A HOXA9 gene methylation detection reagent and its application

Abstract

The present invention discloses a HOXA9 gene methylation detection reagent and its application, wherein the reagent comprises a forward primer sequence as shown in SEQ ID NO.28 and a reverse primer sequence as shown in SEQ ID NO.30. The HOXA9 gene methylation detection reagent prepared by the scheme of the present invention can not only use tissue as a detection sample, but also quickly perform breast cancer examination by taking blood from the subject, avoiding radiation damage to the subject caused by X-ray photography, etc., improving the sensitivity and specificity of breast cancer examination, and providing a simple, fast and effective examination method for breast cancer examination.

Description

Technical Field

[0001] The present invention belongs to the field of gene diagnosis, and in particular relates to a HOXA9 gene methylation detection reagent and application thereof. Background Art

[0002] Breast cancer is a phenomenon in which breast epithelial cells proliferate uncontrollably under the influence of a variety of carcinogenic factors. The symptoms of early breast cancer are not obvious and are very easy to be ignored. The main methods for screening breast cancer currently include breast palpation, imaging examinations, and laboratory tests. Generally, breast palpation is suitable for patients with obvious breast lumps. Imaging examinations mainly include mammography, breast ultrasound, and breast magnetic resonance imaging. Among them, mammography is suitable for breast examinations of screening population-level diagnostic patients, breast ultrasound is suitable for all people suspected of breast lesions, and breast magnetic resonance imaging is mainly suitable for breast cancer screening for people who have difficulty detecting or diagnosing lesions with mammography and ultrasound and high-risk groups. Laboratory tests include biochemical tests and tumor marker tests.

[0003] Some early breast cancers are negative on palpation, and the diagnosis requires the combination of imaging and histopathological examination results; mammography does not show dense breasts and masses near the chest wall well, and the examination has radiation damage, so it is not the first choice for young female patients, especially those who have plans to have children in the near future; breast magnetic resonance imaging has moderate specificity, a high false positive rate, and is not satisfactory for the display of smiling calcified lesions. In addition, this method takes a long time to examine and is expensive; tumor marker examination is not suitable for screening and diagnosis of breast cancer because of its low sensitivity to local lesions and can also be elevated in some benign diseases and malignant tumors of other organs. Therefore, there is still an urgent need for a simple, efficient method that can accurately detect breast cancer. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a HOXA9 gene methylation detection reagent.

[0005] The present invention also provides an application of the methylation detection reagent.

[0006] The present invention also provides a method for detecting DNA methylation of the HOXA9 gene.

[0007] According to one aspect of the present invention, a HOXA9 gene methylation detection reagent is provided, the reagent comprising a forward primer sequence as shown in SEQ ID NO.28 and a reverse primer sequence as shown in SEQ ID NO.30.

[0008] In some embodiments of the present invention, the HOXA9 gene methylation detection reagent detects the sequence of the HOXA9 gene modified by a conversion reagent; the conversion reagent refers to a reagent that converts unmethylated cytosine in DNA into uracil while leaving 5-MeC substantially unaffected.

[0009] In some embodiments of the present invention, the conversion reagent includes hydrazine salts, bisulfites (such as sodium bisulfite, etc.), bisulfites (such as sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.), or one or more compounds that can produce hydrazine salts, bisulfites, and bisulfites under appropriate reaction conditions.

[0010] In some embodiments of the invention, the conversion reagent is bisulfite.

[0011] In some embodiments of the present invention, the HOXA9 gene methylation detection reagent detects a sequence modified by bisulfite.

[0012] In some embodiments of the present invention, the detection region of the HOXA9 gene targeted by the detection reagent for HOXA9 gene methylation is the HOXA9 gene or its promoter region.

[0013] In some embodiments of the present invention, the detection region of the HOXA9 gene targeted by the detection reagent for HOXA9 gene methylation is the CG-enriched region or the non-CG-enriched region or the CTCF (CTCF-binding sites) region of the HOXA9 gene.

[0014] In some embodiments of the present invention, the detection region targeted by the HOXA9 gene methylation detection reagent is a CG-enriched region or a CTCF (CTCF-binding sites) region of the HOXA9 gene.

[0015] In some embodiments of the present invention, the detection region of the HOXA9 gene targeted by the HOXA9 gene methylation detection reagent is a sequence as shown in SEQ ID NO. 11. The selection of the HOXA9 gene detection region will affect the detection efficiency of tumors. The primers designed according to the CG-enriched region of the HOXA9 gene have obvious differences in the detection results of primers designed in different regions.

[0016] In some embodiments of the present invention, the reagent further comprises a fluorescent probe sequence such as a nucleotide sequence shown in SEQ ID NO.29.

[0017] In some embodiments of the present invention, the fluorescent group labeled with the detection probe is VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED or TexasRed, etc.; the quenching group can be TAMRA, BHQ, MGB or Dabcyl.

