CN102994636B - Molecular marker, kit and method for detecting clinical stages of patient with ovarian cancer - Google Patents
Molecular marker, kit and method for detecting clinical stages of patient with ovarian cancer Download PDFInfo
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- CN102994636B CN102994636B CN201210465726.3A CN201210465726A CN102994636B CN 102994636 B CN102994636 B CN 102994636B CN 201210465726 A CN201210465726 A CN 201210465726A CN 102994636 B CN102994636 B CN 102994636B
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- 206010033128 Ovarian cancer Diseases 0.000 title claims abstract description 38
- 206010061535 Ovarian neoplasm Diseases 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title abstract description 12
- 239000003147 molecular marker Substances 0.000 title abstract 4
- 101150047731 MTDH gene Proteins 0.000 claims abstract description 20
- 239000002773 nucleotide Substances 0.000 claims abstract description 16
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims description 13
- 239000003550 marker Substances 0.000 claims description 10
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- 230000003321 amplification Effects 0.000 abstract description 5
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Abstract
The invention discloses a molecular marker for detecting clinical stages of a patient with ovarian cancer. The nucleotide sequence of the molecular marker is shown as SEQ ID NO:1, and is characterized in that single nucleotide polymorphism G>A exists at a 231st site of the molecular marker compared with a normal MTDH gene. A kit for detecting the clinical stages of the patient with ovarian cancer comprises a pair of specific primers with sequences of SEQ ID NO:2 and SEQ ID NO:3. A single nucleotide polymorphism method for detecting whether a sample has an MTDH gene in vitro comprises the following steps of: amplifying the MTDH gene of the sample by the specific primers of the MTDH gene, thus obtaining an amplification product; and performing sequence determination on the amplification product so as to detect whether the amplification product contains the single nucleotide polymorphism 231G>A. The method provided by the invention is simple, accurate and rapid, is high in specificity, and has a great significance to effective early diagnosis, early treatment and individual prevention and treatment of a high risk group or susceptible individuals, and patients with ovarian cancer.
Description
Technical field
The present invention relates to a kind of for the identification of the ovarian cancer patients molecule marker of clinical stages, test kit and detection method thereof, single nucleotide polymorphism (the single nucleotide polymorphism that relates to particularly MTDH gene, SNP) and with the dependency of ovarian cancer, and detect method and the test kit of these SNP, belong to molecular biology and medical field.
Background technology
Ovarian cancer is the female reproductive system tumour that lethality rate is the highest, global new cases 225500 people in 2008, dead 140200 people.Most ovarian cancer patients onsets concealments, symptom is not obvious, and in late period during discovery, poor prognosis, without desirable treatment means.The similar patient of clinical factor such as middle discovery clinical stages, histological type, age of onset even if work clinically, it also has very large difference to the reactivity of chemotherapy and clinical prognosis thereof, thereby causes the undesirable for the treatment of.Therefore inquire into the biological mechanism that development occurs ovarian cancer, find the molecule marker of ovarian cancer patients molecule parting, ovarian cancer patients is implemented to effective individuation control significant.
Therefore, the molecule marker of ovarian cancer patients molecule parting is badly in need of further finding in this area, and exploitation detects ovarian cancer method, the test kit of clinical stages, and relevant medicine.
Summary of the invention
For above-mentioned prior art, present inventor is through research deeply and widely, the SNP of a large amount of candidate genes is measured and analyzed, find that first MTDH and ovarian cancer patients clinical stages are closely related, can be used as the molecule marker of ovarian cancer patients clinical stages detection or molecule parting, therefore, the invention provides a kind ofly for the identification of ovarian cancer patients test kit and the method thereof of clinical stages, and whether vitro detection sample there is the method for the single nucleotide polymorphism of MTDH gene.
