TWI329743B - - Google Patents

Description

1329743 IX. Description of the invention: [Technical field to which the invention pertains] Qian Ming side------------------------------------------------------------------------- Cervical cancer is one of the leading causes of death among women in the world and in Taiwan. According to the World Health Organization (WHO) statistics, cervical cancer is the second leading cause of cancer death among women worldwide, second only to breast cancer; ^ Qian shot cervical blue The examination is the best method of the Lions Palace. There are two main methods for screening the liver and the palace. - It is the most common Pap smear (paps), and the other is HPVtesting; Pap smear is to remove the secretion from the neck of the scorpion, microscopically weave the epithelial cells in the detachment. "Can there be cancerous lesions, + stage _ cervical cancer; ❿ Qing test is reverse transcriptase The chain reaction was given a polymemse chain reaction (RT-PCR) to examine the presence of human papmoma virus (HPV) virus genes. However, because of the Pap smear, it is necessary to rely on the doctor's sampling, the examiner/pathologist to interpret the wipes >1, in addition to prone to high false negative rate (High false negative) and delay the diagnosis and treatment of precancerous lesions. Moreover, the labor cost required is too high, which has difficulty in promotion for many developing countries; on the other hand, human papilloma virus test has two sensitivities' but it is easy to cause high falsehood. The high rate (High fajse p0sitive rate) not only makes patients worry, but also wastes many medical resources on the follow-up examination of false positive patients; therefore, 'how to improve the accuracy and convenience of cervical cancer test methods is a promotion. One of the important topics in cervical cancer testing. In the etiology of cervical cancer, human papillomavirus (HPV) infection is the most significant risk. 12 2 κ"Λ"·^*·····*ννν·'·· ;- ................ risk factor; zurHausen’s 2002 report shows that “high risk Ε6/Ε7 oncoprotein produced by human papillomavirus (Ηρν) It will interact with the tumor suppressor gene /753, causing abnormal cell cycle regulation; in fact, human papillomavirus (HPV) DNA can be detected in all cases of cervical cancer. However, infection with human papillomavirus (HPV) is a necessary condition for cervical cancer 'but not enough to cause cervical cancer; about 60% of low-grade squamous intraepithelial lesions , LSIL) will regress, v 30% will persist, 5-10% will develop into high-grade squamous intraepithelialles (HSIL) 'only less than 1% Will become cervical cancer. The persistent infection of hpv and the amount of virus (v iral lo ad) may be the determinants of the development of highly squamous intraepithelial lesions (HSIL) and cancer; however, the molecular mechanisms of cervical cancer development remain to be confirmed. Other factors, such as environmental and genetic changes, may also play an important role in the deterioration of cervical keratinocytes; and whether or not initiated by HPV, genetic changes have caused genomic instability to be considered for a long time. An important mechanism of cervical cancer development, cytogenetic studies show that 'non-rand〇m chromosomal changes are present in cervical cancer cells; in addition, several molecular genetic studies have identified some Locations of loss of heterozygosity (LOH) often occur, which may be associated with tumor suppressor genes (TSGs) when cervical cancer occurs. - genomic deletions are considered to be important factors in tumor formation. For a long time, we have been accustomed to the idea that the coding in the genome depends on the four bases of ATCG. Knudson proposed double-invasive theory as early as 1975. (two-hit theory), indicating that mutations or deletions accompanying some homologous tumor suppressor genes may cause or cause cancer; however, other information affecting phenotype may be present in the modified test group 5- In 5-methylcytosine, 5-methylcytosine was found in the palindrome of mammalian cells. 6 1329743 Sequence 5'-CpG-3' in the mammalian cells except for some Outside the region called CpG islands (CGIs), most CpG dinucleotide pairs are methylated' CpG islands refer to a large number of regions of approximately 1000 test pairs (1Kb). GC- and CpG-, usually located near the gene, are found near the promoter of a widely expressed gene. The methylation of cytosine occurs after DNA synthesis, from a thiol donor 3_adenosylmethionine, SAM) to transfer the monomethyl group to the 5th carbon position of cytosine. The 'enzyme reaction is carried out by DNA methyltransferase (DNMTs), which is the main methyltransferase of mammals. It is responsible for the replication of the hemimethylated site and post-rep丨Icative restoration) is called hypermethylation, which is called maintenance methylation; on the contrary, DNMT3A and DNMT3B are considered to be mainly responsible for the new position of methylation, and a type of methylation is called The steps of v〇methyla|;i〇n).

Loss of methylation of CpG dinuclear acid (l〇ss methylati〇n), meaning general low methylation, the first epigenetic abnormality in cancer cells; however, in the past few years Studies have shown that certain sites (eg, some tumor suppressor genes) are highly methylated (slte-sPeciflc hypermethylation) associated with loss of function, which may provide selective advantages in cancer generation; The hypermethylation of CpG islands on the sub-region can be accompanied by histone modification (9) gamma mificdificati〇n) followed by successive gene silencing to cause chromatin modification rem〇deiing; In addition to chromosomal deletions and gene mutations, epigenetic silencing of tumor suppressor genes via hypermethylation of promoters is also common in human cancers. Recent epidemiological studies have shown that the concentration of serum folate (a major source of methyl) is associated with HPV infection and clearance; in the metabolism of the methylation cycle (methycycle), the gene for the enzyme Genetic polymorphisms have also been reported on the cervix. . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ··· ..... 1329743 a... The development of intradermal lesions; as with the concept of supergene evolution, studies on the association between DNA thiolation and cervical cancer are also prevalent, DNA methylation of cervical cancer Study days and increases, showing the possibility of using f-based as a screening for cervical cancer; due to the nature of genetic and environmental interactions, the degree of methylation of tumor suppressor genes varies with different genes and different ethnic groups, different Diseases also have different methylatorphenotypes; however, the methylation phenotype of cervical cancer and its association with HPV genotypes are still unknown, and what specific * genes are present in cervical cancer Methylation, and how many genes are needed to meet clinical application needs These problems are still issues that need to be confirmed in the future. It can be seen that there are still many shortcomings in the above-mentioned methods for screening cervical cancer, which is not a good designer, but the witch is to be improved. • The inventors of the present invention have made improvements and innovations in view of the above-mentioned shortcomings derived from the above-mentioned uterine cancer screening screening methods, and have suffered for many years. After the study, I finally succeeded in developing and developing this method of cancer screening. SUMMARY OF THE INVENTION φ The object of the present invention is to provide a method for screening for cervical cancer as a first screen of cancer screening. The second object of the present invention is to provide a cervical cancer. Screening method, in addition to being used as a first-line cervical cancer test, it can also be used as a second-line cervical cancer test for HPV testing to achieve more accurate cervical cancer. Screening effect. Another object of the present invention is to provide a method for diagnosing cancer, which can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (eg, nest cancer, liver cancer) to assist abnormal examination. Diagnosis of the body. A method for screening cancers that can achieve the purpose of the above-mentioned neoplasms, is the state of methylation of the 1329743 target gene in the somatic cells, as a screening indicator for the presence or absence of cancer, and the method includes the following steps: * Step 1 Providing a test subject; Step 2 detecting a methylation status of a cpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene is obtained by SOX, PAX, LMX1A, NKX6-1, WT1, and 0NECUT1 Composition;

Step 3 determines whether the specimen has cancer or precancerous lesions based on the presence or absence of the target gene thiolation state. The tested specimens are Pap smear, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells, and the like. The method for detecting the state of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), sulfite sequencing (BS), microarrays, mass specfr〇meter, denaturing high-performance liquid dirormtagraphy (DHPLC) The target gene SOX1 has the nucleotide sequence shown by SEQ ID No: 1. Wherein the target gene PAXI has a nucleotide sequence as shown in SEQ ID No. 2, wherein the target gene LMX1A has the nucleotide sequence as shown in SEQ ID No: 3. Wherein the target gene NKX6-1 has a nucleotide sequence as shown by sEQED No: 4. Wherein the target gene WT1 has a nucleotide sequence as shown in SEQ tt. No. 5: wherein the target gene ONECUT1 has a nucleotide sequence as shown in SEQ ID No: 6. The present invention enters the money supply-seed cervical paving The method for detecting is to detect the methylation status of the 1329443 target gene in the recipient cell as a screening index for the presence or absence of cervical cancer, and the method comprises the following steps: Step 1 provides a test subject; Step 2 detects the test The methylation status of the CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene consisting of S0X1, PAX1, LMX1A, NKX6-;! 'WT1 and 0NECUT1; and step 3 according to the target gene methyl Whether or not the specimen has cervical cancer and precancerous lesions is determined by the presence or absence of the state. The test subject is a Pap smear, blood, urine, cervical epithelial cells, and the like. The test subject is an abnormal Pap smear. Among them, the test specimen was a cervical cancer cell which was positive for PV testing by human papillomavirus (0〇3). The methylation status of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ) 'bisulfite sequencing (BS), microarrays, mass spectrometry, denaturing high-performanee liquid ehr_tography (DHPLC). It is claimed that the target gene S0X1 has a nucleotide sequence β as shown in SEQ ID No: 其中, wherein the target gene ΡΑΧ 1 has a nucleotide sequence as shown in SEQ ID No: 2, wherein the target gene LMX1A has SEQ! The nucleotide sequence β shown by dNo: 3 wherein the target gene ΝΚΧ 6-1 has the nucleotide sequence as shown in SEQ ID No. 4. Wherein the target gene WT1 has a nucleotide sequence as shown in SEQ ID NO: 5. Ϊ329743 wherein the target gene 0NECUT1 has a nucleotide sequence β as shown in SEQ ID NO: 6. The present invention further provides a method for screening for ovarian cancer by detecting the state of target soil thiolation in the test subject cells. As a screening target for the presence or absence of a nested cancer, the method comprises the following steps: Step 1 provides a test subject; Step 2 detects a CpG sequence methylation status of at least one target gene in the genomic DNA of the test subject The target gene consists of S〇xi, PAXi, and LMX1A; and step 3 determines whether the sample has ovarian cancer or precancerous lesions based on the presence or absence of the methylation status of the target gene. The test subject is ascites, blood, urine, and the like. The methylation status detection method of the CpG sequence of the target gene is methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), coke filling acid sequencing (pyrosequencing) wherein the target gene SOX1 has the nucleotide sequence shown as SEQ ID No: 1. Wherein the target gene PAX1 has the nucleotide sequence as shown in SEQ ID No. 2. Wherein the target gene LMX1A has a nucleotide sequence as shown in SEQDD No. 3. The present invention further provides a method for screening for liver cancer by detecting a state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of liver cancer, the method comprising the following steps: Step 1 provides a test sample 1329743 Step 2 detecting the methylation status of cpG sequence of at least one target gene in the genomic DNA of the test subject 'The target gene is composed of SOX NKX6-1; and Step 3 according to the methylation status of the target gene Whether or not the sample has liver cancer and precancerous lesions. The test subject is ascites, blood, urine, feces, gastric juice, bile, and the like.

