CN111363819A - Method for jointly detecting and diagnosing breast cancer by utilizing ddPCR technology - Google Patents
Method for jointly detecting and diagnosing breast cancer by utilizing ddPCR technology Download PDFInfo
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Abstract
The invention discloses a method for jointly detecting and diagnosing breast cancer by utilizing ddPCR technology, which specifically comprises the following steps: s1, extracting genome DNA and miRNA in a sample, S2, modifying and purifying and quantifying DNA sulfite, carrying out reverse transcription on the miRNA into cDNA, S3, respectively carrying out ddPCR by using the purified DNA genome as a template and specific primers as primers to detect the methylation state of MASPIN, S4, calculating the methylation degree W of an upstream regulatory region CpG island of a MASPIN coding sequence of the sample to be detected, and carrying out combined analysis with the miR-142 content in the sample. The method for jointly detecting and diagnosing the breast cancer by utilizing the ddPCR technology can realize the joint detection of two indexes, namely the methylation state of the maspin gene and the miR-142 content, so as to evaluate the basis of whether a patient is changed with the breast cancer, has more accurate data and more reliable result, can be widely applied to early screening of the breast cancer, and effectively assists in judging the malignancy degree and whether the breast cancer is transferred.
Description
Technical Field
The invention relates to the technical field of biology and disease diagnosis, in particular to a method for diagnosing breast cancer by detecting the methylation state of a gene maspin and the relative content of miR-142 by combining a ddPCR (polymerase chain reaction) technology and methylation specific PCR (polymerase chain reaction).
Background
Currently, the most common imaging methods for breast cancer screening are molybdenum target X-ray and ultrasound. The mammary gland molybdenum target is called mammary gland molybdenum target X-ray radiographic examination, also called molybdenum palladium examination, is the first choice and the simplest and most reliable noninvasive detection means for diagnosing mammary gland diseases at present, has relatively little pain, is simple and easy to operate, has high resolution and good repeatability, can be used for comparing the retained images before and after, is not limited by age and body form, and is used as the conventional examination at present. The kit is characterized in that the kit can detect breast lumps which cannot be touched by doctors, particularly large breasts and fat type breasts, the diagnosis performance can reach 95 percent, and for T0-stage breast cancer (negative in clinic) which is only expressed by little micro calcification, the breast cancer can be detected and diagnosed early only by soft X-ray examination, the diagnosis sensitivity to the breast cancer is 82-89 percent, and the specificity is 87-94 percent. But the molybdenum target of the breast has low sensitivity to dense breast with higher tumor risk. The ultrasonic examination is simple and easy to implement, and is sensitive to X-ray examination of infiltrative breast cancer compared with molybdenum target. With the rapid development and application of new ultrasonic imaging technologies such as ultrasonic elastography, ultrasonic radiography, ultrasonic three-dimensional imaging and the like, ultrasonic examination plays an increasingly important role in the early diagnosis of breast cancer, and with the deep research on breast cancer, the existing detection and diagnosis technologies such as molybdenum target X-ray, ultrasonic examination and the like still have the following obvious short plates:
(1) the occurrence of early in situ breast cancer cannot be detected, i.e. it is difficult to detect when no significant tumor is formed or the tumor mass is small in the early stages of breast cancer occurrence.
(2) It is difficult to predict whether breast cancer will metastasize.
(3) Early metastasis of breast cancer is difficult to detect.
In a word, the existing detection technology can not meet the requirements of a clinician on preoperative pathological histology grading and estimation of tumor molecular typing of a tumor patient.
Loss of control of proliferation and resistance to apoptosis/necrosis are considered markers for many cancer types, including breast cancer. After the primary tumor has formed, metastasis may occur whereby cells acquire the ability to migrate, invade surrounding tissues, enter the blood and lymphatic system, then extravasate and colonize secondary sites. Recurrence at the site of metastasis is a major cause of death in breast cancer patients.