[0018] In some embodiments of the present invention, the detection reagent for HOXA9 gene methylation further contains a detection reagent for an internal reference gene; preferably, the internal reference gene is GAPDH.

[0019] In some embodiments of the present invention, the detection reagent containing the internal reference gene contains the primer pair shown in SEQ ID NO.6 and SEQ ID NO.7, and the probe of SEQ ID NO.8.

[0020] In some embodiments of the present invention, the detection reagent of HOXA9 gene methylation further includes DNA polymerase, dNTPs, Mg 2+ ions, buffers; preferably, DNA polymerase, dNTPs, Mg 2+ Ions and buffers are used for amplification of the HOXA9 gene.

[0021] In a second aspect of the present invention, there is provided an application of the above-mentioned HOXA9 gene methylation detection reagent, wherein the application is an application in the preparation of a breast cancer diagnostic reagent or kit.

[0022] In some embodiments of the present invention, the detection sample of the HOXA9 gene methylation detection reagent is selected from breast tissue, blood, serum or plasma.

[0023] In a third aspect of the present invention, a method for detecting DNA methylation of the HOXA9 gene for non-disease diagnosis purposes is provided, comprising the following steps:

[0024] S1, treating the sample to be tested with bisulfite, bisulfite or hydrazine salt to obtain a modified sample to be tested;

[0025] S2. Using the above-mentioned methylation detection reagent, detect the methylation status of the HOXA9 gene on the sample to be tested modified in step S1.

[0026] In some embodiments of the present invention, the detection in step S2 is performed using methylation-specific polymerase chain reaction or real-time fluorescent quantitative methylation-specific polymerase chain reaction.

[0027] According to the embodiments of the present invention, at least the following beneficial effects are achieved: the HOXA9 gene methylation detection reagent prepared by the scheme of the present invention can not only use tissue as a detection sample, but also can quickly detect breast cancer by taking the blood of the subject, avoiding the radiation damage caused to the subject by X-ray photography, etc., improving the sensitivity and specificity of breast cancer detection, and providing a simple, fast and effective detection method for breast cancer detection. The present invention detects and diagnoses cancer through methylation levels. More and more studies have confirmed that methylation changes are early events in the process of tumorigenesis, and detecting methylation abnormalities makes it easier to find early lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0029] Figure 1 is an amplification curve diagram of the primers designed for region 1 in Example 1 of the present invention;

[0030] Figure 2 is an amplification curve diagram of the primers designed for region 3 in Example 1 of the present invention;

[0031] Figure 3 is an amplification curve diagram of the primers designed for region 2 in Example 1 of the present invention;

[0032] Figure 4 This is an amplification curve diagram when the amount of MgCl2 added to the reaction substrate in Example 2 of the present invention is 0.5 μL;

[0033] Figure 5 This is an amplification curve diagram when the amount of MgCl2 added to the reaction substrate in Example 2 of the present invention is 1.8 μL;

[0034] Figure 6 This is a graph showing the peak time and detection status of the HOXA9 gene methylation detection reagent in Example 3 of the present invention for standard plasmids of different concentrations;

[0035] Figure 7 is the ROC curve of the detection reagent for HOXA9 gene methylation in Example 4 of the present invention in all tissue specimens;

[0036] Figure 8 This is a statistical result diagram of cancer and adjacent tissue detection in Example 4 of the present invention;

[0037] Fig. 9 This is a statistical result diagram of cancer and adjacent tissue detection in Example 4 of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] Using methylated DNA as a detection target has obvious advantages. Compared with protein markers, DNA can be amplified and easily detected. Compared with mutation markers, the sites where DNA methylation occurs are all located in specific sites of the gene, generally in the promoter region, which makes detection easier and more convenient. In order to complete the present invention, the inventors screened out the best detection gene HOXA9 from hundreds of candidate genes through multiple rounds of screening, and the GAPDH gene was used as an internal reference gene, and the primers and probes designed for detection were used for real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescent quantitative methylation-specific PCR, qMSP) detection.

[0040] Example 1 Design and optimization of primers and probes for HOXA9

[0041] The preparation method of the detection reagent for HOXA9 gene methylation is as follows: find the genomic fragment of the HOXA9 gene in the database (as shown in SEQ ID NO.1), convert all Cs other than CpG into Ts, and the sequence is shown in SEQ ID NO.2. Analyze the CpG island situation, and after a large number of screenings, obtain suitable regions (positive regions as shown in SEQ ID NO.9-11) for primer probe design. The designed primers and probes are the primer probes used for HOXA9 gene methylation amplification, and the designed detection primer probes are used for real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescent quantitative methylation-specific PCR, qMSP) detection. The GAPDH gene is used as an internal reference gene, and the negative sequence of the HOXA9 gene is shown in SEQ ID NO.32. Specifically, it includes the following contents:

