞7.08且FAM19A4基因的△Ct值＞8.94且JAM3基因的△Ct值＞11.62，则判定结果为阴性；
b.满足内标通道有S型扩增曲线，且Ct值≤33，PAX1基因检测△Ct值≤7.08，或FAM19A4基因检测△Ct值≤8.94，或JAM3基因△Ct值≤11.62，三基因至少一个通道检测满足相应△Ct值要求，则判定结果为阳性；
c.若内标通道无S型扩增曲线或Ct值＞33，则判定结果无效，建议重新提取样本进行检测。
本文中应用了具体个例对发明构思进行了详细阐述，以上实施例的说明只是用于帮助理解本发明的核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离该发明构思的前提下，所做的任何显而易见的修改、等同替换或其他改进，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种用于宫颈高级别病变和宫颈癌早期检测的组合物，其特征在于，包括FAM19A4基因甲基化引物、JAM3基因甲基化引物和PAX1基因甲基化引物和1对内参基因GAPDH引物，引物序列如下：
    FAM19A4基因甲基化正向引物F：SEQ ID NO.1，
    FAM19A4基因甲基化反向引物R：SEQ ID NO.2；
    JAM3基因甲基化正向引物F：SEQ ID NO.3，
    JAM3基因甲基化反向引物R：SEQ ID NO.4；
    PAX1基因甲基化正向引物F：SEQ ID NO.5，
    PAX1基因甲基化反向引物R：SEQ ID NO.6；
    内参基因GAPDH正向引物F：SEQ ID NO.7，
    内参基因GAPDH反向引物R：SEQ ID NO.8。
  2. 根据权利要求1所述的用于宫颈高级别病变和宫颈癌早期检测的组合物，其特征在于，还包括FAM19A4基因的甲基化位点对应的Taqman探针、JAM3基因的甲基化位点对应的Taqman探针、PAX1基因的甲基化位点对应的Taqman探针和内参基因GAPDH对应的Taqman探针，探针序列如下：
    FAM19A4基因探针：FAM-SEQ ID NO.9-BHQ1；
    JAM3基因探针：ROX-SEQ ID NO.10-BHQ2；
    PAX1基因探针：CY5-SEQ ID NO.11-BHQ2；
    内参基因GAPDH探针：VIC-SEQ ID NO.12-BHQ1。
  3. 根据权利要求1所述的用于宫颈高级别病变和宫颈癌早期检测的组合物，其特征在于，还包括FAM19A4基因甲基化封闭引物、JAM3基因甲基化封闭引物和PAX1基因甲基化封闭引物，封闭引物序列如下：
    FAM19A4基因甲基化封闭引物1：SEQ ID NO.13-C3spacer；
    FAM19A4基因甲基化封闭引物2：SEQ ID NO.14-C3spacer；
    JAM3基因甲基化封闭引物1：SEQ ID NO.15-C3spacer；
    JAM3基因甲基化封闭引物2：SEQ ID NO.16-C3spacer；
    PAX1基因甲基化封闭引物1：SEQ ID NO.17-C3spacer；
    PAX1基因甲基化封闭引物2：SEQ ID NO.18-C3spacer。
  4. 根据权利要求1所述的用于宫颈高级别病变和宫颈癌早期检测的组合物，其特征在于，进行一次检测时，100μmol/L的FAM19A4基因正向引物F加入量为0.10-0.50μL，100μmol/L的FAM19A4基因反向引物R加入量为0.10-0.50μL，100μmol/L的JAM3基因正向引物F加入量为0.10-0.50μL，100μmol/L的JAM3基因反向引物R加入量为0.10-0.50μL，100μmol/L的PAX1基因正向引物F加入量为0.10-0.50μL，100μmol/L的PAX1基因反向引物R加入量为0.10-0.50μL， 100μmol/L的内参基因GAPDH正向引物F加入量为0.01-0.50μL，100μmol/L的内参基因GAPDH反向引物R加入量为0.01-0.50μL。
  5. 根据权利要求2所述的用于宫颈高级别病变和宫颈癌早期检测的组合物，其特征在于，进行一次检测时，100μmol/L的FAM19A4基因探针加入量为0.05-0.50μL，100μmol/L的JAM3基因探针加入量为0.05-0.50μL，100μmol/L的PAX1基因探针加入量为0.05-0.50μL，100μmol/L的内参基因GAPDH探针加入量为0.01-0.10μL。
  6. 根据权利要求3所述的用于宫颈高级别病变和宫颈癌早期检测的组合物，其特征在于，进行一次检测时，100μmol/L的FAM19A4基因封闭引物1加入量为0.50-1.00μL，100μmol/L的FAM19A4基因封闭引物2加入量为0.50-1.00μL，100μmol/L的JAM3基因封闭引物1加入量为0.50-1.00μL，100μmol/L的JAM3基因封闭引物2加入量为0.50-1.00μL，100μmol/L的PAX1基因封闭引物1加入量为0.50-1.00μL，100μmol/L的PAX1基因封闭引物2加入量为0.50-1.00μL。
  7. 一种用于宫颈高级别病变和宫颈癌早期检测的试剂盒，其特征在于，包括权利要求1-6任一项所述的用于宫颈高级别病变和宫颈癌早期检测的组合物。
  8. 根据权利要求7所述的用于宫颈高级别病变和宫颈癌早期检测的试剂盒，其特征在于，还包括阳性质控品和阴性质控品。
  9. 根据权利要求7所述的用于宫颈高级别病变和宫颈癌早期检测的试剂盒，其特征在于，还包括PCR反应液，所述PCR反应液包括Taq DNA聚合酶、dNTPs、Mg 2+和10×DNA聚合酶buffer。
  10. 根据权利要求9所述的用于宫颈高级别病变和宫颈癌早期检测的试剂盒，其特征在于，进行一次检测时，1U/μL的Taq DNA聚合酶加入量为0.3-0.8μL，25mmol/L的dNTPs加入量为2.0-4.0μL，1.5mmol/L的Mg 2+加入量为3.0-5.0μL，10×DNA聚合酶buffer加入量为4.0-6.0μL。
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