CN107868827A - Septin9 target gene DNA methylation assay primers and the kit for detecting people's septin9 gene methylations - Google Patents

Septin9 target gene DNA methylation assay primers and the kit for detecting people's septin9 gene methylations Download PDF

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CN107868827A
CN107868827A CN201711264908.3A CN201711264908A CN107868827A CN 107868827 A CN107868827 A CN 107868827A CN 201711264908 A CN201711264908 A CN 201711264908A CN 107868827 A CN107868827 A CN 107868827A
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septin9
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李同恩
罗玺
师鸿翔
陶然
刘拓
朱兵
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Abstract

The invention discloses a kind of septin9 target genes DNA methylation assay primer and the kit of detection people's septin9 gene methylations, primer includes the qualitative detection target gene Septin9 forward primer F and the reverse primer R without Loop with a Loop.In kit in addition to including above-mentioned primer, also include the Blocker sequences for being used for the reference gene primer pair for detecting sample quality, Taqman MGB probes and a PNA structure, kit application method is simple, as a result easy interpretation, while the pollution that stopped pipe reaction zone comes is also few.Compared with using specific primer and the simple method being identified using probe, preferable specificity can be brought by introducing the primer with Loop, the overall specificity of detection can be greatly improved by adding the Blocker of the PNA structures of closing non-specific sequences simultaneously, be appropriate for the early screening of plasma of colorectal cancer dissociative DNA.

Description

Septin9 target gene DNA methylation assay primers and detection people's septin9 gene methyl The kit of change
Technical field
The invention belongs to methylated genes molecular diagnosis field, and in particular to septin9 target gene DNA methylation assays draw Thing and the kit for detecting people's septin9 gene methylations.
Background technology
The carcinoma of the rectum is one of most common malignant tumour, and it is Colon and rectum that the U.S. in 2004, which there are about 146940 diagnosing patients, Cancer, 56730 are died from the disease, and the incidence of disease of domestic colorectal cancer is also in ascendant trend year by year, men and women's property Colorectal Cancer Rate occupies the 3rd.5 years survival rates of early stage colorectal cancer patients are about 80%, because colorectal cancer early stage is often without special disease Shape, on easy delay treatment opportunity, be only 10% or so to survival rate during late period.Before 2000, some American-European countries have just existed Promote the examination of colorectal cancer people at highest risk in the whole nation.
Show that north america 5 years survival rates of straight colorectal cancer patients are about 60%, and Chinese according to World Health Organization's statistics Corresponding only 32%.It is the order of severity of straight colon cancer life cycle and disease, by stages related, because China is in the diagnosis of straight colon cancer Level, common people's early diagnosis consciousness etc. weakness, more than 80% is middle and advanced stage patient, and early diagnosis ratio is only 11.8%.And have The early stage straight colorectal cancer patients of effect can make straight colon cancer morbidity reduce by 60%, and case fatality rate reduces by 80%.
At present, straight colon cancer non-invasive inspection methods are very low for the examination effective force of disease, since it is desired that suffering from Person collects the experiment of fecal sample fecal occult blood or Fecal Immunochemical fluoroscopic examination every year, and false positive rate is high, and as a result stability is poor, Overall examination rate is less than 3%.It is the gold of straight colon cancer inspection although sigmoidoscopy or colonoscopy recall rate are higher Standard, but need patient to do cleaning INTESTINAL CLEANSING, can produce the feared state of mind by anesthesia and invasion of privacy, patient, check Situations such as bleeding or infection, overall compliance is poor, and examination rate is less than 5%.Therefore, high specific, high sensitivity, compliance Good, non-intrusion type straight colon cancer early diagnosis technology is urgently to be resolved hurrily.