The methylation status of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (*) (DHPLC), coke filling Acid pyrosequencing. Wherein the target gene S0X1 has a nucleotide sequence as shown in SEQ ID No: 1 wherein the target gene NKX6-1 has the nucleotide sequence as shown in SEQ ID No. 4. • [Embodiment] Example 1 Materials and Methods 1. Test Materials The test materials contained a series of complete cervical lesion samples, including normal samples (η = 45), low-grade squamous cell intraepithelial lesions (LSIL, n = 45). Highly squamous cell intraepithelial lesion (HSIL, n = 58), squamous cell carcinoma (SCC, η = 109); the test material additionally contains a series of complete ovarian tumor samples 'including ovarian benign tumor samples (η = 36), ovarian marginal tumor samples (η = 6), ovarian malignant tumor samples (η = 122); all cervical samples and ovarian-like 12 samples were taken from the gynecological tumor tissue bank of the Taipei Military Academy General Hospital' The genomic DNA of each sample was extracted with Qiagen DNA kit, and DNA was quantified by Pic〇Green fluorescence absorption method, and the quality of DNA was detected by gel electrophoresis. In addition, hepatocyte samples contained normal liver cell samples (η = 13), chronic hepatitis (n = 15), cirrhosis (n = 40), liver cancer (hepatocellular carcinoma, HCC, η = 54); all livers The cell samples were taken from the general surgical tumor tissue bank of the Taipei Three Military General Hospital. The genomic DNA of each liver cell sample was also extracted with the Qiagen DNA kit and the DNA was quantified by PicoGreen fluorescence absorption method, and the DNA quality was detected by gel electrophoresis. 2. Differential Methylation Hybridization (DMH) using CpG island microarrays (DMH) Take DNA from 30 cervical cancer tissue samples and mix them together, and take a normal Pap smear. The DNA of the sample is mixed together, and the DNA of the sample is digested with restriction enzymes, ligated to linkers, followed by methylation-sensitive restriction enzymes ΖζσαΠ and /Ul Enzyme sentence, then the DNA is used as a template for PCR for 20 cycles of amplification, and labeled with camp light dye, the DNA of normal Pap smear sample is fluorescent dye Cy3 Marked, the DNA of the cervical cancer tissue sample was labeled with the fluorescent dye Cy5; the labeled sample DNA was used as a probe, and the CpG island microarrays containing the 8,640 CpG island tags (CpG island microarrays) were used. Hybridization reactions were identified using the CGI database (http://derlab.med.utoronto.ca/CpGIslands/) to identify selected CpG islands. The microarray data was analyzed in the circular-features mode of the GenePix 6.0 software, marking the repeatedly selected clones, and filtering to remove unacceptable features; the ratio of Cy5 to Cy3 was greater than The loci of 2.0 is high in mixed cervical cancer tissue samples. • • 2. · · · · · · · . . . . . . 1329743 degrees of methylated gene, thus accepting a gene with a ratio greater than 2.0. 3. Bisu丨fite modification, methylation-specific polymerase reaction (MSP) and sulfite sequencing (BS) using Chemicon Modification kit (DNA m〇dificati〇n咏Chemicon, Temecula, CA) for sulfite modification: genomic DNA of 1 gg sample • (genomic DNA)' chemical modification of genomic DNA with sodium sulfite in single-stranded In DNA, ^ all non-thiolated beetles will undergo deamination and turn into urinary pain bites, while methylated punctures will not be modified 'still maintaining a 5-methyl cellulite bite; Finally, the reacted sample DNA was dissolved in 70 μL of 55 ° C in TE buffer to perform methylation-specific PCR (MSP) and normal DNA of human peripheral blood. Sulfate modification - using 'as a control group with a non-methylated promoter sequence; and human normal DNA

SssI methyltransferase (methyltransferase, New England Biolabs, Beverly, MA) was treated to obtain a positive control group with a methylated dual gene. _ Take the genomic DNA of bg after sulfite modification, and the DNA of the control group and the control group, and perform methylation-specific PCR amplification with MSP primer. The MSP primer is divided into two types. The MSP primer (U) can specifically recognize the unmethylated gene sequence, and the other is the MSP primer (M) which can specifically recognize the methylation gene sequence. The MSP primer sequence of each target gene is shown in Table 1; methylation The total volume of the specific PCR reaction was 25 μl, including Ιμΐ modified plasmid DNA, each primer was 1.5 μm, 0.2 mmol/L dNTPs, and 1 unit Gold DNA polymerase (Applied Biosystems, Foster City, CA); The mixed reactants were placed at 95 ° C for 5 minutes, followed by dissociation at 95 ° C for 30 seconds, appropriate primer bonding 14 Ϊ 329743 temperature bonding for 30 seconds, 72 ° C synthesis for 30 seconds for the cycle The dissociation, bonding, and synthesis steps were repeated for a total of 35 cycles, followed by a reaction at 72 ° C for 5 minutes. The amplified product was electrophoresed and analyzed by 2.5% phospholipid interception containing ethidium bromide (EtBr) and observed under ultraviolet light.

Table 1 Methylation-specific PCR (MSP) MSP primer sequence gene name primer type primer sequence Μ positive stock (F') 5, CGTTTTTTTTTTTTCGTTATTGGC 3r (SEQ ID No: 7) SOX1 anti-share (R') 5 , CCTACGCTCGATCCTCAACG 3r (SEQ ID No: 8) U Positive Strand (F) 5, TGTTTTTTTTTTTTTGTTATTGGTG 3f (SEQ ED No: 9) Anti-Rand (R') 5, CCTACACTCAATCCTCAACAAC 3' (SEQ ID No: 10) Μ 股股(F) 5 , TTTAGAAGCGGGCGGGAC 3' (SEQ ID No: 11) LMX1A anti-strand (R〇5, CCGAATCCAAACACGCG 3, (SEQ ED No: 12) υ 股 (F) 5, GAGTTTAGAAGTGGGTGGGATG 3f (SEQ ID No: 13) Anti-share (R 〇5f CAACCAAATCCAAACACACAAAAC 3' (SEQ ID No: 14) Μ 股 股 (F) 5, TTGTAGCGGCGGTTTTAGGTC (SEQ ID No: 15) ONECUT1 Anti-Stock (R') 5, GCCAAACCCTTAACGTCCCG 3' (SEQ ED No: 16) U (F,) 5, GATTGTAGTGGTGGTTTTAGGTTG 3' (SEQ ID No: 17) Anti-strand (R,) 5f caccaaacccttaacAtcccaatac V (SEQ ID No: 18) Μ 股 (F) 5, TATTTTGGGTTTGGGGTCGC 3' (SEQ ID No: 19) DA V1 anti-share (Ι〇5f CCCGAAAACCGAAAACCG 3' (SEQ ID No: 20) rAAl U positive stock (F) 5f GTTTATTTTGGGTTTGGGGTTGTG 1' (SEQ ID No: 21) Inversion (R) 5, CACCCAAAAACCAAAAACCAC 3r (SEQ ID No: 22) Μ 股 股 (F') 5f CGTGGTCGTGGGATGTTAGC 3' (SEQ ID No: 23) NKX6.1 Anti-Shares (R〇5 , ACAAACAACGAAAAATACGCG 3, (SEQ ID No: 24) υ 股 股 (F) 5, GTGTGGTTGTGGGATGTTAGTG 3' (SEQ ID No: 25) Antifungal 5f CAACAAACAACAAAAAATACACAAC 3' (SEQ ID No: 26) Μ 股 ( (F) 5, TGTTGAGTGAATGGAGCGGTC 2' (SEQ ID No: 27) WT1 anti-strand (R·) 5, CGAAAAACCCCCGAATATAAACG 3, (SEQ ID No: 28) υ 股 (F) 5, GTTGTTGAGTGAATGGAGTGGTTG 3' (SEQ ID No: 29) R) 5, AATTACAAAAAACCCCCAAATATAAACAC 3, (SEQ ID No: 30) The primer type Μ represents the MSP primer that can specifically recognize the methylation gene sequence. The primer type ϋ represents the MSP primer that can specifically recognize the unmethylated gene sequence. 15 Ϊ329743 All samples were subjected to at least two independent sulfite modification and methylation-specific PCR 'in the same sample using the MSP primer that can specifically recognize the methylation gene sequence (in the pcR reaction performed) If the PCR product cannot be synthesized more than twice, the sample is considered to be unmethylated; the PCR product amplified by the MSP primer (M) which specifically recognizes the methylation gene sequence is selected into the PCR4-T0P0 vector (Invitrogen) , Carlsbad, CA) 'Select at least 5 independent clones for sulfite sequencing (BS) 'The sulfite sequencing (bs) used in the primers shown in Table 2, using 377 automatically Sequencer (Applied Biosystems, Foster City, CA) for sulfite sequencing " Table II sulfite sequencing (BS) used in the sequence name of the primer S0X1