The present invention is achieved by the following technical solutions:
A kind of for the identification of the ovarian cancer patients molecule marker of clinical stages, its nucleotide sequence is as shown in SEQ ID NO:1, it is compared with normal MTDH gene, difference is: the 231st exists single nucleotide polymorphism a: G>A, the base of the 231st comprises bases G and two kinds of situations of base A, when being bases G, is normal MTDH gene, when being base A, be the MTDH gene of sudden change:
gctacaaatt agtaggaaaa gaatagtgcc gcccacgggc tttccaactt ttgaattctg 60
gggactaaca acagagagca agagtgaatg agccaaacga cgcaaacgtg ccctcgccag 120
gctggcaact ggtaggcacg cagtgtatta gttgccctgg agagaaacac cctggagaga 180
aatacccaat ttgtgaacta aacctaggtc ccggaagaac agtgttcggc rtcagactcc 240
231, r=G or A
gttgagctca ttctggaagg atccaactgg cgccaccagg gagaaaaagc gattccacct 300
caataacact ccagaaaaag gcatgaagag ccctatacct gccagggcga ctttgaccta 360
gacccggtga cccggttcct agcgctgcag ccctacccgc cccccgcccg cccccgcctt 420
gcacggagcc cctcctctgt actcattcgt tgcgccacgt ctcctaactc tgcgccacca 480
gccaccccgc gaaggcgtcc accaattaac ccctcccagc ttctggtcta cagtaacggg 540
tccccaacgc cgcgtcttaa ccaggcccga accgaccacc gccaaccttc cctgacacgc 600
ctttgcccca cccgaccccg cccgccccca cgtgacgccc acgccccgcc cactggcgcc 660
cacgtgaccc acggtcgtcc ccgcgcgcgg cgtggatcgc ggcccaagcc gccattgttc 720
cgccgaggga ggacagcggg gcctggcgct ggcgccgaga cgccgcttag cggccgccac 780
tggagacact ccctcccgcc tcccgggtct cctggcggcg gcggagtgag gctgacagcg 840
gggaacctgg gagacccctc cgccctcccc gcggtggcag cggccgatcc ccggctccgg 900
cgcgagggac ggccgcgatg cgctcggcct gaggttaccc ggcccggccc ttcctcgctt 960
ccctcgacta ttccactgcg tctccgcgcc ccggcgtcat cctgcgagtc cctctgacgg 1020
gagggaagat ggctgcacgg agctggcagg acgagctggc ccagcaggcc gaggagggct 1080
cggcccggct gcgggaaatg ctctcggtcg gcctaggctt tctgcgcacc gagctgggcc 1140
tcgacctggg gctggagccg aaacggtacc 1170
A kind of for the identification of the ovarian cancer patients test kit of clinical stages, comprise Auele Specific Primer, this primer is a pair of, the sequence with SEQID NO:2 and SEQ ID NO:3, this primer be can be specific the primer of amplification MTDH gene, can amplify specifically the sequence of length 200～2700bp.
Further, test kit also comprises PCR reaction solution, and PCR reaction solution is by dNTP, Mg
2+, Taq enzyme and Buffer form.In PCR reaction solution, each component is conventional with magnitude relation, for one of ordinary skill in the art, is common practise.
Whether vitro detection sample there is a method for the single nucleotide polymorphism of MTDH gene, and step is as follows:
(1) with the MTDH gene of MTDH gene-specific primer amplification sample, obtain amplified production; Described primer is a pair of, has the sequence of SEQ ID NO:2 and SEQ ID NO:3;
(2) amplified production is carried out to sequencing, detect in amplified production whether have following single nucleotide polymorphism: 231G>A; The sequence of 231G>A sudden change Position Number based on SEQ ID NO:1.
The method that the clinical stages of ovarian cancer patients individuality or molecule parting are detected or diagnosed, step is as follows:
Detect this individual MTDH gene, transcript and/or albumen, and compare with normal MTDH gene, transcript and/or albumen, there are differences and just show that this patient clinical is by stages for early stage possibility is higher than normal ovarian cancer patient.Described difference refers to whether there is single nucleotide polymorphism: 231G>A; The sequence of 231G>A sudden change Position Number based on SEQ ID NO:1.
Of the present invention for the identification of the ovarian cancer patients molecule marker of clinical stages, test kit and method thereof, and whether vitro detection sample there is the method for the single nucleotide polymorphism of MTDH gene, detect simple, accurate, quick, high specificity, implements effectively early to examine, early controls with individuation control significant for high risk population or susceptible individual and ovarian cancer patients.For the biological mechanism of inquiring into the development of nest carcinogenesis, find the molecule marker of ovarian cancer patients molecule parting, ovarian cancer patients is implemented to effective individuation control significant.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The detection of embodiment 1 MTDH transgenation and with the ovarian cancer patients association analysis of clinical stages
1.1 research object
Choose ovarian cancer patients 145 examples, treatment is made a definite diagnosis in Shandong Qilu Hospital year April in September, 2008～2011, and clinical data obtains from case history.Most subjects is Donors in Shandong Province.Get subject's peripheral blood 1.5ml, in-80 ℃ of stored refrigerated.All subjects all require informed consent according to Ethics Committee of Shandong Qilu Hospital.
1.2 DNA extraction
By conventional phenol chloroform method, extract experimenter's peripheral blood DNA, specific as follows:
(1) getting 400ul blood is added in 1.5ml centrifuge tube.
(2) add 800ul PBS, mix, 3500g, 15min, abandons supernatant; Come again.
(3) add 400ul lysate, 37 ℃, 1h.
(4) add Proteinase K 4ul, concentration 200ug/ml.
(5) 55 ℃ of water-baths digest spend the night (4-12h).