It is higher that research shows that Septin9 gene methylations have in colorectal cancer and part precancerous lesion peripheral blood in patients Recall rate, in recent years, by blood plasma detect based on septin9 (SEPT9) gene methylation test for colorectal cancer early stage Diagnosis provides more promising screening method.Septin9 genes are located at autosome 17q25.3, as highly conserved GTP Associated proteins are widely present in human cell.Clinical screening experiment so far proves that the SEPT9 genes that methylate are Colon and rectums The specific biomarkers of early stage during carcinogenesis.In the early stage of colorectal cancer, the DNA for the SEPT9 genes that methylate Peripheral circulation blood is released to from the tumour cell of necrosis or apoptosis, by the water that methylates for detecting peripheral blood SEPT9 genes The flat risk that can be determined that colorectal cancer.Due to only needing to extract peripheric venous blood, therefore its patient compliance is higher.Utilize Molecular Detection means, there is non-invasive, detection sensitivity height to the early screening of colorectal cancer.Many DNA are emerged in recent years The detection technique to methylate, saving your breath also, there have to be ten several.Two classes can be substantially divided into：The DNA methylation assay and full genome of specific site The methylation analysis of group.Wherein the DNA methylation assay of specific position mainly includes methylation status of PTEN promoter (MS-PCR), sulfurous Sour hydrogen salt processing is sequenced again afterwards, methylation sensitive high-resolution solubility curve is analyzed etc..For the PCR of methylation-specific (Methylation-specific PCR, MSP) detection method, it is easy to operate, time-consuming shorter, as a result it is easy to analyze.
Common at present primer and kit based on the detection of Human colorectal carcinoma specific methylation, it is common that be directed to The promoter region of SEPTIN9 genes devises a pair of PCR primers for being used for the detection of Human colorectal carcinoma specific methylation, to auxiliary Diagnosis colorectal cancer is helped to have certain effect, but its specificity and sensitivity still have much room for improvement.
The content of the invention
The technical problem to be solved by the invention is to provide a kind of septin9 mesh of the new detection primer with Loop Mark gene methylation detection primer and detect the kit of people's septin9 gene methylations,
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：A kind of septin9 target genes methylate Detection primer, including target gene septin9 primer pairs, its nucleotide sequence are as follows：
Septin9 forward primers F with a Loop sequence：
5'-GGGGGCGTT-GATGCTCTTCCGATCT-TTTTCGTCGTTGTTTTT-3' SEQ ID NO.1
Septin9 reverse primers R：5'-GAAGGTGGGTGTTGGG-3' SEQ ID NO.2.
The reagent of detection people's septin9 gene methylations containing above-mentioned septin9 target genes DNA methylation assay primer Box.
Kit also includes being used for the reference gene primer pair for detecting sample quality, and its nucleotide sequence is as follows：
Reference gene ACTB forward primers F：5'-GGTAGGGAGTATATAGGTTG-3' SEQ ID NO.3
Reference gene ACTB reverse primers R：5'-TTTTTTGGATTGTGAATTTG-3' SEQ ID NO.4.
Kit also includes two Taq Man-MGB probes, respectively target gene septin9 gene probes and internal reference base Because ACTB probes, its nucleotide sequence are as follows：
Septin9 gene probes：5'-GTCGGATTTCGCGGTT-3' SEQ ID NO.5
Reference gene ACTB probes：5'-TTTTTTATTAGGTTGTGGTT-3' SEQ ID NO.6
5' ends mark fluorescent reporter group R, the 3' end mark quenching group of each Taqman MGB probes for being used to detect Q, and the color that the fluorescent reporter group of two probes is shown is different.
The Taqman MGB probe nucleotide sequence 5' end flag F AM of detection people's septin9 genes, 3' ends mark BHQ1；Detect reference gene ACTBTaqman MGB probe nucleotide sequence 5' ends mark VIC, 3' ends mark BHQ1.
The Blocker primers of the kit also PNA structures including detection probe non-specific binding, nucleotide sequence is such as Under：
Blocker sequences：5'-TTATGTTGGATTTTGTGGTTAATGTGTAG-3' SEQ ID NO.7.