[0042] 1. Selection of detection area

[0043] Since the methylation status and distribution of the same gene are not uniform, for the same gene, the diagnostic detection efficiency of methylation primers and probe detection systems designed in different regions is different for the same sample and the same tumor. Sometimes, the selected region is not suitable, resulting in no diagnostic effect on the tumor. After repeated research and comparison, the inventors have found that the regional sequences of some HOXA9 are shown in Table 1:

[0044] Table 1

[0045]

[0046]

[0047] 2. Design different primer and probe sequences for the three regions:

[0048] (1) Two forward primers (P1F1, P1F2), three reverse primers (P1R1, P1R2, P1R3), and three probe sequences (FP1-1, FP1-2, FP1-3) were designed for region 1. The sequences are shown in Table 2.

[0049] Table 2

[0050]

[0051]

[0052] (2) Three forward primers (MCF1, MCF2, MCF3), three reverse primers (MCR1, MCR2, MCR3), and four probe sequences (MCFP-1, MCFP-2, MCFP-3, MCFP-4) were designed for region 2. The primer sequences are shown in Table 3.

[0053] Table 3

[0054]

[0055]

[0056] (3) Two forward primers (P3F1, P3F2), two reverse primers (P3R1, P3R2), and one probe sequence (FP3-1) were designed for region 3. The primer sequences are shown in Table 4.

[0057] Table 4

[0058]

[0059] Plasmid pUC57-Amp-HOXA9 (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) containing the methylation sequence shown in SEQ ID NO.2 was used as a template for amplification. The amplification system is shown in Table 5, the reaction procedure is shown in Table 6, and primers designed for regions 1, 2, and 3 were used for amplification. Only the primer probe sequences shown in Table 7 can be amplified to obtain an S-shaped curve.

[0060] Table 5

[0061]

[0062]

[0063] Table 6

[0064]

[0065] Table 7

[0066]

[0067]

[0068] The primers and probes designed for the screened regions 1, 2 and 3 were used for fluorescence quantitative PCR amplification. The amplification results were as follows: Figure 1-3 As shown in the figure, it can be seen that the results of primers designed in different regions are significantly different. Among them, the amplification results obtained by the primers and probes designed for region 3 are the best. Therefore, the primers and probe sequences designed for region 3 and the primers and probe sequences designed for the GAPDH internal reference gene are selected as detection reagents for HOXA9 gene methylation (as shown in Table 8) for subsequent experiments.

[0069] Table 8

[0070]

[0071]

[0072] Example 2 Optimization of amplification system

[0073] The entire amplification system was optimized by optimizing the concentration of the reaction substrate MgCl2.

[0074] Reaction system: 0.5 μL and 1.8 μL of the reaction substrate MgCl2 with a concentration of 25 mM were added to the solution system shown in Table 5 as the experimental group, and the internal standard group was the internal reference gene. The primer probe group used the forward primer P3F2, reverse primer P3R1, and probe FP3-1 designed in region 3.

[0075] The experimental results are as follows Figure 4-5 As shown, Figure 4 This is the result of adding 0.5 μL 25 mM MgCl2 to the reaction system. Figure 5 The result diagram of adding 1.8 μL 25 mM MgCl2 to the reaction system shows that the added concentration of the reaction substrate MgCl2 has a significant effect on the amplification effect. The present invention optimizes the addition concentration of the reaction substrate MgCl2 to achieve a better amplification effect.

[0076] Example 3 Sensitivity measurement

[0077] The sensitivity test of the HOXA9 gene methylation detection reagent obtained in the above example comprises the following steps:

[0078] (1) Dilute the pUC57-Amp-HOXA9 standard plasmid to 10 3 copies / reaction tube, 10 2 copies / reaction tube, 10 1 copies / reaction tube.

[0079] (2) The standard plasmids of each concentration in step (1) were amplified using the HOXA9 gene methylation detection reagent in the above example. The amplification system is shown in Table 5, and the amplification program is shown in Table 6.

[0080] The detection results of the HOXA9 gene methylation detection reagent for the peak time and detection of standard plasmids with different concentrations are as follows Figure 6 As shown in the figure, it can be seen that the detection limit of the fluorescence quantitative PCR method established by the detection reagent for HOXA9 gene methylation is 10 1 copies / reaction tube.

[0081] Example 4 Detection of breast cancer by HOXA9 gene methylation reagent

[0082] This embodiment provides a HOXA9 gene methylation detection reagent, comprising the primer probes shown in Table 8.

[0083] The experimental process of the HOXA9 gene methylation detection reagent for breast cancer detection is as follows:

[0084] 1. DNA extraction

[0085] Tissue specimens from patients diagnosed with breast cancer and patients without breast cancer were collected, and DNA was extracted using a kit purchased from Sangon Biotech Co., Ltd.