LMX1A 0NECUT1 primer type primer sequence positive stock (F') 5, · GTTGTTTTYGGGTTTTTTTTTGGTTG 3, (SEQ ID No: 31) anti-strand (R1) 5, A-TTTCTCCTAATACACAAACCACTTACC 3f (SEQ ED No: 32) positive stock (F') 5 ,TAGTTATTGGGAGAGAGTTYGTTTATTAG 3f (SEQ ED No: 33) anti-strand (R1) 5, CTACCCCAAATCRAAAAAAAACAC 3,_(SEQ ID No: 34) positive (F) 5, GAGTTTATTTAAGTAAGGGAGG 3f (SEQ ID No: 35) anti-share (R1) 5 , CAACTTAAACCATAACTCTATTACTATTAC 3, (SEQ ID No: 36)

PAX1 Stock (F) V GTGTTTTGGGAGGGGGTAGTAG 3f BS1 Anti-Stock (R') 5f CCCTCCCRAACCCTACCTATC 3f Stock (F,) 5, GATAGAAGGAGGGGGTAGAGTT 3, BS2 Anti-Stock (R·) 5, TACTACCCCCTCCCAAAACAC 3, NKX6.1 WT1

Stock (F1) 5f GGTATTTTTGGTTTAGTTGGTAGTT 3' BS1 Anti-Stock (R1) 5, AATACCCTCCATTACCCCCACC 3f Stock (F) 5' GGTGGGGGTAATGGAGGGTATT 3' BS2 ,, Anti-Stock (R1) 5, CCTAAATTATAAATACCCAAAAAC 3, Stock (F') 5, GTGTTGGGTTGAAGAGGAGGGTGT 3' anti-strand (R1) 5, ATCCTACAACAAAAAAAAATCCAAAATC (SEQ ID No: 37) (SEQ ID No: 38) (SEQ ID No: 39) (SEQ ID No: 40) (SEQ ID No: 41) (SEQ ID No: SEQ ID No. : 42) (SEQ ID No: 43) (SEQ ID No: 44) (SEQ ID No: 45) 3' (SEQ ID No; 46) IV, via 5,-aza-2,-deoxycytidine (5 '-aza-2'-deoxycytidine) treatment's re-expression of methylation genes in cervical cancer cell lines 16 1329743 First in the HeLa cervical cancer cell line, methylation-specific PCR (MSP) testing may have The methylation status of highly methylated genes and the selection of genes with methylation. The cervical cancer cell line was treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxyCytidine (Sigma Chemical Co.) for 4 days to make the cell strain not originally expressed by methylation. Re-exhibition and analysis of gene expression by RT_PCR; use

Qiagen RNeasy kit (Qiagen, Valencia, CA) extracts total RNA (total RNA) and adds DNase I to remove DNA contamination: 1 pg total RNA per sample is Superscript Π • reverse transcriptase and 6 The cDNA random synthesis was performed by random hexamer (Invitrogen); the synthesized cDNA was PCR amplified by PCR master mix reagents kit (Applied Biosystems) and placed in a thermocycler (GeneAmp 2400 PE, Applied Biosystems). In the middle reaction, the amplified cDNA was analyzed by RT-PCR, and the RT-PCR primers used for each target gene are shown in Table 3. Table 3 Sequence of MSP primers used in RT-PCR

Gene Name Primer Type Primer Sequence SOX1 Positive Strand (F,) 5, AGACCTAGATGCCAACAATTGG 3, (SEQ ID No: 47) Anti-Stock (R,) 5, GCACCACTACGACTTAGTCCG 3' (SE〇ID No: 48) LMX1A Stock (F) 5, GCTGCTTCTGCTGCTGTGTCT 3r (SEQ ID No: 49) anti-strand (R〇5, ACGTTTGGGGCGCTTATGGTC 31 (SEQ ED No: 50) ONECUT1 positive stock (F') 5, CAAACCCTGGAGCAAACTCAA 3' (SEQ ID No: 51) anti-share (R1 5, TGTGTTGCCTCTATCCTTCCC (SEQ ED No: 52) PAX1 positive (F·) 5, CCTACGCTGCCCTACAACCACATC 37 (SEQ ID No: 53) Anti-shares (ϊ〇5, TCACGCCGGCCCAGTCTTCCATCT 3r fSEOIDNo: 54) NKX6.1 Stocks (F) 5, CACACGAGACCCACTTTTTCC 3f (SEQ ID No: 55) anti-strand (R) 5; CCCAACGAATAGGCCAAACG 3f (SEQ ID No: 56) WT1 positive (F) 5, GCTGTCCCACTTACAGATGCA 3' (SEQ ID No: 57) anti-share (R| 5, TCAAAGCGCCAGCTGGAGTTT 31 (SEQ ID No: 58) 17 .·· J·.- *······*·. *4··* · 1329743 5. Detection of human papillomavirus (HPV) Detection of human papillomavirus (HPV) DNA in squamous cell carcinoma (SCC) by LI consensus PCR and reverse line blot Exceeding the results of the analysis of the hybridization technique, the sequence of the novel human papillomavirus (novel HPV type) was confirmed by DNA sequencing. VI. Statistical analysis Data analysis by statistical software SAS version 9.1, gene methylation and clinical The relationship between the φ number (including the HPV state) is calculated using the f test and Fisher's exact test, and the age is calculated and adjusted by the logistic regression model. The odds ratio (ORds) with HPV infection and the 95% confidence interval (CI), the alpha level of statistical - ^ significance is set to = 0.05; and the calculation uses HPV and methyl Markers are used to diagnose the sensitivity and specificity of cervical lesions. Example 2 Screening of cervical cancer methylation index genes Screening for differential methylation hybridization (DMH) by CpG. island microarrays to screen out cervical squamous cell carcinoma (SCC) The gene with high methylation; CpG island microarrays showed that there were 216 points between the cervical cancer tissue sample and the normal Pap smear sample with differential methylation, and the sequence repeat was removed. After that, '26 gene promoter regions CpG islands (promoter CGIs) were obtained. These gene promoters were sequenced and analyzed, and six genes were selected, including: S0X1 (SEQ ID No: 1), PAX1 (SEQ ID No: 2), LMX1A (SEQ ID No: 3), NKX6. -1 (SEQ ID No: 4), WT1 (SEQ ID No: 5), and 〇NECUTl (SEQ Liao) No: 18 ···················································· 1329743 6), the detailed information is shown in Table 4; as shown in Table 4, these six genes are important transcription factors in the development process, s〇xl, ρΑχι, LMX1A, Shun 6 WT1 respectively It is important for the development of the brain, the roofplate, the extremities, the scorpion and the genitourinary tract. 0NECUT1 is important for the performance of liver and pancreatic genes, but few studies have reported that these genes are involved in cancer. Table 4 identifies the methylated genes in cervical cancer cells using CpG island microarrays. w m A UniGene Known gene function No. S0X1 NM_005986 13q34 Gene name Chromosome localization Gene full name