Maspin is a serine protease inhibitor, a member of the serpin family of protease inhibitors, and acts as a tumor suppressor, and Maspin has been shown to inhibit cell motility, invasion, and metastasis. Loss of maspin gene expression is a common event in breast cancer, leading to increased invasive potential and the spread of metastatic disease. The loss of maspin gene expression in breast cancer cells is not due to loss or rearrangement of the maspin gene, but rather due to factors that regulate maspin expression being disrupted during cancer progression. Aberrant cytosine methylation of CpG dinucleotides in tumor suppressor gene promoters is often associated with their oncogenic effects and changes in chromatin structure during cancer progression and their transcriptional silencing. Studies have shown that DNA hypermethylation leads to silencing of the maspin gene in human breast cancer.
miR-142 is one of miRNA family members with abnormal expression in various malignant tumors and is supposed to play an important role in the occurrence and development of the malignant tumors. Research shows that miR-142 expression in breast cancer tissues is lower than that in paracancerous tissues, and miR-142 expression in breast cancer cells MCF-7, MDA-MB-231 and MDA-MB-468 is lower than that in normal mammary epithelial cells HBL-100. The results show that the abnormal expression of miR-142 is related to the occurrence of breast cancer, and can play a role in inhibiting cancer genes in the occurrence of the breast cancer.
The technology uses a method for diagnosing the breast cancer by combining a ddPCR technology with methylation specificity PCR detection gene maspin methylation state and miR-142 relative content as a basis for detecting the breast cancer, and compared with the traditional diagnosis method, the method has the advantages of more accurate data and more reliable result, and compared with the published diagnosis method, the method has more reliable result.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for diagnosing breast cancer by utilizing ddPCR technology combined detection, which has more accurate data and more reliable result compared with the traditional diagnosis method, and has more reliable result compared with the published diagnosis method.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for diagnosing breast cancer by utilizing ddPCR technology combined detection specifically comprises the following steps:
s1, extracting genome DNA and miRNA in the sample: extracting total DNA in a sample by adopting a commercial genome extraction kit, extracting total miRNA in the sample by using a commercial miRNA extraction kit, determining the concentration of the DNA and the miRNA, and storing at-80 ℃ for later use;
s2, DNA sulfite modification and purification quantification, and miRNA is reversely transcribed into cDNA: dissolving 2 mu g of genome DNA in 50 mu L of triple distilled water, adding 5.5 mu L of NaOH, carrying out denaturation at 37 ℃ for 20min, then respectively adding newly prepared 30 mu L of hydroquinone and 520 mu L of sodium bisulfite, carrying out water bath at 50 ℃ for 16h, then purifying the DNA by a commercial DNA purification kit through a column, dissolving the DNA in 50 mu L of sterilized deionized water after the DNA passes through the column, then adding 5 mu L of NaOH for desulfurization, 1 mu L of glycogen, carrying out ethanol precipitation, re-dissolving the DNA in 20 mu L of TE solution, and then storing the DNA at-20 ℃ for later use;
s3, ddPCR with purified DNA genome as template, specific primers as primers, respectively, to detect MASPIN methylation status, ddPCR with cDNA after miRNA reverse transcription as template, miR-142 specific primers as primers, to detect miR-142 content in the sample, PCR reaction performed for a total volume of 20 μ Ι _, with 8 μ Ι _ diluted sample template, 10 μ Ι _, 2 × ddPCR Supermix (without dUTP), 1 μ Ι _, 20 × primer mix of targets (FAM), 1 μ Ι _, 20 × reference primer mix, after PCR amplification, the sample was placed in a droplet reader, which analyzed each droplet separately using QuantaSoft detection system, raw fluorescence amplitude data was extracted from droplet reader using QuantaSoft, QuantaSoft software counted PCR and PCR droplet negatives, and target DNA concentration in the raw sample was determined by Poisson statistics, in PCR, target molecules were randomly distributed into droplets, in any single color, there was one or more than one drop, one drop was counted as positive and PCR droplet negative, and if there was not more than one drop, the target molecule was found to be a drop, the target molecule was not found to be a drop, the occurrence was not found to be a negative, the same as if there was not a drop, the target molecule was not found to be a drop, the target molecule present, the target molecule was not found to be a drop, the target molecule present, the target molecule was not found to be a drop was not present, the drop was not present, the threshold, the drop was not present, the threshold, the negative, the drop was not present, the negative drop was not present, the negative drop was not present.
S4, calculating the methylation degree W of the CpG island in the upstream regulatory region of the maspin coding sequence of the sample to be detected, and carrying out combined analysis with the miR-142 content in the sample.
Preferably, the concentration of NaOH added for the denaturation in the step S2 is 2mol/L, and the concentration of hydroquinone is 10 mmol/L.
Preferably, the concentration of sodium bisulfite in the step S2 is 3mol/L, and the pH of the sodium bisulfite is 5.0.