[0086] 2. DNA modification

[0087] EZ DNA Methylation-Gold Kit from ZYMO RESEARCH BiotechnologyTM (D5002) instructions for bisulfite modification.

[0088] 3. Amplification and detection

[0089] The liquid preparation system is shown in Table 9.

[0090] Table 9 Liquid Preparation System

[0091]

[0092]

[0093] The reaction procedure is shown in Table 10.

[0094] Table 10

[0095]

[0096] 4. Test results

[0097] Sample information: There were 23 breast tissue samples in total, including 0 normal tissue samples, 15 cancer tissue samples, 3 breast cancers of unclear classification, and 8 pairs of cancer and adjacent normal control samples.

[0098] Experimental results: The ROC curve of the HOXA9 gene methylation detection reagent prepared by the present invention in all tissue specimens is as follows: Figure 7 As shown in the figure, it can be seen that when predicting patients diagnosed with breast cancer, the AUC value is 0.865, the best limit corresponds to a sensitivity of 91.7%, and a specificity of 75%. The statistical results of cancer and adjacent tissue detection are as follows Figure 8 and Fig. 9 As shown in Table 11, breast cancer was diagnosed when the △Ct value was less than 16.143. The results showed that the △Ct value of cancer tissue was significantly lower than that of adjacent adjacent tissue (p<0.027).

[0099] Table 11

[0100]

[0101]

[0102] Studies have shown that the loss of the HOXA9 gene plays an important role in the occurrence of breast cancer. The HOXA9 gene is highly methylated in breast cancer tissue, and in the breast tissue and blood of patients with different degrees of lesions, the methylation level increases with the increase in the degree of lesions. Gene methylation testing only requires taking blood from the subject and does not cause damage to the subject's body. At the same time, HOXA9 gene methylation has high sensitivity and specificity in breast cancer and can better monitor the progression of breast cancer.