Sex determining DNA binding region Y-box 1 Transcription factor activity PAX1 ] >4M_006192 20pll.2 Paired box gene 1 DNA binding LMX1A 1 SfM_l 77398 Iq22-q23 LIM homeobox transcription factor 1 alpha Transcription factor activity Zinc ion binding NKX6-1 1 νίΜ_006168 4q21.2-q22 NK6 transcription factor related locus 1 Transcription factor activity ONECUT1 ϊ ^_004498 15q21.1-q21.2 One cut domain family member 1 Transcription factor activity Transcriptional activator activity WT1 1 Like a 024426 11ρ13 Wilm's tumor 1 Transcription factor activity Zinc Ion binding CpG sequence analysis of about 500 bp nucleotides before and after the transcription start point (+1) of each gene is shown in Figure 1. The CpG sequence in each gene is indicated by "|" and is targeted for each gene. Design MSP primers (as shown in Table 1) and sulfite sequencing (BS) primers (as shown in Table 2), methylation-specific PCR (MSP) and sulfite sequencing (BS) The position of the synthesized fragment is also indicated in Figure 1. Methylation-specific PCR (MSP) was then performed on the mixed uterine __sample (3G _ 合 混合 mixed normal smear smear sample 00 samples), in order to methylation of these six genes Whether the different _samples have differences, the results shown in Figure 2, these 6 genes in the mixed uterus _ sample transfer in the methylation phenomenon ((4) two 2 shed outside the mixed normal cervical smear There is no thiolation phenomenon in the sample (as shown in Figure 2). The sample of the cervical cancer tissue of the patient is tested. Take 4 cervical cancer tissue samples: this (m T3, take 4 normal samples (10) , N2, N3, N4) perform f-based specificity. The R (MSP) knife is used to uniquely identify the MSP primer (U) of the unmethylated gene sequence, and the specific-identification-based gene-sequence touch MSP The primer (10) was used to perform the f-based specific pcR division. The results are shown in Figure 3. These six genes have methylation in individual cervical cancer tissue samples (Fig. 3, 3, 5, 7). In the column) / the same gene in the normal sample is less than the occurrence of thiolation phenomenon (as shown in Figure 9, paragraph 9, U, 13, 15); As described above, these six genes were used as screening methylation index genes for cervical cancer. • Example 3 Correlation between DNA methylation and gene expression in cervical cancer cell lines. Whether the expression of the gene is regulated by DNA f-based treatment, treatment of HeLa cervical cancer cells with 10 μΜ of DNA methyltransfer inhibitor 5,-aza-2,-de〇xycytidine (AZC) (Sigma Chemical Co.) After 4 days, the methylation-specific PCR (MSP) was used to examine the demethylation of the above six gene promoters; respectively, the MSP primer (U) which can specifically recognize the unmethylated gene sequence, and the specificity The Msp primer (Μ), which recognizes the thiolated gene sequence, was subjected to thiolation-specific PCR (MSP). The results are shown in Figure 4a. HeLa cervical cancer without treatment of 5'-aza-2'-deoxycytidine (AZC) In the cell line (AZC-), all of the six target genes have methylation (as shown in Figure 1A, the first ruin) 'and> (undetected unmethylated gene 20 (Figure 4A) In column 2), it can be detected in the treatment of 5'-aza-2'-deoxycytidine (AZC) for 4 days in HeLa cervical cancer cell line (AZC+). To the unmethylated target gene (shown in Figure 4a, block 4), showing the cervical cancer cell line treated with the 5'-aza-2'-deoxycytidine (AZC) Some of the above 6 target genes have been methylated. The performance of these 6 genes in HeLa cervical cancer cell lines was analyzed by RT-PCR. The results are shown in Figure 4B, after 5'-aza-2. The mRNA of these six target genes was detected in the cell line treated with '-deoxycytidine (AZC) (as shown in Figure 6 b, block 6), without 5'-aza-2'-deoxycytidine ( In the AZC) cell line, no mRNA of any target gene was detected (as shown in column 5 of Figure 4B). From the results, it can be seen that these six target genes are indeed in cervical cancer cells. DNA methylation regulates its gene expression. When the gene has methylation, the expression of the gene is inhibited. After the thiolation is removed, the target gene begins to manifest. In addition, the sulfite sequence (BS) was used to analyze whether the target gene has hypermethylation in the HeLa cervical cancer cell line. The results are shown in Figure 5, without 5'-aza-2'-de. In the xycytidine (AZC)-treated cell line (Fig. 5A), the number of highly methylated samples of the target gene was higher than that of the leg S'-azaldeoxyeytidine (A2C)-treated strain (Fig. 5B); Sulfite sequencing (BS) was analyzed in cervical squamous cell carcinoma (scc) and normal samples. The results are shown in Figure 6. In the cervical squamous cell carcinoma (scc) (Figure 6A) sample, the target gene The number of samples with methylation was also significantly higher than that of the normal sample (Fig. 6B). Example 4: Methylation analysis of target genes in clinical cervical samples, see Table 5, normal samples, low-grade squamous cell intraepithelial lesions, 1 call, high-scale squamous epithelial, Qianqing L), and miscellaneous wealth ( Scc) The average age of the sample is 21 -..Λ,.·.·.,..···. 1329743 11·3'39·7±9.6, 46·4± 14.4 and 53.3± ι〇9 years old ( ^7<0.05); the ratio of positive for the high-risk Hpv dna of the sample was: 214% of the normal sample, 47.7% of the low-grade squamous cell intraepithelial lesion (LSIL) sample. 'Highly squamous cell intraepithelial lesion (HSIL) sample 59 3%, 88.9% of squamous cell carcinoma (SCC) samples. The results showed that patients with Hpv were more likely to have different severity of cervical lesions (the odds ratios of LSIL, HSIL, and SCC samples were 3J, 5, 2, 29.9; 95% confidence intervals were 1.1-8.3, 2.1- 13.0, 11.5-77.7). In the cervical lesion samples of different severity, the methylation-specific PCR (MSp) was used to analyze the rhodamine state of the target gene, the methylation status of the target gene and the presence or absence of human papillomavirus (HPV). The results of the analysis are shown in Table 5. The six genes of SOX, pax, LMX1A, ΝΚΧ6·4, WT1 and ONECUT1 have high frequency methylation in squamous cell carcinoma (SCC). The proportion of methylation in cancer (SCC) samples was: 8丨5%, 94 4%, 89.9%, 80.4%, 77.8〇/〇, and 20.4〇/〇; and in the normal cervical samples, each gene methyl The ratios are: 2.2. /〇, 〇%, 6.7%, 11.9%, 11.1%, and 〇% (p 〇〇1); therefore, these 6 genes are methylated in squamous cell carcinoma (scc) samples compared to normal cervical samples. The situation is obviously much higher. The frequency of methylation of B NKX6-1 gene was 53 3% in LSIL samples, 55.1% in the positive samples, and 80.4% in SCC samples. Statistical results showed that patients with gene methylation were suffering from the disease. The risk of squamous cell carcinoma (scc) is higher (the odds ratio is 29,85%, and the confidence interval is 10.4-85.2). The frequency of methylation of the PAX1 gene was 2.3% in the LSIL sample, 42.1/〇 in the HSIL sample, and 94 4〇/〇 in the SCC sample; statistical results showed that patients with ρΑχι gene thiolation 'Dangerous squamous cell intraepithelial lesion L) and squamous cell carcinoma (scc) wind 22 T329743 risk compared to raj (HSIL and see samples have an odds ratio of >999.9; 95% confidence interval is < 0_ 1 ->999.9). The frequencies of SOX1, LMX1A and 0NECUT1 in the precancerous lesions (prec lesions) were very low, but the frequency of methylation in η muscle samples and scc samples increased significantly. 9 tear and 81 s% 'secret and 89 9% ' 7 4 〇 /. And 2〇为; δ10 results show that patients with s〇xl, LMX1A or 〇necuti gene methylation, Luo has a higher risk of squamous cell carcinoma (SCC) (the odds ratio of the three) They are respectively 20.2.124 5, 7 3 ; -· 95% confidence intervals are 25.8-999.9, 33.G-47G], 2.0-25.9). The frequency of methylation of the WT1 gene increases with the severity of the lesion. The frequency of wti gene methylation in normal samples is 111%, which is 2% in the sample and 42.1% in the Qingyi sample. In the SCC sample, it was 77 8%. The statistical results showed that patients with gene thiolation had a higher risk of high-grade squamous cell intraepithelial lesion (HSIL) and squamous cell carcinoma (scc). The odds ratios are 6 7 and 27 9 respectively; 95% confidence intervals are 2·219 8 , 9.8-78.9) » Diagnostic performance of DNA methylation indicators Analyze the sensitivity of DNA methylation (sens out and specificity, To determine whether the target gene can be used as a biomarker for cervical cancer screening and cervical cancer screening, the results are shown in Table 6. The HPV test is used to screen the sample for sensitivity and specificity of squamous cell carcinoma (SCC). They were 83.1% and 85.5% respectively (the 95% confidence interval was 77.6-88.5 and 79.6-91.4 respectively); and the methylation status of the five genes of SOX, PAX, LMX1A, NKX6-1 and WT1 were analyzed for screening. The presence or absence of squamous cell carcinoma (SCC), the methylation status of each gene against squamous cells The sensitivity of cancer (SCC) is 77.8%-94.4%, and its specificity is 88.1%·1〇〇%; when combined with HPV test and individual methylation index genes to detect disease (combined parallel 23 testing, CPT) (4) If the HPV job is required to be a positive or negative result, the cervical cancer screening result of the test sample is positive, and the sensitivity is between 97.2% and 98.2. 'Times at 66.7%·795%; when combined with the conversion test, CST) HPV test and individual methylation index genes, meaning that the first test, and HPV test positive samples for each indicator base - methyl The splicing test has a sensitivity of 69_4%-85.0%, and the specificity of all tests is 1%. When using both high-grade squamous cell intraepithelial lesions L) and squamous cell carcinoma (scc) diagnostic criteria, the sensitivity and specificity of screening for HPIL or SCC by HPV s was 75.0% and 85.5%, respectively. (The 95% confidence interval is 7〇·2-79_8 and 79.6-91.4 respectively); and the thiolation status of the five genes of SOX, PAX, LMX1A, ΝΚΧ6-1, and WH is analyzed to check whether the sample has HSIL or 50:: The methylation status of each gene is 1^1]^ or 8(:(: the sensitivity is between 57.4% and 76.2%, and the specificity is between 88.1%_100%; when the HpV test is combined with Individual methylation indicator genes to detect disease (CPT), the sensitivity can be increased to 85.8%-94.9%; and when the sequential combination (CST) RPV test and individual methylation indicator genes, the specificity of all tests 100%; when combined with (CPT) HPV test and s〇xl, PAX, LMX1A three methylation status test to screen samples for squamous cell carcinoma (SCC), the sensitivity is up to 1 〇〇%, and when the same method is used to check the presence or absence of HSIL or SCC in the sample, the sensitivity is 93.4%. In the individual thiolation index gene screening In the results of squamous cell carcinoma (SCC), the sensitivity of screening for the squamous cell carcinoma (scc) in the sample by the methylation status of the PAX1 gene alone is the highest, and its sensitivity is up to 94.4% (the 95% confidence interval is 95%). 90.0-98.8) Similarly, the sensitivity of screening for samples with or without HSIL or SCC in the methylation status of ραχϊ gene can reach 76.2% (95°/. confidence interval is 69.7-82.7), while the two tests are specific. Sex is 1%. 24 1329743

The order w disc hacker AdH ¥, hungry ^ 硪 S-SE ^ 醵 tm * Ψ 赛 赛 赛 韧 骚 屮 屮 HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP 1.3-8.6) 3.1 (1.1-8.3) 32/54 (59.3%) 5.3 (2.1-13.3) 5.2 (2.1-13.0) 96/108 (88.9%) 29·: (11.3-75.8) 29.9 (11.5-77.7) /Κ0.0001 ONECUT1 0/45 (〇%) 3/45 (6.7%) 4/54 (7.4%) 2.3 (0.5-10.8) 2.3 (0.5-10.8) 22/108 (20.4%) 7.4 (2.1-25.7 ) 7.3 (2.0-25.9) ^=0.001 WT1 5/45 (11.1%) 9/45 (20.0%) 2.0 (0.6-6.5) 2.7 (0.8-9.3) 24/57 (42.1%) 5.8 (2.0-16.9) 6.7 (2.2-19.8) 84/108 (77.8%) 28.0 (10.0-78.8) 27.9 (9.8-78.9) ^<0.0001 NKX6-1 5/42 (11.9%) 24/45 (53.3%) 8.5 (2.8- 25.5) 9.6 (3.1-30.4) „ 27/49 (55.1%) 9.1 (3.1-27.0) 9.6 (3.2-29.1) 86/107 (80.4%) 30.3 (10.6-86.5) 29.8 (10.4-85.2) ^< 0.0001 LMX1A 3/45 (6.7%) 6/45 (13.3%) 2.2 (0.5-9.2) 2.2 (0.5-9.7) 8/50 (16.0%) 2.7 (0.6-10.7) .2.7 (0.7-10.9) 98/ 109 (89.9%) 124.7 (33.1-469.9) 124.5 (33.0-470.1) ρ<0.0001 PAX1 0/41 (〇%) 1/44 (2.3%) 24/57 (42.1%) >999.9 (<0.1 - >999.9) >999.9 (<0.1->999.9) 101/107 (94.4%) >999.9 (<0.1 ->999.9) >999.9 (<0.1->999.9) ρ<0.0001 SOX1 1/45 (2.2%) 2/45 (4.4%) 2.0 (0.2-23.4) 3.1 (0.3-36.7 5/54 (9.3%) 4.5 (0.5-39.9) 5.1 (0.6-45.9) 88/108 (81.5%) 193.5 (25.2-1000) 200.2 (25.8-999.9) ^<0.0001 due to normal (n = 45) LSIL (n = 45) odds ratio (95% confidence interval) odds ratio * (95% confidence interval) HSIL (η = 58) odds ratio (95% confidence interval) odds ratio * (95% confidence interval) SCC (η = 109) Winning ratio (95% confidence interval) Winning ratio* (95% confidence interval) Statistics