Kit also includes PCR reaction solutions, and PCR reaction solutions include archaeal dna polymerase, dNTPs, Mg2+。
Detect the dissociative DNA that To Template is human plasma extraction.
The beneficial effects of the invention are as follows：Kit provided by the invention is easy to operate, result interpretation is simple, to used Instrument is without particular/special requirement.And whole PCR processes use stopped pipe form, make result more accurate, avoid cross pollution.This is additional Enter reference gene ACTB detection primer and corresponding Taqman MGB probes, tied by probe and methylated DNA fragments specificity Close, and then methylation sites are further accurately detected.Simultaneously using PCR specific primers and Taqman with Loop Methylation sites are identified MGB probes, have higher sensitivity, accuracy.There is choosing, also use and tied based on PNA The Blocker of structure unless the interference of methylation fragment, strictly controls the amplification of non-specific fragment, examines this kit to go It is more preferable to survey specificity.
Embodiment
The present invention is described in further detail with reference to embodiment：
Septin9 target genes DNA methylation assay primer of the present invention, including target gene primer pair, its nucleotide sequence is such as Shown in lower：
Target gene Septin9 forward primers F with Loop：5'-GGGGGCGTT-GATGCTCTTCCGATCT- TTTTCGTCGTTGTTTTT-3'(SEQ ID NO.1),
Target gene Septin9 reverse primers R：5'-GAAGGTGGGTGTTGGG-3'(SEQ ID NO.2).
PCR (Methylation-specific PCR, MSP) method of the invention based on methylation-specific, and innovate Property introduce Loop sequences, devising correlation for -1000bp to -1500bp promoters this fragment methylation sites draws Thing.The methylation of this section of sequence is of a relatively high, possesses important clinical detection meaning.
Present invention also offers a kind of kit for detecting people's septin9 gene methylations, including above-described primer. It is prepared into kit, use more convenient to carry.
For the quality of detection template, the kit of above-mentioned detection septin9 gene methylations, in addition to reference gene ACTB detection primer pair, its nucleotide sequence are as follows：
Reference gene ACTB forward primers F：5'-GGTAGGGAGTATATAGGTTG-3'(SEQ ID NO.3),
Reference gene ACTB reverse primers R：5'-TTTTTTGGATTGTGAATTTG-3'(SEQ ID NO.4).
First, reference gene detection can carry out quality control to sample；Second, increase the detection of internal reference, can combine The detection of target gene, increase the accuracy of detection.
The kit of above-mentioned detection people septin9 gene methylations, in addition to two Taqman-MGB probes, respectively mesh Probe and reference gene (ACTB) probe, its nucleotide sequence for marking gene septin9 are as follows：
Target gene septin9 probes：5'-GTCGGATTTCGCGGTT-3'(SEQ ID NO.5),
Reference gene ACTB probes：5'-TTTTTTATTAGGTTGTGGTT-3'(SEQ ID NO.6)；
5' ends mark fluorescent reporter group R, the 3' end mark quenching group Q of two probes, and the fluorescence report of two probes It is different to accuse the color that group is shown.
The kit of above-mentioned detection people septin9 gene methylations, target gene septin9 gene probe nucleotide sequences 5' ends flag F AM, 3' ends mark BHQ1；Reference gene (ACTB) probe nucleotide sequence 5' ends mark VIC, 3' ends mark BHQ1.By the mark of different fluorescence, to reach the purpose for distinguishing detection.
In order to avoid unnecessary non-specific amplification, the kit of above-mentioned detection people septin9 gene methylations, also wrap A Blocker primer based on PNA structures is included, nucleotide sequence is as follows：
The Blocker primers of PNA structures：5'-TTATGTTGGATTTTGTGGTTAATGTGTAG-3'(SEQ ID NO.7)
Blocker sequences are completely artificial synthesized using PNA (peptide nucleic acid) progress, and the characteristic in its abiotic source ensure that it not It can be identified by the biology enzyme in organism, both add the specificity of Blocker combinations, ensure it without amplification again.