Preferably, the concentration of NaOH added for desulfurization in the step S2 is 3mol/L, and the concentration of glycogen added is 20 mg/ml.
Preferably, the experiment is performed in step S2 with a 10 μ L RT reaction system: 1 μ g of RNA, 1 μ L of 5M mirnarart primer, 2 μ L, RtaseMix2 μ L of 5X reverse transcription buffer, and RNase-freeH2And supplementing 10 mu L of O, mixing the system uniformly, performing instantaneous centrifugation, and storing at-20 ℃ for later use.
Preferably, the RT reaction procedure is: 60min at 42 ℃ and 10min at 70 ℃.
Preferably, the thermal cycling conditions in step S3 include an activation period of 10 minutes at 95 deg.C, followed by a two-step thermal profile of 40 cycles including denaturation at 94 deg.C for 30 seconds and co-annealing and extension at 55 deg.C for 1 minute, with the enzyme of the last cycle being inactivated at 98 deg.C for 10 minutes.
Preferably, in step S4, when the copy number is 30% ≦ W and the relative copy number of miR-142 is less than 1, the sample to be tested is likely to have breast cancer, and W ═ maspin methylated copy number/(maspin methylated copy number + maspin unmethylated copy number).
Methylation-specific PCR is a simple, specific and sensitive way to detect single-gene methylation. The basic principle is to treat genomic DNA with sodium bisulfite, whereby unmethylated cytosine is converted to uracil and methylated cytosine is not, and then to amplify the same nucleotide sequence of the gene to be tested using specific primers.
The use of droplet digital pcr (ddpcr) is a new method for absolute quantification of nucleic acids. The method uses a water-oil emulsion droplet system, where the sample is fractionated into 20000 droplets. Each nanoliter-sized droplet forms a partition that separates template molecules that have substantially the same function as the individual wells in the plate where the PCR reaction takes place. In any single droplet, at least one target molecule is present or not present at all. The number of positive droplets then reflects the abundance of the target molecule in the sample. ddPCR uses similar assay reagents and workflow as used in standard simulation systems, but calculates the total number of individual target molecules in digital format without the use of a calibration curve. Sample distribution and data collection at the end of the reaction offers the advantage of direct and independent nucleic acid quantification without the need for calibration curves, providing more accurate and reproducible data than real-time PCR.
(III) advantageous effects
The invention provides a method for jointly detecting and diagnosing breast cancer by utilizing a ddPCR (double-stranded polymerase chain reaction) technology. Compared with the prior art, the method has the following beneficial effects:
(1) the method for jointly detecting and diagnosing the breast cancer by utilizing the ddPCR technology can realize that the methylation of the CpG island in the gene promoter region is usually detected by applying the methylation specific PCR (MS-PCR) technology at present, the high sensitivity of the method enables the method to be used for methylation detection of trace templates in serum or plasma samples, the method is a method which is mostly adopted at home and abroad at present, the MSP method is a qualitative detection method rather than a quantitative detection method, and is a very sensitive technology, and the characteristic enables the method to detect extremely trace samples.
(2) The method for jointly detecting and diagnosing the breast cancer by utilizing the ddPCR technology carries out the quantification of nucleic acid molecules by a novel mode through the droplet-type digital PCR, compared with the traditional PCR and the quantitative PCR, the precision, the accuracy and the sensitivity of the result are better, the quantification result does not depend on a Ct value any more, the initial concentration of a target sequence is directly given, the absolute quantification in the real sense is realized, a droplet generator forms a reaction system containing the nucleic acid molecules into droplets with thousands of nano-scale, wherein each droplet does not contain the nucleic acid target molecules to be detected or contains one to a plurality of nucleic acid target molecules to be detected, each droplet is taken as an independent PCR reactor, after the PCR amplification, a droplet analyzer is adopted to detect each droplet one by one, the droplet with a fluorescent signal is judged to be 1, the droplet without the fluorescent signal is judged to be 0, this technique is therefore called "digital PCR" and ultimately, based on the poisson distribution principle and the proportion of positive droplets, the analysis software can calculate the concentration or copy number given the target molecule to be detected.
(3) According to the method for jointly detecting and diagnosing the breast cancer by utilizing the ddPCR technology, the two technologies are combined to detect the methylation state of maspin, so that the qualitative and quantitative detection can be realized, a PCR system and a PCR program are optimized, and then specific primers are combined, so that the accuracy of a detection result is ensured.