S(N 1329743

u •ί赛XS 堞•Μ Μ 箸ψϋ Specificity (95% confidence interval) (79.6-91.4) (93.0-100.0) (63.3-89.1) (100.0-100.0) (100.0-100.0) (66.8-92.2) ( 100.0-100.0) (85.1-100.0) (57.8-85.1) (100.0-100.0) (80.7-99.3) (55.8-84.2) (100.0-100.0) (78.3-97.9) (52.4-80.9) (100.0-100.0) Winning Ratio 85.5 97.6 76.2 100.0 100.0 Ί9.5 100.0 92.9 71.4 100.0 90.0 70.0 100.0 88.1 66.7 100.0 Sensitivity (95% confidence interval) (70.2-79.8) (49.8-65.0) (80.4-91.2) (42.9-58.3) (69.7-82.7 (85.0-94.3) (58.0-72.5) (59.3-74.0) (84.5-94.1) (50.8-66.2) (65.4-79.5) (91.4-98.3) (51.3-66.7) (58.2-72.7) (85.8-94.8) (46.3-61.5) Winning ratio 75.0 57.4 85.8 50.6 76.2 89.6 65.2 66.7 89.3 58.5 72.4 94.9 59.0 65.5 90.3 53.9 Specificity (95% confidence interval) (79.6-91.4) (93.0-100.0) (63.3-89.1) (100.0- 100.0) (100.0-100.0). (66.8-92.2) (100.0-100.0) (85.1-100.0) (57.8-85.1) (100.0-100.0) (80.7-99.3) (55.8-84.2) (100.0-100.0) (78.3 -97.9) (52.4-80.9) (100.0-100.0) Squamous cell carcinoma (see) The odds ratio is 85.5 97.6 76.2 100.0 100.0 79.5 100.0 92.9 71.4 100.0 90.0 70.0 100.0 88.1 66.7 100.0 Sensitivity (95% confidence interval) (77.6-88.5) (74.2-88.8) (95.6-100.0) (63.8-80.7) (90.0-98.8) (95.6-100.0) (78.3-91.8) (84.3-95.6) (95.7-100.0) (73.3-88.1) (72.9-87.9) (95.6-100.0) (62.4-79.6) (69.9-85.6) (94.1-100.0) (60.8-78.1) Winning ratio m 00 81.5 98.1 72.2 94.4 98.1 85.0 89.9 98.2 80.7 80.4 98.1 71.0 77.8 97.2 69.4 Test method Individual test Individual test CPT CST Individual test CPT CST Individual test CPT CST Individual test CPT CST Individual test CPT CST Biomarker HPV SOX1 PAX1 LMX1A NKX6-1 WT1 vlxm+ ( Γεοοοο.κ)<ν·69 (ε·ζ,64·68) 寸·ε6 (SOOOOK) S9 (ΟΌΟι-ΟΌΟι) 0001Hdo IXOS+ >s •Ο.Ιν'ν^ι,Λ.ι.ο^ ι.,^.ϋ-Λ,Λ-Λ,ν,'ΛΑ.,ι.'. ·' ·.·.«! 1329743 Example 5 Methylation analysis of target genes in ovarian tumor samples

Methylation-specific PCR (MSP) was used to analyze the methylation status of target genes in ovarian tumor samples. The methylation status analysis results of the target genes are shown in Table 7. The SOX and PAX1 were analyzed in each ovarian tumor sample. The methylation status of the three genes of LMX1A showed that the three genes, SOX, PAX1 and LMX1A, were not methylated in all ovarian benign tumors and ovarian marginal tumor samples; The frequency of methylation of these three genes increased significantly. The frequency of methylation of S〇X1 gene was 55 7〇/〇, the frequency of 曱γ gene thiolation was 49.2%, and the frequency of methylation of LMX1A gene was It is 32 8%.

Sample 0/36 (0.0%) 0/36 (0.0〇/〇) Table VII #methylation status analysis of target genes in nest tumor samples, gene tumors (4)) 0/6 (0.0%) 0/6 (0.0〇 /〇) 0/6 (〇·〇%) 68/122 (55.70/0) 60/122 (49.2〇/〇) 40/122 (32.8%) Statistics卩<0.0001 P< 0.0001 p< 〇.〇〇 〇! Example 6 Methylation analysis of target genes in hepatocyte samples. Methylation-specific PCR (MSP) analysis of methylation status of target genes in hepatocyte samples. The table continues, in the positive sputum cell sample, the SOX1 gene f-based is 77%, and the frequency of methylation of the SOX1 gene with abnormal lesions is greatly increased. In the chronic hepatitis sample, liver The frequency of methylation of 'SOX1 gene in hardened samples and liver cancer samples was 33 3%, 27 5%, and 53%, respectively. In addition, the frequency of 曱6] gene thiolation in the normal liver fines (ι〇%) was also significantly lower than the frequency of ΝΚΧ6-1 gene thiolation in the liver cancer 27 1329743 sample (57〇/〇). _ 一_Π ---- Normal liver cells (η=13) - S0X1 ΝΚΧ6-1 1/13 (7.7%) 1/10 (10%) Chronic hepatitis (η= 15) 5/15 (33.3%) Liver Hardening (η=40) 11/40 (27.5%) • Liver cancer (η=54) 29/54 (53.7%) 12/21 (57%) Statistics ρ = 0.005 Ρ < 0.05 - Cancer diagnosis provided by the present invention The method of comparing with the prior art has the following advantages: 1. The method for screening cancer provided by the present invention is to use the degree of methylation of a specific gene in the sample as a diagnostic indicator for the presence or absence of cancer, and to use it. Comparing the Pap smear and the HPV test method, the sensitivity and specificity of the cancer diagnosis method of the present invention are both better than the above two methods. In addition to screening for first-line cervical cancer, 'Hpvtesting' test can also be combined or used as a second line of cervical cancer. Subject 'in order to achieve more accurate results of cervical cancer screening. 3_ The method for cancer diagnosis provided by the present invention can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (such as ovarian cancer and liver cancer) to assist in the diagnosis of abnormal samples. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment is not intended to limit the scope of the invention, and the equivalent of 28 丄j/y/43 is not deviated from the spirit of the invention. Or, for example, the equivalent embodiment of the change in the degree of methylation of each target gene in the test subject, etc., should be included in the patent scope of the present application. In summary, the method of cancer diagnosis provided in this case is innovative, and can improve the above-mentioned functions more than the conventional method of cervical cancer. It should be fully in line with the new and progressive legal requirements. 'Maily filed an application according to law, and it is expected that your application for the invention patent application will be 'invented by the invention. [Simplified description of the schema] • ® - For each target _CPG sequence analysis used in the cancer screening method of the present invention, those having CpG sequences in each gene are indicated by "|"; each gene is introduced into the fragment by "-" Labeling; the position of the synthetic sulfite sequencing (BS) primers is indicated by "(a)"; the second is the target gene for the cancer screening of the present invention, and the cervical cancer sample in Kunming (3) One sample was mixed) the results of the methylation-specific PCR _> analysis in the normal uterus record (mixed sample) of Weihe; the sputum column was a mixed normal Pap smear sample (10 samples) Mixed), column 2 is a mixed cervical cancer tissue sample (3 samples _ mixed) 'column 3 is the negative control group (negative c〇ntr〇1), the fourth block is the positive control group (four) ^ control) Column 5 is a blank control group (water); Figure 3 is a target gene for the cancer screening method of the present invention, in a side cervical cancer tissue sample and an individual normal Pap smear sample, The result of the basic-specific pcR (MSP) analysis, Dingb T 2. T3 and T4 represent 4 individual cervical cancer tissue samples. Nl, Ν2, Ν3, Ν4 represent 4 individual normal samples, and the column indicating υ indicates MSP primers that can specifically identify the unmethylated gene sequence. (U) the result of methylation-specific pCR (MSp), the column indicating the Μ indicates the result of f-based specific PCR (MSP) with the Msp primer (M) which can specifically recognize the methylation gene sequence; 29 Figure 4A shows the target genes used in the cancer screening method of the present invention, in HeLa cervical cancer cell lines (AZC-», 1st and 2nd), which are not treated with 5', aza-2'-deoxycytidine, And the results of methylation-specific PCR (MSP) analysis in HeLa cervical cancer cell lines (A2C+, 3rd and 4th) treated with 5,-aza-2'-deoxycytidine; The methylation specificity j>cr (MSP) results from the MSP primer (U) that specifically recognizes the unmethylated gene sequence. The marker indicates that the msp primer specifically recognizes the methylation gene sequence ( Μ) Results of thiolation specific PCR (MSP); Figure 4B is for the cancer screening method of the present invention Each of the target genes in HeLa cervical cancer cell lines without treatment of s'-aza-2'-deoxycytidine (AZC-, column 5), and HeLa cervix with 5'-aza-2'-deoxycytidine treatment In the cancer cell line (AZC+, column 6), the results of RT-PCR analysis; ^ Figure Α is the target gene used in the cancer screening method of the present invention, without processing 5'-aza-2'- The results of sulfite sequencing (BS) analysis in the HeLa cervical cancer cell line of deoxycytidine; Figure 5B shows the target genes used in the cancer screening method of the present invention, in the treatment of 5'-aza-2 Results of sulfite sequencing (BS) analysis in HeLa cervical cancer cell lines of '-deoxycytidine; Figure 6A shows the target genes used in the cancer screening method of the present invention, in cervical squamous cell carcinoma (SCC), the result of sulfite sequencing (BS) analysis; and FIG. 6B is the sulfite sequencing (BS) in the normal sample for each target gene used in the cancer screening method of the present invention. The result of the analysis. [Explanation of main component symbols] Sequence Listing <110> Lai Hongzheng <120> - A method of cancer screening. <160> 58 <210> 1'