(6) add the saturated phenol of Tris of 400ul, mix and shake 10min.
(7) 5000g, 15min, water intaking phase.
(8) add 100ul NaAc, 800ul dehydrated alcohol ,-20 ℃, 20min.
(9) 12000g, 5min, abandons supernatant.
(10) 600ul 70% ice ethanol, 12000g, 5min, abandons supernatant, repeats once.
(11) 12000rpm is centrifugal, 10 minutes.Abandon supernatant, dry.Be dissolved in 100 μ l TE.
(12) measure concentration and purity, packing, frozen in-80 ℃.
1.3 primers, pcr amplification and order-checking
From GenBank, download MTDH genome sequence (Gene ID:92140; Nucleotide:AC_000140.1GI:157734173), with primer premier5.0 design primer.Concrete primer details are in Table 1.
Table 1 primer sequence table
| Primer title | Sequence | SEQ ID NO： |
| Upstream primer | 5'CTGGCAACTGGTAGGCACGC 3' | 2 |
| Downstream primer | 5'GAGGGACTCGCAGGATGACG 3' | 3 |
Pcr amplification condition: 94 ℃ 3 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) * 35,72 ℃ 7 minutes, 10 ℃ of insulations.Pcr amplification product is 1255bp.
Pcr amplification product is delivered the order-checking of Shanghai Bo Shang biotech company, sequencing result application software Meglign 7.0 and Chromas 2.33 test and analyze, this enforcement has adopted backward sequencing, by analysis, finds to exist following SNP:231G>A.
1.4 SNP somatotype and association analysiss
Application card side (X
2) check the distribution in subject site is carried out to statistical study; When the expectation number of a cell is less than 5, applying Fisher rigorous examination analyzes.All P values are bilateral probability, think and have remarkable statistical significance when P<0.05.Applying the dependency of unconditional logistic regression analysis between ill to genotype frequency, gene frequency and ovarian cancer assesses, calculate its odds ratio (Odds ratio, OR) and 95% credibility interval (confidence interval, CI), and apply SPSS17.0 software (SPSS Inc.Chicago, Illinois, USA) carry out statistical study.
Found that rs16896059 (231G>A) site in SEQ ID NO:1 and the clinical stages significant correlation of ovarian cancer patients, A allelotrope carrier (GA+AA) is that early stage possibility is larger clinical stages, can be used as the ovarian cancer patients molecule marker of clinical stages.Detailed results is in Table 2.
The dependency of table 2 231G>A loci gene type and ovarian cancer patients clinical stages
Embodiment 2 ovarian cancer patients clinical stages detection kit
Therefore due to the sudden change of 231G>A in SEQ ID NO:1 and the clinical stages height correlation of ovarian cancer patients, can take again patient's DNA based on this sudden change design MTDH gene-specific primer and expand detection as template.
Prepare a test kit (100 person-times), it contains material as shown in table 3:
Table 3
Extract experimenter's peripheral blood 2ml, ordinary method is extracted DNA.Utilize ovarian cancer patients clinical stages detection kit to carry out PCR reaction, reaction product is checked order, utilize Meglign 7.0 and Chromas 2.33 softwares test and analyze sequencing result.
Detected result contains rs16896059 (231G>A) ovarian cancer patients clinical stages is that early stage possibility is higher than normal ovarian cancer patient.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various modifications or change to the present invention, but the equivalent form of value of these changes or modification drops in limited range of the present invention equally.
Claims (2)
1. Auele Specific Primer, in the application for the preparation of identifying in the ovarian cancer patients test kit of clinical stages, is characterized in that: described Auele Specific Primer is a pair of, and its sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3; Be used for identify ovarian cancer patients clinical stages molecule marker, its nucleotide sequence is as shown in SEQ ID NO:1, it is compared with normal MTDH gene, difference is: the 231st exists single nucleotide polymorphism a: G>A, the base of the 231st comprises bases G and two kinds of situations of base A, when being bases G, is normal MTDH gene, when being base A, it is the MTDH gene of sudden change.
2. application according to claim 1, is characterized in that: in described test kit, except Auele Specific Primer, also comprise PCR reaction solution, PCR reaction solution is comprised of dNTP, Mg2+, Taq enzyme and Buffer.
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Non-Patent Citations (2)
| Title |
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| Identification of Novel Variants of Metadherin in Breast Cancer;Xianqiang Liu et al;《Plos One》;20110308;第6卷(第3期);e17582:1-6，参见材料和方法部分、表2和表3 * |
| Xianqiang Liu et al.Identification of Novel Variants of Metadherin in Breast Cancer.《Plos One》.2011,第6卷(第3期),e17582:1-6，参见材料和方法部分、表2和表3. * |
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