(4) The method for jointly detecting and diagnosing the breast cancer by utilizing the ddPCR technology is used for evaluating the basis of whether a patient is changed with breast cancer or not by jointly detecting the methylation state of the maspin gene and the miR-142 content, has more accurate data and more reliable result, can be widely applied to early screening of the breast cancer, and effectively assists in judging the malignancy degree and metastasis of the breast cancer.
Drawings
FIG. 1 is a test result chart of a practical example of the present invention;
FIG. 2 is a comparison of the gene sequences of the maspin CpG islands of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-2, an embodiment of the present invention provides a technical solution: a method for diagnosing breast cancer by utilizing ddPCR technology combined detection specifically comprises the following steps:
s1, extracting genome DNA and miRNA in the sample: extracting total DNA in a sample by adopting a commercial genome extraction kit, extracting total miRNA in the sample by using a commercial miRNA extraction kit, determining the concentration of the DNA and the miRNA, and storing at-80 ℃ for later use;
s2, DNA sulfite modification and purification quantification, and miRNA is reversely transcribed into cDNA: mu.g of genomic DNA was first dissolved in 50. mu.L of triple distilled water, 5.5. mu.L of NaOH was added, denaturation was carried out at 37 ℃ for 20min, then newly prepared 30. mu.L of hydroquinone and 520. mu.L of sodium bisulfite were added, respectively, and water bath was carried out at 50 ℃ for 16h, after which the DNA was column-purified using a commercial DNA purification kit,after passing through the column, the DNA was dissolved in 50. mu.L of sterilized deionized water, then 5. mu.L of NaOH was added for desulfurization, 1. mu.L of glycogen was added, and then precipitated by ethanol, redissolved in 20. mu.L of TE solution, and then stored at-20 ℃ for later use, the concentration of NaOH added for denaturation was 2mol/L, and the concentration of hydroquinone was 10mmol/L, the concentration of sodium bisulfite was 3mol/L, and the pH of sodium bisulfite was 5.0, the concentration of NaOH added for desulfurization was 3mol/L, and the concentration of glycogen was 20mg/ml, and the experiment was carried out in a 10. mu.L RT reaction system: 1 μ g of RNA, 1 μ L of 5MmiRNART primer, 2 μ L, RtaseMix2 μ L of 5X reverse transcription buffer, and RNase-freeH2And supplementing 10 mu L of O, mixing the system uniformly, performing instantaneous centrifugation, and storing at-20 ℃ for later use, wherein the RT reaction procedure is as follows: 60min at 42 ℃ and 10min at 70 ℃;
s3, ddPCR with purified DNA genome as template, specific primers as primers, respectively, for detecting MASPIN methylation status, ddPCR with cDNA after miRNA reverse transcription as template, miR-142 specific primers as primers, for detecting miR-142 content in the sample, PCR reaction performed for a total volume of 20 μ Ι _, with 8 μ Ι _ diluted sample template, 10 μ Ι _, 2 × ddPCR Supermix (without dUTP), 1 μ Ι _, 20 × primer mix of targets (FAM), 1 μ Ι _, 20 × reference primer mix, after PCR amplification, the sample was placed in a droplet reader, which analyzed each droplet separately using QuantaSoft detection system, raw fluorescence amplitude data was extracted from droplet reader using QuantaSoft, QuantaSoft software counted PCR and PCR droplet negatives, and target DNA concentration in the raw sample was determined by Poisson statistics, in PCR, target molecules were randomly distributed into droplets, in any single color, there was one or more than one Poisson droplet, one positive and PCR droplet negative was counted, and if there was not more than one Poisson, the target molecule was not able to be detected by a single PCR cycle, if there was not more than one cycle, no PCR reaction occurred, no PCR reaction was not less than 1S, no more than 1S, no PCR cycle, no more than a PCR cycle, no PCR cycle was determined to occur, no PCR cycle, no more than a PCR cycle, no cycle was able to occur, no cycle was able to be a PCR cycle, no cycle was able to occur, no cycle was determined to be a cycle, no cycle was able to occur, no cycle was able to occur.
S4, calculating the methylation degree W of the CpG island in the upstream regulation and control region of the maspin coding sequence of the sample to be detected, and carrying out joint analysis with the content of miR-142 in the sample, wherein when the methylation degree W is more than or equal to 30% and the relative copy number of miR-142 is less than 1, the sample to be detected is likely to have breast cancer, and W is the methylation copy number of maspin/(the methylation copy number of maspin + the unmethylated copy number of maspin).