In PCR amplification procedures, the primer of purpose fragment is not easily with the genetic fragment that does not methylate and weight occurring The genetic fragment of sulphite conversion combines, and occurs breaking through amplification, and then produces non-specific amplification band, influences final to sentence Read result.Therefore, this kit adds additional the Blocker primers of PNA structures in amplification system, it is therefore an objective to make detection mesh The primer for marking gene is only combined with methylation fragment, further improves PCR amplification sensitivity and specific amplification.
The kit of above-mentioned detection people septin9 gene methylations, in addition to PCR reaction solutions, the PCR reaction solutions include Archaeal dna polymerase, dNTPs, Mg2+.Add PCR reaction solutions after, kit can a step complete quantitative fluorescent PCR, reduce pollution wind Danger, makes testing result more accurate.
The kit of above-mentioned detection people septin9 gene methylations, the DNA for the blood plasma extraction that detection object is behaved.With blood Dissociative DNA is starched as detection object, wound, convenient detection will not be caused to tissue.
The present invention is for -100bp extremely -1500bp promoter fragments design DNA methylation assay the primers of septin9 genes, spy It is different in nature strong, it can effectively detect the methylation status of septin9 genes.
MSP methods carry out due to the particularity of sequence primer discrimination may being caused bad when design of primers, easily There is the situation of mispairing in the other positions of gene in generation.Now if adding one section of special inhuman source at 5 ' places of primer Sequence, the artificial special Loop sequences of one section of introducing so that primer PCR second wheel begin to it is specific with and meanwhile contain The region for having special Loop sequences and target sequence reacts, then the specificity reacted will greatly improve, and add Taqman MGB probes specific can also be made a distinction to methylation fragment so that the specificity of whole detection is obtained for Improve.
Illustrated below by way of the mode to specific implementation to strengthen understanding of the technical staff to the present invention program.
In example, all biomaterials and reagent are the common material of biological field, can be obtained by normally buying approach .It is prepared by the kit of embodiment 1
The primer and probe sequence contained in kit：
Target gene Septin9 forward primers F：
5'-GGGGGCGTT-GATGCTCTTCCGATCT-TTTTCGTCGTTGTTTTT-3'
Target gene Septin9 reverse primers R：5'-GAAGGTGGGTGTTGGG-3'；
Reference gene ACTB forward primers F：5'-GGTAGGGAGTATATAGGTTG-3',
Reference gene ACTB reverse primers R：5'-TTTTTTGGATTGTGAATTTG-3'；
Target gene septin9 gene probes：FAM-GTCGGATTTCGCGGTT-MGB-BHQ1；
Reference gene ACTB probes：VIC-TTTTTTATTAGGTTGTGGTT-MGB-BHQ1；
PNABlocker sequences：5'-TTATGTTGGATTTTGTGGTTAATGTGTAG-3'；
(1) the primed probe mixed liquor (single reaction) of pre- mixed detection septin9 gene methylations：
Target gene Septin9 forward primers F (100 μ Μ, 0.6 μ L)；Target gene Septin9 reverse primers R (100 μ Μ、0.6μL)；Reference gene ACTB forward primers F (10 μ Μ, 0.72 μ L)；Reference gene ACTB reverse primers R (10 μ Μ, 0.72μL)；Target gene septin9 gene probes (10 μ Μ, 1.2 μ L)；Reference gene ACTB probes (10 μ Μ, 0.24 μ L)； PNA Blocker(100μΜ、0.6μL)。
(2) DNA PCR expand MIX (single reaction)：
Archaeal dna polymerase Mix, containing archaeal dna polymerase, dNTPs, Mg2+, Buffer, totally 25.32 μ L；
The kit of embodiment 2 detects septin9 gene methylations
1) 20 plasma samples are taken to carry out dissociative DNA extraction, dissociative DNA extracts kit is TIANGEN Serum/ Plasma Circulating DNA Kit；
2) dissociative DNA extraction is completed, and is carried out bisulfite conversion to the DNA extracted, is obtained purified DNA, turn It is Centrix Technology Ltd. to change kit from health；
3) pcr amplification reaction system is prepared：
Target gene Septin9 forward primers F (100 μ Μ, 0.6 μ L)；Target gene Septin9 reverse primers R (100 μ Μ、0.6μL)；Reference gene ACTB forward primers F (10 μ Μ, 0.72 μ L)；Reference gene ACTB reverse primers R (10 μ Μ, 0.72μL)；Target gene septin9 gene probes (10 μ Μ, 1.2 μ L)；Reference gene ACTB probes (10 μ Μ, 0.24 μ L)； PNA Blocker (100 μ Μ, 0.6 μ L), archaeal dna polymerase Mix25.32 μ L.