[0103] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict. Sequence Listing <110> Hunan Hongya Gene Technology Co., Ltd. <120> A HOXA9 gene methylation detection reagent and its application <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2900 <212> DNA <213> Artificial Sequence <400> 1 gcacatcccg ctggggcaaa tgagcctcct gcatggggtc tctggccgaa gtgcagaact 60 ctgctgaact ggttaggaaa ggcagtcagg cctcggacac aatggaaccc tggcagacag 120 acgcacagac agtcacttaa aattgcacgc agtaaaactt tggctcgccc tcccctccga 180 gaccttcctt tctcctactc tgtcctcttg tcccccttct ccttcctccc actctcaaaa 240 cgctgtatag aatgaaattt ggaaacaggt ctccttgcca cgcagaggga aagcatgctg 300 ccttgtgttc tgtagcaaga ttaggattcc tctgtcccgt tcactgactt cgtctttctt 360 tcccaacctg tccctctacg ccccccactc cttatttaac cttcctggaa ggccttcgga 420 gctgggcaag ccgtcagggc gccctaaggc cgctgatcac gtctgtggct tatttgaata 480 atctgtcatg gggacccttg tggcccgggt cgcccgcagc ctcatcttgg caggatttac 540 gccgccactg gccgaaggca agaagtggaa ggaatcggcc gtctcccca gcgtcccagc 600 tccggctgcc ctggctgccg ccgctcacgg acaatctagt tgtacaaaag gctctctggg 660 ctgcactgct ttcgaagaac ggcccaaagt atctcggtcc tgggcctggg cagccaagga 720 gaggggcggc cagtcttggc tcgtcccgaa gtgcccgccc cgccccctct cgctgcagca 780 gccgcctcct ctcccgtagc cctgcgggcc gctcttcact gctctccaga cttggggccc 840 tatctgaggc gtcccaaaca ccaacttctg gctcctggcc ccaactcgag aggcttccag 900 cgaggacgaa ggcaggctcg agaaacct ggcgggccag cagatccggg aggccggggct 960 ggaggcggcg gcggatttga agggaggaga cacttactgg gatcgatggg gggcttgtct 1020 ccgccgctct cattctcagc attgttttca gagaaggcgc cttcgctggg ttgtttttct 1080 ctatcaactg gaggaacc aaagcatag tcagtcaggg acaaagtgtg agtgtcaagc 1140 gtgggacagt caccccttct ggccgacagc ggttcaggtt taatgccata aggccggctg 1200 gagggcaagc ccgcgaagga gagcgcaccg ggcgtgggct ccagccagga gcgcatgtac 1260 ctgccgtccg gcgccgccgc cgccacgggc gcctgggggt gcacgtaggg gtggtggtga 1320 tggtggtggt acaccgcagc gggtacagcg ttggcgcccg ccgcgtgcac tgggttccac 1380 gaggcgccaa acaccgtcgc cttggactgg aagctgcacg ggctgaagtc ggggtgctcg 1440 gccagcgtcg ccgcctgccg gggaggctgg cccagggtcc ccggcgcata gcggccaacg 1500 ctcagctcat ccgcggcgtc ggcgcccagc aggaacgagt ccacgtagta gttgcccagg 1560 gccccagtgg tggccatcac cgtgcccagc gcctggcccg cccggcccga cccacggaaa 1620 ttatgaaact gcagatttca tgtaacaact tggtggcacc gggggggaag tacagtcacc 1680 taataagttg ccggcgcccg cgcccccatt ggccgtgcgc gtcacgtgcc cgtccagcag 1740 aacaataacg cgtaaatcac tccgcacgct attaatggtc cgatgttttg cagtcataat 1800 ttttatagca aaagccatat gtttttatgt aaagggatcg tgccgctcta cgatggggtt 1860 tgttttaatt gtggccaacg acgattaaaa gatcaatct agccttgtct ctgtactctc 1920 ccgtctcccc cccatacac acactctta agcggactat ttatatcac attaatcac 1980 gccatcaaga aggcgggggt cccgcgtgcg agtgcggcca gcggagcccc tcacataaaa 2040 ttagacaata attgaagcca taaaaaagca gccaaatcgc attgtcgctc tactgtattt 2100 aaatctatat ttatgatatt tcataaggag ttattgttc agaagccaca caggctggcg 2160 ggaagtcgga aacgaccac agatcgttt gcctcgccgt ggctcccagc tgtaaaaatt 2220 tacgaggact tggaaggtt agactgttgt gtttggttgg cgagctccct gtaaataatc 2280 cctgcggtcc ccgggagagg cgagtttacc cgcggccgcc ctcgaaagt caattcac 2340 gcaggatccg tccaaacgg agccgccgcc gccctacca gggcactcca ggcagggacc 2400 ggccgctcag ggagtaccgc gggtgtaggt cccacagct acccgctgg agcgaggggc 2460 gccccgggca cccttaatt cgccttttgct acgaggaccc cacggaggag ctggccagga 2520 gggagcggcc agccgccacc agggcgaagg ttttgaggc ctggttggtt gtgcggcgcg 2580 ctcggtcccc ggccctcgac cccacgcaca cgcgcgccca gcccgccttt ctcatcagct 2640 ggcaatcagg attcccaggc gcaggcggct ggcgacccag ccctgtgctc cagcctcaga 2700 ggctctaacc atgagcgctg caagcctggt tgcgctccgt gaatcccagc tggggaaaaa 2760 actacaagtg gcatgaatgg aaggcaagtt cggtttggga aaaggcagcc tcgcctaaga 2820 gaccccgcag ctccggaacc tgggaggccc gcaccgatgt ggcctgtccc ggggccgcgt 2880 gagcctttca gggctccttc 2900 <210> 2 <211> 5500 <212> DNA <213> Artificial Sequence <400> 2 tgggttaggt tggtaggatt tcgcgggggt ggcgggcggt gcgaggatgt agggttttgt 60 ttttttgtaa gtttttaaaa tgtttgtttg tatggttacg ttgggtcggt ggggttttta 120 tttttttttt tagagtttgt ttagaaggcg gtttttagtt ttttattttg ttattttttt 180 tttttttttt ttattaagtt aacgaaggtt taggtttggg aattttttcg ggggtattaa 240 ggtgagagtg gtttttttat tgttattggt tagattagat tagtttttta ggggtttttt 300 cgtcgtttcg tatatagata taaggtcggg agtttggttt tttcgtcggg ttttttagtt 360 ttttttcgtc gttaggtagt tggtttgttt gggagagttt agttcggcgg attggttgtg 420 gtaggtttcg tttttgaaag ttagttagag gtaaaattgg ggttttttt tattatttta 480 attcgtttt atttagagat tttatatagg tttttttgat ttagaagaaga aggtgtaaag 540 agtagattcg taaaaaatag aaaagaatta atatatttta tttggtaaaa agttaaatat 600 tattttaata taataatttg gttagtaggt tttgaggtaa ttattgtaaa atatatagtg 660 taagtttagt ttgatggaaa tttagattt attaaggata taaatttata gtagtttaat 720 ggcggtttta tagtgtataa tttatatta ataaaattaa tttcgttata