The miR-142 primer sequence is as follows:
Hsa-miR-142RealtimeF：GTACCGTCATAAAGTATAAA
Hsa-miR-142RealtimeR:AGTGCAGGGTCCGAGGTATTC
Hsa-U6RealtimeF:CTCGCTTCGGCAGCACA
Hsa-U6RealtimeR:AACGCTTCACGAATTTGCGT
the gene sequence of MASPINCPG island is as follows:
CATTTCCTTCAGGATGTATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTAC AGTATAACAATTAGTGTAGGTATCTGACTTATAGAAATGCAAAGCTAAAC TGTAGTCGGCTTGAGATCCATAGTTAGGTCTTATTCTGGGTCTTGCCCAG GGTCTGACACACTCAAAAAATGCTTATTCAGTCAATCACAGACTCCAAT CAAAATGTCCAGTTAAAACAGGGAAGCAGAGTAGAAACCTATTGAATGC AGAAGACTTTATTGAGGCCACTCTTTCCTAAAATTAATCAGCAGGCATGT TCTCCAGAAACTAGTTTGAAATTCTGTAATTTAAAAAATGTGAACTGCTT GTATAACTCCATCCTCTCCCACTTTTCTGTTTTCTAGTGTCTTTTTAGGTA GAATTCTAAAGAACTTAAAATTTTGGCTGCCGTGGAAAAGAATATTACTG GGAAATGGGTCTTCTCAGTCAAATAGAATCACAGTTTTAATACTCTTTCC ACTGAATGGGAACCACTTTTTTTTTTCCTGCAATTTTGCCTAAATCAACA GTGTGTTTTGTCAATGATGTTACACCTGAGACTTGTGAGTCCTTGCCTTA CTTGGGTTCAATGAGAGGGTGAGAACATTCACGTACAGGGTCTGACCTC CAGAGATACTTCAACAAGTGAAAGCTTTTGACAGGAAATGTTCTGTTTA ATCTTTTGTTAACATATCTGTCCAATATACCTTGTATCATCCCAGCTCTGG ACCTCCGTTCTCAGGCTTAAATGGTCTCCTGATGTCCAATGATGCTGCCT CCATGATCCTTCCCCAGTCATAGCCAGACTGAAAAGGTCACACCCATCT CTGAACTACTTTTGTCATTTGCACCTCTCTTAAGTCCCTTATTACATGCCT CCAGATACTTTCTTTTGTTTCTTGTCTTCCTACTGTACCCTTTGGGGAGAG GGAACATGTCTTATAGCCCCAGTCGAATGGTGCCTCTAATTTTCACCCAG TTAGAATCACCCAAAGGCATTTCACTTTGCTAGAACTTCCTTGGGTATTG TTGAATTCCTGTCATTGGAAATGTTTCTGCCCCGGTTGAGCAAGCACTGA ACAGGAGTGTTTTAGAGGGAATTAAAACTTCAAAAAGGAGAATAGAGA GGCTCTACCATTTAAATGCAAGTCGCTTGAAATCTGTAGACCACAGTAGC AAGCACGAAGCAGGTATTCAATAAATGTGGTGAATTCAATGTCAATAACA TGCTCCAGTCTTTTGTCCACTATTCCCCGAGCCTACCTAACCAATGGGCA AAGTGGTACTAATTGCCATCATTAGGAAGCACATGCTATGTGTCAGAAAG TATGTAAGAATTGTCTCTTCATCCATCTCTGTCTATATCTCATTTTATCGAT CTTATTGACAAATGAGAACTATATATAGATCTCATTTTACAGATGAGGAAT CTGAAGATAAAGGGATTAAGCAATTTGCTCAGGATTTCTCAAGCAATGA GTGGTTAGACTTAGTTTGCAAATTTATGACTCTGCTCCCAAATTCTCATTT TCCCTCCTACAGCTGGATAATAGAATTTTTAAGAAGCATCCTTCCACACC TCCTCACCTATGACCTTCCCTCCACTTGCTCCACTGATGCCTAAAATTCT GTTTTTGGAGTTCACTTAGTTTATGACTTTGATTCTGGTTAACATTAAAAT ACCCAGGACATGGGGGAGGGGAAGCTGTTATCAATCTTAGTTTAGTCAG