≪ 211> 2300 < 212 > DNA < 213 > person who is wise species (Homo sapiens) < 400 > 1 tctccacaag cactctcatc tcagggtgcc tcggggaggg cagaggagga atacttgggc 60 tcaaaagtcg tctttggacc actttcagat cagtctggtg gaaacggtag tgtgagcgct 120 atgctacttg agactgcgtt tgaaaatctc tttcacgttc ccaaatcaaa gccactttga 180 ggtttaagaa tgataaccac aggtgaatgc cttactcttt ccacgagcca ggcctttcct 240 ttgcatgacg cagaccggcg gctgagaccc gtcctgaggc cccgcctttc attcggttta 300 tgtggccccg cgcagttcac gcagttttct ccgttcttag gcaaccagct cgtgggaaat 360 tcactccaga aaagcgtgcg ccatatcatt attttgcgat tcaacaaact ttttcaactg 420 ttgttcagga actagattcc agatagatct tgttgtgttc ggccttccta gaaattcctt 480 ttccagagga agaagatccg ggttgggaag agtgcgtgac tatggccccg ggctcattga 540 aagactcctc cccacaaaag tttagggctg atactaaacg aagctatgga gccatgccca 600 cacaaatact ccccctttac ccgaattgtg Gggcctggac tgtgaaggcc ttcgctgcaa 660 gagcgggcac tggcgaactt cagtgcacgc cgcggccgag aacggatgca gggcggggga 720 tggctgagcc gcctgatcct tgcagagaac ctgcaggggc cctcggaggg tatcccctgc 780 g tccaaggga gcgcccctcc ttttcagcac tcgggggaac tgagggcgac gtgccagccc 840 cgcactcaac tttccccttc cctgcaggca cagagttggc cggcgggggc agaggaggag 900 ctgggtctcc actgcgcccg tttaaacctg gccaggggct gcgtttcctc cccccacccc 960 acgacgatcc tttcttagtc ttcgcttttc aacccaatcg ttaatcattc ggaacgcgcg 1020 ggcggggagc ggcgaggagg gcgagctcgg ggttcgccgc cgccgccgcc gccgcgcgcg 1080 cgcgctcagg aagcggtgtg gctgtcaccc cctcccgggc ctcctccccc ctccttcctg 1140 ctttgctccc cctccttcct cccctcctcc ccgctccgcc gcccgcgccc agtgtatcta 1200 ctccctcccc acgtcactcg ccagcgcgcc atgcaaatca ccgccgccgc cggctcccat 1260 tggccgcggc gcgctcattt aatggcagcc cgggcccggc gtatggctgc tgggccccgc 1320 gcgccgccgg ccccgcgtgc gcctccgctc cgagcgcacg gccccgggca ggcagcgggc 1380 agcccatccc gggctcggcg gccccggctc tccggccctc tccgcgagcc cgcgctcctc 1440 ccgctgtccc cgggcccctc cctggctgca ccgtaatcgc cccctgcagg cccccctgcg 1500 cctccccccc cccgccactg gcgcctggct tcccccgggc acctgggacc agcacatgcc 1560 cagcgcacgc ggcgcgccgc cctgctagaa gttgcagcct ccgagttgga ggccgctgag 1620 gaccgagcgc aggaggaagg agacagcgcg cagcggcggc cggcgaggag acagcacacc 1680 ccgggccggg cccagcgcac cgctcccggc cccaaaagcg gagctgcaac ttggccacga 1740 ctgcacctgt ttgcaccgct ccgccgaggg cgcctgggct gcggtggcgg cgaagacggc 1800 1329743 gaccccgacc gtcggcctct ttggcaagtg gtttgtgcat caggagaaac tttccacctg 1860 cgagccgaac cggcgccgag tgcgtgtgtt tctgcctttt tttgttgtcg ttgcctccac 1920 ccctccccat tcttctctcc gctaggaccc ccccgccccc gtctcactcc gtctgaattc 1980 ctctccgtct ccctcccacc ccggccgtct atgctccagg ccctctcctc gcggtgccgg 2040 tgaacccgcc agccgccccg 'atgtacagca tgatgatgga gaccgacctg cactcgcccg 2100 gcggcgccca ggcccccacg aacctctcgg gccccgccgg ggcgggcggc ggcgggggcg 2160 gaggcggggg cggcggcggc ggcgggggcg ccaaggccaa ccaggaccgg gtcaaacggc 2220 ccatgaacgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc caggagaacc 2280 ccaagatgca caactcggag 2300

<210> 2 <211> 1200 <212> DNA

≪ 213 > person who is wise species (Homo sapiens) < 400 > 2 agaaagacga ggccaggcca cctgggatag agtggtgcga gattccaccg ccagggagaa 60 aggaacttgt ccttcagacc tagagctgga gctatgcatt tggcctcccg ccggtcgcgc 120 ttggggacag gagggcgccg gatctatgcg ccccttagca gcagatcggg atccttttgc 180 ctcctgcccc ttctgtcact gcttggagag ggatgagttc tggtggctgg gcctggctgt 240 aggagacagg atttggaccg tgcccctctc gcatcaccga aatcaccccc actattccaa 300 gagtggttgg ctattaaacg tgaagatttc ctgagagaag gattgaggac ctggccagga 360 atgggacaca agttccgcct tgtgtcttcc tgacaggagc cctgcaccgc gctggacgct 420 caccttgaca ctcccagcca gctggggtac tgatcccacc cttccccggc cgctgccccg 480 ggagtgggga ggtagagaga gccacacccg aaacaccttt ccacgataaa cttttattct 540 ctatcttatt attaatggtg gcggaaataa aactaaaacc aaaacgaaaa cgagtactag 600 tactaacaca ctaatcaatt tgagatgact tccccctcat tcccaaagct agaggaggaa 660 gggggctgaa aggggctcag agcagtggaa ggtcccaggc ccagctgggg ttgggacgtg 720 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tagaggggtg ggttccgggg 780 gggtggtgga agagggccga ggggggagga tagaaggagg gggtagagtt tcagggcggg 840 gaggggggcg ctggggcgca gtgacgggaa ccaatgagct gccaactcgc gcgtctccgg 900 cgtgactgcc gagattgacg tggaggacac gtcaaattga ttcccgcacg ctgcagcctc 960 ccggtcagac gaatttctcc caatcggatg aagttcaccc tgggcctggg gtcgcgggcg 1020 tggagagtgt cctgggaggg ggcagcagcg gcggcggcag gccctggagc gggcggcagc 1080 gcgctccgct gccgcgcaca gcgcgtctcc agcccgcggc tgggccgccg cggctctcgg 1140 ctctcgggcg ccctccctct atgcctctca cgcggcggcg gcggcgccca agctctcccg 1200

<210> 3 <211> 2350 <212> DNA <213> Human genius (Homo sapiens) 2 1329743 <400> 3

agccccccgc ccagcgcacc ccaccccctc tccgccgccg cgcacgcagc gccgcggccc 60 ctgtcagaag ctgcaggatc cgccccggcg aagcagggcc gactcgcacc caggaccctg 120 ggcctctgcc ttccctccta 'gccttggaga agcaactggc cctctcctcc cgctgaggag 180 cgacgcgggc tggtaggacg tcccgggaag gccggcagct cgcgaccacg tcccggccca 240 gcctgggcgc gccgaggagc agagccagcg gccggcgttc gctccggctc cctccccggc 300 gctccgaagc cgagggcggc tcctccggct gcagtctcgg gggcgacgcc ttcccgggca 360 gaagcttcca gcagcgctcc gcaacttctc tctgctccag tcactgggag agagctcgcc 420 taccaggtaa gtaaggctgc ccgtgcctag gctgtggctc gggcgggcgt gtttctgaaa 480 gttgacttga aatgcatcca agagtgagcc gggccagccg gggctcctcc ccgaggcgac 540 tcttttgctt ctgcagacat tcccggcact ggccgactgg cgggaggacc tccccgcgcg 600 ccccgcacac cggctcctgc gcgcacccca acagagcgca gcgccaggag tccagaagcg 660 ggcgggacgc cctccgggtc ccttacagtg ccccttctcg acctggggca ggtgagggcc 720 gcaacggggc ggctgggacg cgggattgca aaccccatcg tcccgcgtgc ctggacccgg 780 tcgctcgagg gagggtaccc actctttata tacccaatat accccagtag ctgcgttcct 840 gcagagacgt Cccgagggc c caccttcgta taggttgggg cggagtcgga ttcgggatgg 900 aaaacctggg gcaagggatg taggtggggg tgaggggggc aggagaagga gaaacgcagt 960 tggggggcgg aggcctaagt acataacgtg ttgacttcaa gtgaaatcag atcagccaga 1020 gcagttcgct gtgactgatc tctcctccca ccctacattc tcttggctgg accctatcct 1080 cctggctgat tctggtcgcc ctggacactc cctcagttct ttcccaggag tgcggtggct 1140 gctggcgccg agtcccagcg ggcacggacg tcagacgcat cgtttcttct cctctacagg 1200 tcctcccggc ccggcccgaa catgctggac ggcctaaaga tggaggagaa cttccaaagc 1260 gcgatcgaca cctcggcctc cttctcctcg ctgctgggtg agtgttcagg ccgtgcgtcc 1320 tgggcgcact ctctttccgc ttggcgctga.gctctggagc cccgctctct gggacctggt 1380 ccgcgatagg gaagctagcg cccctcttca tacactaaat cactatctgt 1440 ccgtcagtgc ttgtgggtcg tccctaccca aataaatcca acaagccgcc ccaggcctca 1500 cgcactgggc accgaattcc ccaaagccgc gaggggcggg cgagcttgtt cgtaggcgtc 1560 tgagtggcaa gtgattaaaa atacccaggg ctggattttt aatctcggag ctgatcgacg 1620 tctcataaat gccgccctct tctcgcggcc tagaggcaat agcatccgag acccgaggcc 1680 tggagcgccc aagttcgagg tgagccccat Aggctt ctct cccccaccaa ctccagcccc aatttcagcc 1740 atgggcaagg ccgagagaga cttttctggg ccagtaggca acgcagcgcg gggattagac 1800 cgcgcggctg ggccctaggc tccgcgttaa ttaactggag ctggatgtcg ggtcctgtgg 1860 gtcccccctc acaactcctt tgcagcgaca gaggaggagc agcgagtcaa ggaccccgga 1920 agagtgtcca cacacgggtc ttgcagagct gaagccagag attctggtta cggctccccc 1980 acccactctg cccttggagc ttttcaagtt tgggaagccc gtattttatt ttattttatt 2040 ttatttattt atttttagca ggagggagtt ttccttctgt cctttaaacc tcttgcctct 2100 ttcactcgga accgctagag cggcatgaat Gtggaggatg agacagttct gctggagaaa 2160 ccaaatccag tgaggaatct ccccaccccc aacaccctag atgaaacgaa atcacgtgca 2220 ctgagcgccc ctctttgaac ccccctttcg ggaacttttg gacttgccca gcctcggaag 2280 agagcggaag ccagagcagg cagtacccgg gcgggcagcg ggtgcggatt tatgtataca 2340 gcgggcgtcg 2350 3