4) PCR amplification programs are as follows：
First amplification stage：95 DEG C of pre-degeneration 3min；
Second amplification stage：94 DEG C of denaturation 15s, 60 DEG C of annealing extension 35s, 45 circulate；
Signal collection, 60 DEG C of collection FAM of phase III, VIC signals.
5) Analysis of test results
20 samples are detected using above-mentioned reaction system, can accurately detect 3 positive samples, target gene Ct The difference of value and reference gene Ct values is respectively less than 6, illustrates that septin9 methylates.Remaining 17 negative samples are not expanded The difference of increasing curve, target gene Ct values and reference gene Ct values is all higher than 6, illustrates that septin9 does not have to methylate.Interpretation side Method, such as table 1.
The result interpretation of table 1 refers to
The ct value results of sample Sentence read result
△ Ct (Ct target gene-Ct reference genes)≤6 SETP9 methylates
△ Ct (Ct target gene-Ct reference genes) ＞ 6 SETP9 does not methylate
6) performance verification of finished product kit
The finished product kit completed using preparing carries out the detection of product precision, stability, according to the bar of above-mentioned setting Part carries out properties of product checking, using the sample of said extracted, if the difference of target gene Ct values and reference gene Ct values is respectively less than 6, illustrate that septin9 methylates.If the difference of target gene Ct values and reference gene Ct values is all higher than 6, illustrate that septin9 does not have Methylate.
The explanation of above example is only intended to help the main thought for understanding the present invention.For the common of the art For technical staff, on the premise of without prejudice to the invention general principle, appropriate optimization and adjustment can be carried out to the invention, These optimizations and adjustment are fallen within the protection domain of the claims in the present invention.
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Claims (8)

  1. A kind of 1. septin9 target genes DNA methylation assay primer, it is characterised in that including target gene septin9 primer pairs, Its nucleotide sequence is as follows：
    Septin9 forward primers F with a Loop sequence：
    5'-GGGGGCGTT-GATGCTCTTCCGATCT-TTTTCGTCGTTGTTTTT-3' SEQ ID NO.1
    Septin9 reverse primers R：5'-GAAGGTGGGTGTTGGG-3' SEQ ID NO.2.
  2. 2. detection people's septin9 gene methylations containing septin9 target genes DNA methylation assay primer described in claim 1 Kit.
  3. 3. the kit of people's septin9 gene methylations is detected according to claim 2, it is characterised in that also include being used for The reference gene primer pair of sample quality is detected, its nucleotide sequence is as follows：
    Reference gene ACTB forward primers F：5'-GGTAGGGAGTATATAGGTTG-3' SEQ ID NO.3
    Reference gene ACTB reverse primers R：5'-TTTTTTGGATTGTGAATTTG-3' SEQ ID NO.4.