TTAATATAGCGAGTTGCTCTTATTCATTACTCAACAAGCACTTATTGACTG CATACTGTACATCAGGTCTGTACCAAGCTTTGGTGTTATAAAAATGAATG AAGTTTGGCACTTACCTTTAACATTGTTGTAGTCACATATAGGAGAATGT AAGAGATTATTATGAAACAATGTAGTAAATGTACAATAGGGAATTCTAGA TAAGCACAGCAGAGAAGCAACCAGCTCCGTTTCAGGTCCTTTCCTGAG ACTGATTCGGCTGGAAGGGAGTAGGTCCCGCCAAATGAAGAAGCTGTG GGAAGACAGGAGGACAAGAACAGGCTCCACGAAGAGATTTCAGAGCA GAGCTGCGTACTCCTTTTTCTTTTTGTTTCTTTTGCTCTGTCACCCAGGCT GAAGTACAGTGGTTAGCTCACGGCTCACTGCAGCTTTGACCTCCCAGGC TCAAGTGATCCTCTCGTCTCAGCTTTCCAAGTAACTGGGACCACAGGCA TGCATCACCACACTAGGCTATTGTTTTACATTTTTTGTAGAGATGGGGTCT CACCATGTTGCCCAGGTTGGTCTCAAACTCCTGGGCTCAAGCAATCCGC TCACGTCAACCTCCCCAAATGCTGGGATTACAGGCGTGAGCCACCGCGC CAGGCCTGAGTAATCCTAATCACAGGATTTTAAAAAGAAACTTCCTGCG CCACCCATTAAACAATATCTCCTACCAATTTGGTAGTAAATATTTTGCTAA TAGTACCTAATTTTTAGGTAGGCACTGTGTTTATACATATATCCATTCCTTC TTTTTTGATTGTCTTTCTGTTTAATGGGCAGCTACCTCTCTTGGCATCTAG CAGAATGAGCTGCTGCAGTTTACACAAAAAGAATGGAGATCAGAGTACT TTTTGTGCCACCAACGTGTCTGAGAAATTTGTAGTGTTACTATCATCACA CATTACTTTTATTTCATCGAATATTTCACCTTCCGGTCCTGCGTGGGCCGA GAGGATTGCCGTACGCATGTCTGTACGTATGCATGTAACTCACAGCCCCT TCCTGCCCGAACATGTTGGAGGCCTTTTGGAAGCTGTGCAGACAACAGT AACTTCAGCCTGAATCATTTCTTTCAATTGTGGACAAGCTGCCAAGAGG CTTGAGTAGGAGAGGAGTGCCGCCGAGGCGGGGCGGGGCGGGGCGTG GAGCTGGGCTGGCAGTGGGCGTGGCGGTGCTGCCCAGGTGAGCCACCG CTGCTTCTGCCCAGACACGGTCGCCTCCACATCCAGGTCTTTGTGCTCCT CGCTTGCCTGTTCCTTTTCCACGCATTTTCCAGGATAACTGTGACTCCAG GCCCGCA。
application case
Selecting a species as human by using a genereporter tool, selecting a latest database, searching a maspin gene, selecting 3000bp and 5' UTR at the upstream of a promoter to obtain a sequence of an upstream regulation and control region of a maspin gene coding sequence, submitting and analyzing CpG island information in the sequence through a website, wherein the result is shown in figure 1, and the analysis shows that a CpG island exists between 2875 and 2976bp and the length is 102 bp.
According to the obtained information of the CpG island in the maspin upstream regulatory region, two sets of primers aiming at methylated and unmethylated sequences are designed by the code, and are shown in table 1.