attagtattt 780 attttttta attaaaatta agtataaatt ttaggtagta attttttgta tatatgtata 840 gtttcgaatt tttttattgt tcgtttggtg taaaaataat atttaagttt gtttgattat 900 gtatattttt tttaattata tagattatat atataataga taagatagga ttatatagat 960 aatggataga tttaaatgtt cgtattttta aggtggagaa aatgatgaat ttatgtattt 1020 tcgagaatat ttaaaattttt atttttttt attgggaat ttttagtt attttataat 1080 1140 aattgtttt ttttttatt aaataatttg agttttaattt gatttttatt ttttattatt 1200 attaatgggtg tattattag tattagag tttgaattat aaaatataat tattaata 1260 aaatagtg tttaagtatt ttggtagt aatttgtcgt aaattgta aattaatta 1320 attatataga tattaaaatta tattatagt tgtgtattat aaaaggtag 1380 tttttttt ttgttttaa gttaggaata ggtagatttt taaaaata tatataagtt 1440 attatatatta gttattagta ttaatgtttt ttttttttttttttagaaa 1500 atggaaagtt tataatatttt ttttatta accggtaggt tacgagtta gtttgaatag 1560 ggtttgttt ggaaaagagatg tggtttgagg tttagagtcg tttgtgcgg ggatggtgga 1620 ggttagggtg ggggtgagag gcggaagggg gcggatagttttttttt 1680 ttttttagtt tttttttt ttttaataa gtttaaatgg tattattcgt ttttgttcg 1740 gtttttgttg atttttttta tttttatttt gcggttttgg aattagattt tgatttgttt 1800 ttcggtgagg ttgagtagtc gagttatttc gtatttgcgg tttttggtga ggtatatgtt 1860 gaatagaaat ttttttttta gttttagggt ttggtgtttt gtataggggt atcgtttttt 1920 tcgagtggag cgcgtatgaa gttagttggt tgttgggtta tttgcgggga agagaaatat 1980 tgggtttagg agtagaagat gtatatttcg ttggggtaaa tgagtttttt gtatggggtt 2040 tttggtcgaa gtgtagaatt ttgttgaatt ggttaggaaa ggtagttagg tttcggatat 2100 aatggaattt tggtagatag acgtatagat agttatttaa aattgtacgt agtaaaattt 2160 tggttcgttt tttttttcga gatttttttt ttttttattt tgtttttttg tttttttttt 2220 tttttttttt atttttaaaa cgttgtatag aatgaaattt ggaaataggt ttttttgtta 2280 cgtagaggga aagtatgttg ttttgtgttt tgtagtaaga ttaggatttt tttgtttcgt 2340 ttattgattt cgtttttttt ttttaatttg tttttttacg ttttttattt tttatttaat 2400 ttttttggaa ggttttcgga gttgggtaag tcgttagggc gttttaaggt cgttgattac 2460 gtttgtggtt tatttgaata atttgttatg gggatttttg tggttcgggt cgttcgtagt 2520 tttatttgg taggatttac gtcgttattg gtcgaaggta agaagtggaa ggaatcggtc 2580 gtttttttta gcgttttagt ttcggttgtt ttggttgtcg tcgtttacgg ataatttagt 2640 tgtataaaag gttttttggg ttgtattgtt ttcgaagaac ggtttaaagt attcggttt 2700 tgggtttggg tagttaagga gaggggcggt tagtttggt tcgtttcgaa gtgttcgttt 2760 cgtttttttt cgttgtagta gtcgtttttt ttttcgtagt tttgcgggtc gttttttatt 2820 gttttttaga tttggggttt tatttgaggt gttttaaata ttaatttttg gttttggtt 2880 ttaattcgag aggtttttag cgaggacgaa ggtaggttcg agagaaattt ggcgggttag 2940 tagattcggg aggtcggcgt ggaggcggcg gcggattttga agggaggaga tatttattgg 3000 gatcgatggg gggtttgttt tcgtcgttt tatttttagt attgttttta gagaaggcgt 3060 tttcgttggg ttgtttttt ttattaattg gaggagaatt ataagtatag ttagttaggg 3120 ataaagtgtg agtgttaagc gtgggatagt tattttttt ggtcgatagc ggtttaggtt 3180 taatgttata aggtcggttg gagggtaagt tcgcgaagga gagcgtatcg ggcgtgggtt 3240 ttagttagga gcgtatgtat ttgtcgttcg gcgtcgtcgt cgttacgggc gtttggggt 3300 gtacgtaggg gtggtggtga tggtggtggt atatcgtagc gggtatagcg ttggcgttcg 3360 tcgcgtgtat tgggttttac gaggcgttaa atatcgtcgt tttggattgg aagttgtacg 3420 ggttgaagtc ggggtgttcg gttagcgtcg tcgtttgtcg gggaggttgg tttagggttt 3480 tcggcgtata gcggttaacg tttagtttat tcgcggcgtc ggcgtttagt aggaacgagt 3540 ttacgtagta gttgtttagg gttttagtgg tggtattat cgtgtttagc gtttggttcg 3600 ttcggttcga tttacggaaa ttatgaaatt gtagatttta tgtaataatt tggtggtatc 3660 gggggggaag tatagttat taataagttg tcggcgttcg cgtttttatt ggtcgtgcgc 3720 gttacgtgtt cgtttagtag aataataacg cgtaaattat ttcgtacgtt attaatggtt 3780 cgatgttttg tagttataat ttttatagta aaagttatat gtttttatgt aaagggatcg 3840 tgtcgtttta cgatggggtt tgttttaatt gtggttaacg acgattaaaa gattaaattt 3900 agttttgttt ttgtatttt tcgtttttt tatat atatttttta agcggattat 3960 tttatattat aattaattac gttattaaga aggcgcgggt ttcgcgtgcg agtgcggtta 4020 gcggagttt ttatataaaa ttagataata attgaagtta taaaaaagta gttaaatcgt 4080 attgtcgttt tattgtattt aaatttat ttatgatat ttataaggag ttatgtttt 4140 agaagttata taggttggcg ggaagtcgga aacgattaat agattcgttt gtttcgtcgt 4200 ggtttttagt tgtaaaaatt tacgaggatt tggaaaggtt agattgttgt gtttggttgg 4260 cgagtttttt gtaataaatt tttgcggtt tcgggagagg cgagtttatt cgcggtcgtt 4320 ttcgaaaagt taaatttaac gtaggattcg ttttaaacgg agtcgtcgtc ggttttatta 4380 gggtatttta ggtagggatt ggtcgttag gggtatcgc gggtgtaggt ttttatagtt 4440 attcgtttgg agcgaggggc gttcgggtaa tttttaaatt cgttttgtt acgaggattt 4500 tacggaggag ttggttagga gggagcggtt agtcgttatt agggcgaagg ttttgagggt 4560 ttggttggtt gtgcggcgcg ttcggttttc ggttttcgat tttacgtata cgcgcgttta 4620 gttcgtttt tttattagtt ggtaattagg atttttaggc gtaggcggtt ggcgatttag 4680 ttttgtgttt tagtttaga ggttttaatt atgagcgttg taagtttggt tgcgtttcgt 4740 gaattttagt tggggaaaa attataagtg gtatgaatgg aaggtaagtt cggtttggga 4800 aaaggtagtt tcgtttaaga gatttcgtag