  4. 4. the kit of people's septin9 gene methylations is detected according to claim 3, it is characterised in that also including two Taq Man-MGB probes, respectively target gene septin9 gene probes and reference gene ACTB probes, its nucleotide sequence It is as follows：
    Septin9 gene probes：5'-GTCGGATTTCGCGGTT-3' SEQ ID NO.5
    Reference gene ACTB probes：5'-TTTTTTATTAGGTTGTGGTT-3' SEQ ID NO.6
    5' ends mark fluorescent reporter group R, the 3' end mark quenching group Q of each Taqman MGB probes for being used to detect, and The color that the fluorescent reporter group of two probes is shown is different.
  5. 5. the kit of people's septin9 gene methylations is detected according to claim 4, it is characterised in that the detection people The Taqman MGB probe nucleotide sequence 5' end flag F AM of septin9 genes, 3' ends mark BHQ1；Detect reference gene ACTBTaqman MGB probe nucleotide sequence 5' ends mark VIC, 3' ends mark BHQ1.
  6. 6. the kit of people's septin9 gene methylations is detected according to claim 4, it is characterised in that also include detection The Blocker primers of the PNA structures of probe non-specific binding, nucleotide sequence are as follows：
    Blocker sequences：5'-TTATGTTGGATTTTGTGGTTAATGTGTAG-3' SEQ ID NO.7.
  7. 7. the kit of people's septin9 gene methylations is detected according to claim 4, it is characterised in that also anti-including PCR Liquid is answered, PCR reaction solutions include archaeal dna polymerase, dNTPs, Mg2+。
  8. 8. the kit of people's septin9 gene methylations is detected according to claim 4, it is characterised in that detection target mould Plate is the dissociative DNA of human plasma extraction.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055552A (en) * 2018-08-23 2018-12-21 北京迈基诺基因科技股份有限公司 The method and its special complete reagent whether detection Septin9 gene promoter methylates
CN109207592A (en) * 2018-09-26 2019-01-15 人和未来生物科技（长沙）有限公司 Kit and its application for colorectal cancer detection
CN110283913A (en) * 2019-07-21 2019-09-27 广州奥百阕谱生物科技有限公司 RASSF1A gene methylation state detection kit and its application
WO2019233102A1 (en) * 2018-06-06 2019-12-12 苏州唯善生物科技有限公司 Primer and probe set for diagnosis, detection, or screening of colorectal cancer
CN112608991A (en) * 2020-12-21 2021-04-06 广东省妇幼保健院 qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106244724A (en) * 2016-10-26 2016-12-21 北京鑫诺美迪基因检测技术有限公司 The primer of detection septin9 gene methylation and test kit

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106244724A (en) * 2016-10-26 2016-12-21 北京鑫诺美迪基因检测技术有限公司 The primer of detection septin9 gene methylation and test kit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WENTAO XU等: "Accurate and easy-to-use assessment of contiguous DNA methylation sites based on proportion competitive quantitative-PCR and lateral flow nucleic acid biosensor", 《BIOSENSORS AND BIOELECTRONICS》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019233102A1 (en) * 2018-06-06 2019-12-12 苏州唯善生物科技有限公司 Primer and probe set for diagnosis, detection, or screening of colorectal cancer
CN109055552A (en) * 2018-08-23 2018-12-21 北京迈基诺基因科技股份有限公司 The method and its special complete reagent whether detection Septin9 gene promoter methylates
CN109207592A (en) * 2018-09-26 2019-01-15 人和未来生物科技（长沙）有限公司 Kit and its application for colorectal cancer detection
CN109207592B (en) * 2018-09-26 2020-01-10 人和未来生物科技（长沙）有限公司 Kit for colorectal cancer detection and application thereof
CN110283913A (en) * 2019-07-21 2019-09-27 广州奥百阕谱生物科技有限公司 RASSF1A gene methylation state detection kit and its application
CN112608991A (en) * 2020-12-21 2021-04-06 广东省妇幼保健院 qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome
CN112608991B (en) * 2020-12-21 2023-12-12 广东省妇幼保健院 qMS-PCR kit for detecting Prader-Willi syndrome/Angelman syndrome

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