<210> 4 <211> 1250 <212> DNA <213> Human genius (Homo sapiens) <400> 4 aaagagcgga ggagccgcgg agagggtcag tttggccaga ggacaggact tagaagaccg 60 aagcctggga agccgcgaag aaaatgccag agaggagagt caaaggctga gagtgagagg 120 gagagaggga gcgggggtgg ggcgggggtc gccgggcccg tttgcagaag tggactgacg 180 agcggcgccg aaacccagcc gtcagacttt tcacacttag tcttttgttt ttctgtgttt 240 ttcctccccc tttttctttt caatattgca actccagtgc cccgtgggcc agagggcaag 300 gcgggtggag gtgaggacct gggagccgcg gggatccgtg gcactgccct ttctggcgca 360 gcagcccggg gcagcgtggg cggaggaagc ccgcacagag gctagatctc ccgcgggctg 420 gatgcgcttt ctccccgggc acagtgagcg tcgaatgcga atcagccgcg cgaccgaaag 480 agcagagcat cccagtaaga tcagaggagc gccacgggct gcacaaggcg tcctttgaac 540 ctccccaaag aaagcaagcc acccccaccc tccaacttca aagtggagat Tcggcaacta 600 actttgctac aaactctccg gagccagcct gggttttgtt ttgcttattt cccgggggca 660 gaagatgaga agtagcgcac tttgaacagc taggaaaagt gaggaagaga gaatagccag 720 ggatcgaatc taggactcgc ggaacgaaag gactgcctag cccgccggga c gcctgcttt 780 tctcggcgag ctgccgcctc ccgcgtggag ggtttggaca tctctgctgc gcagctaggc 840 gagcaactcc cggcagcggc atttttggtt cagttggcag ctcgcctccg ggcgcgccga 900 gtgcctctcc gctcgcgccc tcggcgcttc cggctcctct gagccccgcg gggggcacca 960 gccagcgccc tcgctgcaag gctacggtct ccggcgtggc cgtgggatgt tagcggtggg 1020 ggcaatggag ggcacccggc agagcgcatt cctgctcagc agccctcccc tggccgccct 1080 gcacagcatg gccgagatga agaccccgct gtaccctgcc gcgtatcccc cgctgcctgc 1140 cggccccccc tcctcctcgt cctcgtcgtc gtcctcctcg tcgccctccc cgcctctggg 1200 cacccacaac ccaggcggcc tgaagccccc ggccacgggg gggctctcat 1250

≪ 210 > 5 < 211 > 1700 < 212 > DNA < 213 > person who is wise species (Homo sapiens) < 400 > 5 tcactgagca ctccaggagt tccccaaact caagggtttt agatattcct aaccgccttc gcccggctta accagaatgg tacccgctcc gggcgccagg cttccctcct cactggaaag cgcctggcgc taactggtgc tatcctcgac ctgccgggct atgctccggc aaactagccg ggaaactaag aaacctcacc aactcccggc cagggccaca tcgtatccaa cggaatatac ctgttttccc tgctgctgac aagtaaacaa cacccaactg ggcagtgctc accctcccct gcaggctttg ggcttaaccg tccaatttta ctactagccg agggacgttc ggcggagtgg tcacccctcc ggcgtttgcc tagaagaatt ggtaggcggc atcgaaatac gctttcagtc 60 120 180 240 300 360 420 4 1329743

ccgacctctg gaacccacaa agggccacct ctttccccag tgaccccaag atcatggcca 480 ctcccctacc cgacagttct agaagcaaga gccagactca agggtgcaaa gcaagggtat 540 acgcttcttt gaagcttgac tgagttcttt ctgcgctttc ctgaagttcc cgccctcttg 600 gagcctacct gcccctccct ccaaaccact cttttagatt aacaacccca tctctactcc 660 caccgcattc gaccctgccc ggactcactg cttacctgaa cggactctcc agtgagacga 720 ggctcccaca ctggcgaagg ccaagaaggg gaggtggggg gagggttgtg ccacaccggc 780 cagctgagag cgcgtgttgg gttgaagagg agggtgtctc cgagagggac gctccctcgg 840 acccgccctc accccagctg cgagggcgcc cccaaggagc agcgcgcgct gcctggccgg 900 gcttgggctg ctgagtgaat ggagcggccg agcctcctgg ctcctcctct tccccgcgcc 960 gccggcccct cttatttgag ctttgggaag ctgagggcag ccaggcagct ggggtaagga 1020 gttcaaggca gcgcccacac ccgggggctc tccgcaaccc gaccgcctgt ccgctccccc 1080 acttcccgcc ctccctccca cctactcatt cacccaccca cccacccaga gccgggacgg 1140 cagcccaggc gcccgggccc cgccgtctcc tcgccgcgat cctggacttc ctcttgctgc 1200 aggacccggc ttccacgtgt gtcccggagc cggcgtctca gcacacgctc cgctccgggc 1260 ctgggtgcct acag cagcca gagcagcagg gagtccggga cccgggcggc atctgggcca 1320 agttaggcgc cgccgaggcc agcgctgaac gtctccaggg ccggaggagc cgcggggcgt 1380 ccgggtctga gccgcagcaa atgggctccg acgtgcggga cctgaacgcg ctgctgcccg 1440 ccgtcccctc cctgggtggc ggcggcggct gtgccctgcc tgtgagcggc gcggcgcagt 1500 gggcgccggt gctggacttt gcgcccccgg gcgcttcggc ttacgggtcg ttgggcggc'c 1560 ccgcgccgcc accggctccg ccgccacccc cgccgccgcc gcctcactcc ttcatcaaac 1620 aggagccgag ctggggcggc gcggagccgc acgaggagca gtgcctgagc gccttcactg 1680 Tccacttttc cggccagttc 1700

≪ 210 > 6 < 211 > 1850 < 212 > DNA < 213 > person who is wise species (Homo sapiens) < 400 > 6 gaaagaaagc cgcggcatgt ggcggccggg tgtacgtctc aaactggggg cctccgcaca 60 cgtccccacc aggcccaaag accaggttcg attccagatt ggagcgtgac tgtgggaagg 120 gcgaaattac tcccgaagct gaattgattt tcaaatctgg aggcgtctct cggggacgcc 180 gggaaaaggg cgtccctagg ggccaagcgg agacccgcgc gccggcgtcc accctgtctg 240 cgtcagtact tctggaaagc aaccgcctcc gggtgcttta gcaggagggc cttgggagta 300 accgcaggga cggcccgagc ctccacggcc ccaccagcac ccggaagtta acatgggtac 360 cctccagggc actccctccg ctctccctcg cccccctcca caggccgagt catcggcgaa 420 gcggacgcac agccttaatt atgagctgag cgggagaagg agccaggcgg cgggggacag 480 tgaggtatgg cccgaactgg gattcttggc actgattact cctctcccca gggcatggat 540 gagaaagggt gggcaagtat gtatctggga ggacgaaggg tgccgggtca Acggccgcca 600 aacggaccca gccctttaag caatctgcac cccaccccac cccaaccccc atcccccaat 660 aggccgtgaa ttcaagggtg ggaaagcgca ctcccagcag ccccccggga aataaagctt 720 agtgggctag agccgaaggg gtgatgacac agtccccagc tccccgggca a Gctgcaccg 780 5

ggaagcagca actggagaga gaggggcgat gtctccaagc acagcactcc agccgctaga 840 agcccgacac gagcgtcccc gggctgggag gacagagccc actcaagcaa gggaggcgag 900 cgagccaggc gcgagtctcc tgggattgca gcggcggccc caggtcgcgc tctgcgccaa 960 tctttcgcac gtgcccgcag ctccctggcc atccagcgcc gcagggaagg cgctgggccc 1020 cctccttcat ttgtaccggg acgccaaggg cctggcgcgc cgcgacctag ggggcggggg 1080 cgggcctcgc gcatgcgcgc tgcgcctggc gggcgtgagg gcgggccgct gcggcggcgg 1140 cggcggcggc taccgaaccg cggccacaga gtctgtaaca gtaacagagc catggctcaa 1200 gctggccagc ggggcgggca ggcagcagac gcggcaggcg cgcgggccgc ggcaggggag 1260 ccggagacct cagaatttta agaaagagag gggcgagagg tggccgaggc gggcgggctg 1320 gggcactgcg ctctcccaac ggcgcggatc ctctttggaa attaatatta aaaaaaaaaa 1380 agccgaggac gcagagggga aggtgggggg taagagggaa ggcgagacac acacacacac 1440 acacacgcac acgcacacac ggacacacac acacggagag agagagagag agagacagag 1500 ccccacagtg agaggaagga aggcaacagt cgccagcagc cgatgtgaag accggactcc 1560 gtgcgcccct cgccgcctct gcctggccac atcgatgttg tgtccgccgc ctgctcgccc 1620 ggatcacga t gaacgcgcag ctgaccatgg aagcgatcgg cgagctgcac ggggtgagcc 1680 atgagccggt gcccgcccct gccgacctgc tgggcggcag cccccacgcg cgcagctccg 1740 tggcgcaccg cggcagccac ctgccccccg cgcacccgcg ctccatgggc atggcgtccc 1800 tgctggacgg cggcagcggc ggcggagatt accaccacca ccaccgggcc 1850 < 210 > 7 < 211 > 24 < 212 > DNA < 213> Artificial Sequence <400> 7 cgtttttttt ttttcgttat tggc 24