tttcggaatt tggaggttt gtatcgatgt 4860 ggtttgtttc ggggtcgcgt gagttttta gggtttttt ttttttttt agttgttatt 4920 tcgggtttcg ttttggttat ttacggggtt cggagattcg gcggagaggt ataaggttta 4980 aagagaggta gttatagttt aaggttaggg ttggaaatta gaacggggag gggtaaagg 5040 gtatcgattt tagtttatt tttgggtttg gttacgttgg ggaagtttat tttttattcg 5100 ttggggtaa attaaaaggt cgtcgttatt tcgttaattg gaaggaaatt ttttttgttt 5160 ttaattttta atagaaagta gcgattttta gaataggggt aattaaattt acgtgtggat 5220 attgtgtttg taatagtgtg ttttttatta gtttatttt ttggcggcga ggttggcggt 5280 ttcgggcgtt tttattttt ttttttttt ttgtttttt tt 5340 attttttttt taaatatttt gatatatttt cggttggggat ttttaaatat tagcgaggtt gttaagtcgc gcggatatcg attgggtgtt tttttttgta ttcgcgtttg gaagaggga 5460 gtggtcgata tcgaatggtt tcgttttgtt <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 cgtgacgcgt acggttaatg g <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <400> 4 cttaataca ccgaaaaaaa aatacaatca c <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <400> 5 cgcggggcgtc ggtaatttat taggtg <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 aaagtttgtg tttagattgt gggtg <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <400> 7 tctcccttaa acttccctac caa 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 actccaaaac tacccaacac cccca 25 <210> 9 <211> 200 <212> DNA <213> Artificial Sequence <400> 9 gagggtaagt tcgcgaagga gagcgtatcg ggcgtgggtt ttagttagga gcgtatgtat 60 ttgtcgttcg gcgtcgtcgt cgttacgggc gtttgggggt gtacgtaggg gtggtggtga 120 tggtggtggt atatcgtagc gggtatagcg ttggcgttcg tcgcgtgtat tgggttttac 180 gaggcgttaa atatcgtcgt 200 <210> 10 <211> 322 <212> DNA <213> Artificial Sequence <400> 10 ttatgactgc aaaacatcgg accattaata gcgtgcggag tgatttacgc gttattgttc 60 tgctggacgg gcacgtgacg cgcacggcca atgggggcgc gggcgccggc aacttattag 120 gtgactgtac ttcccccccg gtgccaccaa gttgttacat gaaatctgca gtttcataat 180 ttccgtgggt cgggccgggc gggccaggcg ctgggcacgg tgatggccac cactggggcc 240 ctgggcaact actacgtgga ctcgttcctg ctgggcgccg acgccgcgga tgagctgagc 300 gttggccgct atgcgccggg ga 322 <210> 11 <211> 200 <212> DNA <213> Artificial Sequence <400> 11 cgagtttatt cgcggtcgtt ttcgaaaagt taaatttaac gtaggattcg ttttaaacgg 60 agtcgtcgtc ggttttatta gggtatttta ggtagggatt ggtcgtttag ggagtatcgc 120 gggtgtaggt ttttatagtt attcgtttgg agcgaggggc gttcgggtaa tttttaaatt 180 cgtttttgtt acgaggattt 200 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 cggcgtcgtc gtcgttacg 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 gttcggcgtc gtcgtcgtta 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 gcgtttgggg gtgtacgtag 20 <210> 15 <211> twenty four <212> DNA <213> Artificial Sequence <400> 15 cgtaggggtg gtggtgatgg tggt 24 <210> 16 <211> twenty four <212> DNA <213> Artificial Sequence <400> 16 gtaggggtgg tggtgatggt ggtg 24 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <400> 17 taccaccacc atcaccacca c 21 <210> 18 <211> twenty one <212> DNA <213> Artificial Sequence <400> 18 ccaacgctat acccgctacg a 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 acccaataca cgcgacgaac 20 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <400> 20 ggttaatggg ggcgcgggcg tc 22 <210> twenty one <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty one gttggacggg tacgtgacgc g 21 <210> twenty two <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty two aacgcctaac ccgcccgacc cg 22 <210> twenty three <211> 30 <212> DNA <213> Artificial Sequence <400> twenty three caaatttcat ataacaactt aataacaccg 30 <210> twenty four <211> 28 <212> DNA <213> Artificial Sequence <400> twenty four ccacgaaaat tataaaacta caaatttc 28 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <400> 25 attaggtgat tgtatttttttttcggtg 28 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <400> 26 cggttaatgg gggcgcgggc gtcgg 25 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <400> 27 aacggagtcg tcgtcggt 18 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <400> 28 gagtcgtcgt cggttttat agggta 26 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <400> 29 tactccctaa acgaccaatc cctacc 26 <210> 30 <211> twenty four <212> DNA <213> Artificial Sequence <400> 30 taactataaa aacctacacc cgcg 24 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <400> 31 aataactata aaaacctaca cccgc 25 <210> 32 <211> 324 <212> DNA <213> Artificial Sequence <400> 32 ttatgattgt aaaatattgg attattaata gtgtgtggag tgatttatgt gttattgttt 60 tgttggatgg gtatgtgatg tgtatggtta atgggggtgt gggtgttggt aatttattag 120 gtgattgtat tttttttttg gtgttattaa gttgttatat gaaatttgta gttttataat 180 ttttgtgggt tgggttgggt gggttaggtg ttgggtatgg tgatggttat tattggggtt 240 ttgggtaatt attatgtgga tttgtttttg ttgggtgttg atgttgtgga tgagttgagt 300 gttggttgtt atgtgttggg gatt 324