TABLE 1 two sets of primer information tables for methylated and unmethylated sequences
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for jointly detecting and diagnosing breast cancer by utilizing ddPCR technology is characterized by comprising the following steps: the method specifically comprises the following steps:
s1, extracting genome DNA and miRNA in the sample: extracting total DNA in a sample by adopting a commercial genome extraction kit, extracting total miRNA in the sample by using a commercial miRNA extraction kit, determining the concentration of the DNA and the miRNA, and storing at-80 ℃ for later use;
s2, DNA sulfite modification and purification quantification, and miRNA is reversely transcribed into cDNA: dissolving 2 mu g of genome DNA in 50 mu L of triple distilled water, adding 5.5 mu L of NaOH, carrying out denaturation at 37 ℃ for 20min, then respectively adding newly prepared 30 mu L of hydroquinone and 520 mu L of sodium bisulfite, carrying out water bath at 50 ℃ for 16h, then purifying the DNA by a commercial DNA purification kit through a column, dissolving the DNA in 50 mu L of sterilized deionized water after the DNA passes through the column, then adding 5 mu L of NaOH for desulfurization, 1 mu L of glycogen, carrying out ethanol precipitation, re-dissolving the DNA in 20 mu L of TE solution, and then storing the DNA at-20 ℃ for later use;
s3, ddPCR with purified DNA genome as template and specific primers as primers to detect MASPIN methylation status, ddPCR with miRNA reverse transcribed cDNA as template and miR-142 specific primers as primers to detect miR-142 content in the sample, PCR reaction with total volume of 20 μ L, sample template with 8 μ L dilution, 10 μ L of 2 × ddPCR Supermix, 1 μ L of 20 × target primer mix (FAM), 1 μ L of 20 × reference primer mix, after PCR amplification, sample is placed in a droplet reader which analyzes each droplet separately using a two-color detection system, quantaSelection extracts raw fluorescence amplitude data from droplet reader, quantaSelection software counts PCR positive and PCR negative droplets and determines target DNA concentration in the raw sample by Poisson statistics, in ddPCR, target molecules are randomly distributed into droplets, in any single droplet, there is zero or more than one target molecule, Poisson 'S negative and PCR negative droplets are counted according to Poisson' S law, if there is a high target molecule distribution, if there is a multiple target molecule distribution, if there is no target molecule distribution, then there is a random, if there is a multiple target molecule distribution is a threshold, if there is no negative, then there is a target molecule distribution is a random, if there is a multiple target molecule distribution, if there is no target molecule distribution, then there is a random, if there is a multiple target molecule distribution is a threshold, if there is a random, then there is no target molecule distribution is a random, if there is a target molecule distribution, if there is a multiple target molecule distribution, if there is a match, then there is a multiple target molecule distribution is a target molecule distribution is.
S4, calculating the methylation degree W of the CpG island in the upstream regulatory region of the maspin coding sequence of the sample to be detected, and carrying out combined analysis with the miR-142 content in the sample.
2. The method for diagnosing breast cancer by using ddPCR technology in combination as claimed in claim 1, wherein: the concentration of NaOH added for denaturation in the step S2 was 2mol/L, and the concentration of hydroquinone was 10 mmol/L.
3. The method for diagnosing breast cancer by using ddPCR technology in combination as claimed in claim 1, wherein: the concentration of sodium bisulfite in the step S2 is 3mol/L, and the pH of the sodium bisulfite is 5.0.
4. The method for diagnosing breast cancer by using ddPCR technology in combination as claimed in claim 1, wherein: the concentration of NaOH added for desulfurization in the step S2 is 3mol/L, and the concentration of glycogen added is 20 mg/ml.
5. The method according to claim 1, wherein the step S2 is performed using 10 μ L RT reaction system including 1 μ g RNA, 1 μ L5M mirnarart primer, 2 μ L5 × reverse transcription buffer, 2 μ L Rtase Mix2 μ L and RNase-free H2Supplementing 10 mu L of O, mixing the system evenly, centrifuging instantaneously,and stored at-20 ℃ for later use.
6. The method for the combined detection and diagnosis of breast cancer using ddPCR technology as claimed in claim 5, wherein: the RT reaction procedure is as follows: 60min at 42 ℃ and 10min at 70 ℃.
7. The method for diagnosing breast cancer by using ddPCR technology in combination as claimed in claim 1, wherein: the thermal cycling conditions in step S3 included an activation period of 10 minutes at 95 deg.C, followed by a two-step thermal profile of 40 cycles including denaturation at 94 deg.C for 30 seconds and combined annealing and extension at 55 deg.C for 1 minute, with the enzyme of the last cycle being inactivated at 98 deg.C for 10 minutes.
8. The method for diagnosing breast cancer by using ddPCR technology in combination as claimed in claim 1, wherein: and in the step S4, when the copy number is not less than 30% and W and the relative copy number of miR-142 is less than 1, the sample to be detected is possibly breast cancer, and W is the methylated copy number of maspin/(methylated copy number of maspin + unmethylated copy number of maspin).
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