<210> 8 <211> 20 <212> DNA <213>Artificial sequence<400> 8 cctacgctcg atcctcaacg 20 <210> 9 <211> 25 <212> DNA <213> 6 1329743 <400> 9 tgtttttttt tttttgttat tggtg <210> 10 <211> 22 <212> DNA <213>artificial sequence <400> 10 cctacactca atcctcaaca ac

<210> 11 <211> 18 <212> DNA <213>Artificial sequence <400> 11 tttagaagcg ggcgggac <210> 12 <211> 17 <212> DNA <213> Artificial sequence <;400> 12 ccgaatccaa acacgcg <210> 13 <211> 22 <212> DNA <213> artificial sequence<400> 13 gagtttagaa gtgggtggga tg

<210> 14 <211> 24 <212> DNA 1329743 <213>Artificial sequence <400> 14 caaccaaatc caaacacaca aaac <210> 15 <211> 21 <212> DNA <213> Sequence <400> 15 ttgtagcggc ggttttaggt c

<210> 16 <211> 20 <212> DNA <213>Artificial sequence<400> 16 gccaaaccct taacgtcccg <210> 17 <211> 24 <212> DNA <213>Artificial sequence<213>;400> 17 gattgtagtg gtggttttag gttg <210> 18 <211> 25 <212> DNA <213>artificial sequence <400> 18 caccaaaccc ttaacatccc aatac <210> 19 V.· J*· , \* ι-V/. 1329743 <211> 20 <212> DNA <213>Artificial sequence<400> 19 tattttgggt ttggggtcgc <210> 20 <211> 18 <212> DNA <213><400> 20

Cccgaaaacc gaaaaccg <210> 21 <211> 24 <212> DNA <213>Artificial sequence <400> 21 gtttattttg ggtttggggt tgtg <210> 22 <211> 21 <212> DNA <213> Artificial sequence <400> 22 cacccaaaaa ccaaaaacca c <210> 23 <211> 20 <212> DNA <213>manual sequence <400> 23 cgtggtcgtg ggatgttagc 1329743 <210> 24 <211> 21 <;212> DNA <213>Artificial sequence<400> 24 acaaacaacg aaaaatacgc g <210> 25 <211> 22 <212> DNA <213>Artificial sequence <400> 25 gtgtggttgt gggatgttag tg <210&gt ; 26 <211> 25 <212> DNA <213>Artificial sequence<400> 26 caacaaacaa caaaaaatac acaac

<210> 27 <211> 21 <212> DNA <213>Artificial sequence <400> 27 tgttgagtga atggagcggt c <210> 28 <211> 23 <212> DNA <213><400> 28 ...J·,* II.!·ν· 1329743 23 cgaaaaaccc ccgaatataa acg <210> 29 <211> 24 <212> DNA <213>Artificial sequence <400> 29 gttgttgagt Gaatggagtg gttg 24

<210> 30 <211> 29 <212> DNA <213>Artificial sequence <400> 30 29 aattacaaaa aacccccaaa tataaacac <210> 31 <211> 26 <212> DNA <213> Sequence <400> 31 gttgttttyg ggtttttttt tggttg 26 <210> 32 <211> 28 <212> DNA <213>Artificial Sequence<400> 32 atttctccta atacacaaac cacttacc 28 <210> 33 <211><212> DNA <213> Artificial sequence 11 1329743 <400> 33 tagttattgg gagagagtty gtttattag <210> 34 <211> 24 <212> DNA <213>Artificial sequence <400> 34 ctaccccaaa tcraaaaaaa acac

<210> 35 <211> 22 <212> DNA <213>Artificial sequence <400> 35 gagtttattt aagtaaggga gg <210> 36 <211> 30 <212> DNA <213><400> 36 caacttaaac cataactcta ttactattac <210> 37 <211> 22 <212> DNA <213> artificial sequence <400> 37 gtgttttggg agggggtagt ag <210> 38 <211> 21 1329743 < 212> DNA <213>Artificial sequence <400> 38 ccctcccraa ccctaectat' c <210> 39 <211> 22 <212> DNA <213>Artificial sequence <400> 39 gatagaagga gggggtagag tt

<210> 40 <211> 21 <212> DNA <213> artificial sequence <400> 40 tactaccccc tcccaaaaca c

<210> 41 <211> 25 <212> DNA <213>Artificial sequence <400> 41 ggtatttttg gtttagttgg tagtt <210> 42 <211> 22 <212> DNA <213><400> 42 aataccctcc attaccccca cc .Ιι.ϊ'.'Λ* ilVlV.i;"..'.'.»»!.!·. ·Λ:ι',-\·.ΐ'.· '·ί-^5 . •όί'"·' 1329743 <210> 43 <211> 22 <212> DNA <213>Artificial Sequence <400> 43 ggtgggggta atggagggta tt •<210> 44 <211> 24 <212> DNA <213> artificial sequence

t <400> 44 cctaaattat aaatacccaa aaac <210> 45 <211> 24 <212> DNA <213>manual sequence <400> 45 gtgttgggtt gaagaggagg gtgt <210> 46 <211> 28 <212> DNA <213>Artificial sequence <400> 46 atcctacaac aaaaaaaaat ccaaaatc <210> 47 <211> 22 <212> DNA <213>Artificial sequence <400> 47 agacctagat gccaacaatt gg <210> 48 <211> 21 <212> DNA <213>Artificial sequence<400> 48 gcaccactac gacttagtcc g

<210> 49 <211> 21 <212> DNA <213>Artificial sequence <400> 49 gctgcttctg ctgctgtgtc t <210> 50 <211> 21 <212> DNA <213><400> 50 acgtttgggg cgcttatggt c

<210> 51 <211> 21 <212> DNA <213>Artificial sequence <400> 51 caaaccctgg agcaaactca a <210> 52 <211> 21 <212> DNA <213> 132973⁄4 <400> 52 tgtgttgcct ctatccttcc c <210> 53 <211> 24 <212> DNA <213>Artificial sequence <400> 53 cctacgctgc cctacaacca catc

<210> 54 <211> 24 <212> DNA < 213 > artificial sequence <4〇0> 54 tcacgccggc ccagtcttcc atct <210> 55 <211> 21 <212> DNA <213> Artificial sequence <400> 55 cacacgagac ccactttttc c <210> 56 <211> 20 <212> DNA <213>artificial sequence <400> 56 cccaacgaat aggccaaacg

<210> 57 <211> 21 <212> DNA » k-s· .· · .k:1329743

<213>Artificial sequence <400> 57 gctgtcccac ttacagatgc a 21 <210> 58 <211> 21 <212> DNA <213> artificial sequence <400> 58 tcaaagcgcc agctggagtt t 21 17

Claims (1)

I1329743 Patent application scope: 1 A method for screening a seed cancer risk, the method comprising the following steps: Step 1 provides a cervical sample sample; Step 2 detects a CpG sequence of a target gene in the cervical sample sample In the assimilation state, the target gene instrument consists of SOX1, PAX1, LMX1A, NKX6-1, WT1 and „0NECUT1; and step 3 if the target gene is methylated, indicating that the specimen has the possibility of cervical cancer The method of cervical cancer screening according to the first aspect of the invention, wherein the cervical sample comprises a Pap smear and a cervical epithelial cell. 3. The child as claimed in claim 1 The method for screening cervical cancer, wherein the cervical specimen is an abnormal smear of the cervix. The method for screening cervical cancer according to claim 1, wherein the cervical sample is A cervical cell sample that is positive for HPV testing (p0SitiVe). (9 5. A method for screening for cervical cancer as described in claim 1, wherein the CpG sequence of the target gene The thiolation state detection method is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) 'sulfite sequencing (bisulfite sequencing, BS), microarrays, mass spectrometer analysis (mass, denaturing high-performance liquid diroimtography (DHPLC), pyrosequencing). The method for screening for cervical cancer according to Item 1, wherein the target gene 1 1329743 S0X1 has a nucleic acid sequence as SEQ ID Na.* 7. The cervical cancer screening as described in the scope of the patent application. The method PAX1 has a nucleotide sequence as shown in SEQ ID No: 2. 8. The method of applying the sub-surface detection of the 丨 述 LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM LM As for the method of cervical blue detection described in the patent application, the NKX6-.1 has the nucleotide sequence as shown in SEQ ID NO: 4. 10. The method of applying the bamboo surface riding as described in item (4) , WT 1 has a nucleotide sequence as shown in SEQ Π) No: 5. 11. The method of screening for cervical cancer according to the scope of claim i, ONECUT1 has a core as shown in SEQ ID No: 6. Glycosidic acid sequence. Wherein the target gene, wherein the target gene is the target gene, wherein the target gene, wherein the target gene