Claims

1. Use of a HOXA9 gene methylation detection reagent in the preparation of a breast cancer diagnostic reagent or kit, characterized in that: The reagent includes a forward primer sequence as shown in SEQ ID NO.28 and a reverse primer sequence as shown in SEQ ID NO.

30.

2. The use according to claim 1, characterized in that: The HOXA9 gene methylation detection reagent is used to detect the sequence of the HOXA9 gene modified by the conversion reagent.

3. The use according to claim 2, characterized in that: The conversion reagent is selected from one or more of hydrazine salts, bisulfite and bisulfite.

4. The use according to claim 3, characterized in that: The conversion reagent is bisulfite.

5. The use according to claim 1, characterized in that: The detection region of the HOXA9 gene targeted by the HOXA9 gene methylation detection reagent is the HOXA9 gene or its promoter region.

6. The use according to claim 5, characterized in that: The detection region of the HOXA9 gene targeted by the HOXA9 gene methylation detection reagent is the sequence shown in SEQ ID NO.

11.

7. The use according to claim 1, characterized in that: The reagent also includes a fluorescent probe sequence whose nucleotide sequence is shown as SEQ ID NO.

29.

8. The use according to claim 1, characterized in that: The methylation detection reagent also includes a detection reagent for an internal reference gene.

9. The use according to claim 8, characterized in that: The internal reference gene is GAPDH.

10. The use according to claim 8, characterized in that: The detection reagent of the internal reference gene contains primers and probes for the internal reference gene; the detection reagent of the internal reference gene GAPDH contains the primer pair shown in SEQ ID NO.6 and SEQ ID NO.7, and the probe shown in SEQ ID NO.

8.

11. The use according to claim 1, characterized in that: The HOXA9 gene methylation detection reagent also includes DNA polymerase, dNTPs, Mg 2+ One or more of ions and buffers.

12. The use according to claim 11, characterized in that: The HOXA9 gene methylation detection reagent contains DNA polymerase, dNTPs, Mg 2+ Ions and buffers are used for amplification of the HOXA9 gene.

13. The use according to claim 1, characterized in that: The detection sample of the HOXA9 gene methylation detection reagent is selected from breast tissue, blood, serum or plasma.

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