CN105177120A - Fluorescent in-situ hybridization probe for quickly detecting TOP2A gene abnormality - Google Patents
Fluorescent in-situ hybridization probe for quickly detecting TOP2A gene abnormality Download PDFInfo
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- CN105177120A CN105177120A CN201510496464.0A CN201510496464A CN105177120A CN 105177120 A CN105177120 A CN 105177120A CN 201510496464 A CN201510496464 A CN 201510496464A CN 105177120 A CN105177120 A CN 105177120A
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Abstract
The invention discloses a TOP2A gene and 17th chromosome specificity probe sequence and detailed description of experiment steps for quick detection. By a quick detection method, fluorescent in-situ hybridization detection efficiency is improved greatly, and clinical diagnosis and treatment are facilitated.
Description
Technical field
The present invention relates to the fluorescence in situ hybridization probe whether a kind of design and devdlop of quick FISH probe, particularly rapid detection TOP2A gene be abnormal.
Background technology
Mammary cancer is one of malignant tumour common in women, occupies first of tumour at its sickness rate of western countries and case fatality rate, in recent years, in the trend that the sickness rate of China also linearly rises.
Clinical study shows, nothing recurrence lifetime (RFS) of the patient with breast cancer of TOP2A gene unconventionality is shortened, and poor prognosis, especially patient with breast cancer's prognosis of TOP2A genetically deficient is poorer.The chemotherapy regimen effect that the patient of TOP2A gene unconventionality accepts anthracene nucleus medicament is better than the normal patient of TOP2A gene, the patient of TOP2A exception uses CEF scheme (endoxan+pidorubicin+Fluracil, wherein pidorubicin is anthracene nucleus medicament) carry out treating and can reduce the risk of recurrence of 65% and the mortality risk of 71%, and the normal patient of TOP2A uses this scheme can only reduce the risk of recurrence of 6% and the mortality risk of 10%.Therefore, the gene appearance detecting TOP2A in patient with breast cancer contributes to judging prognosis and instructs clinical rational drug use.
The method of current detection TOP2A gene comprises: immunohistochemical method (IHC) detects TOP2A protein overexpression, fluorescence in situ hybridization (FISH) detects TOP2A gene copy number, enzyme linked immunosorbent assay (ELISA) detects serum T OP2AECD(Student on E-cadherin) or tumor tissues.Wherein the most practical method is FISH.Because the length of traditional F ISH probe sequence is comparatively large, cause required hybridization time very long (being generally the assorted 16-18 hour that spends the night), can not obtain detected result timely, this is unfavorable for clinical Treatment and diagnosis.FISH probe is improved, shortens hybridization time, improve detection efficiency, be conducive to clinical Treatment and diagnosis.
Be necessary the fluorescence in situ hybridization probe developing a kind of rapid detection TOP2A gene unconventionality.
Summary of the invention
The object of the present invention is to provide a kind of fluorescence in situ hybridization probe of rapid detection TOP2A gene unconventionality.
The technical solution used in the present invention is:
The fluorescence in situ hybridization probe of rapid detection TOP2A gene unconventionality, comprises TOP2A gene-specific probe, No. 17 chromosome-specific probes.
1. the sequence of probe is respectively:
TOP2A gene-specific probe sequence:
| Probe 1 | TGCTCCGCCCAGACACCTACATTG |
| Probe 2 | TAAAGGAAGGTTCAAGTGGAGCTCT |
| Probe 3 | CCTAACCGACGCGCGTCTGTGGAG |
| Probe 4 | GCGGCTTGGTCGGGGGTGGTCTCG |
| Probe 5 | GGGTCCTGCCTGTTTAGTCGCTTTC |
| Probe 6 | GGGTTCTTGAGCCCCTTCACGACCG |
| Probe 7 | TCACCATGGAAGTGTCACCATTGC |
| Probe 8 | GTGACGGGTGAAGCTCTGGGGCT |
| Probe 9 | GGTCAGGCCGGCGACCGGCTTG |
| Probe 10 | CATTCCCGGTTCCGGGGTGATCGT |
No. 17 chromosome-specific probe sequences:
| Probe 1 | ATTTCAGCTGACTAAACAGAAGC |
| Probe 2 | TGGAAAACGAATTATGGTCACAT |
| Probe 3 | TGGTGACGACTGAGTTTAACTCACAG |
| Probe 4 | TTTGGACCACTCTGTGGCCTTCG |
| Probe 5 | TGGAAACGGGATAACTGCACCT |
| Probe 6 | AGGGCTTTGTGGTTTGTGGTGGA |
| Probe 7 | CAGAACCTTCTTCGTGATGTCTGC |
| Probe 8 | CTCCGAAGATGTCTTTGGAAACGG |
| Probe 9 | AGGGGACATGTAGACCTCTTTGAA |
2. above-mentioned probe is for repeatability is high on homologue, the short data records that specificity is good.
3., in the probe of the use in above-mentioned detection kit, the fluorophor that TOP2A gene-specific probe connects is green fluorescence group; The fluorophor of No. 17 chromosome-specific probe connections is red fluorescence group.
4. probe damping fluid used is: 20mM ~ 50mMTris-Hcl(PH8.0 ~ 8.8), 10mMMgcl
2, 10% ~ 30% methane amide.
5. the hybridising region on probe slide is placed in hybridization instrument and carries out sex change hybridization, denaturation temperature is 95 DEG C, and denaturation time is 10min, and hybridization temperature is 42 DEG C, and hybridization time is 20min.
6. the judgement of result, hybridize complete, at fluorescence microscopy Microscopic observation, observe 50 counting cells, add up danger signal quantity in each nucleus and green quantity and ratio calculated (in nucleus Green signal sum/50 nucleus of ratio=50 danger signal sum).If ratio is greater than 2.0, then judge TOP2A gene amplification; If ratio is less than 0.8, then judge TOP2A genetically deficient; If ratio is between 0.8-2.0, then again observe counting, the count results judgement be still between 0.8-2.0 is that TOP2A gene is normal.
The invention has the beneficial effects as follows:
Test kit of the present invention, adopts the short data records fluorescent probe optimized, original FISH was foreshortened to 1 ~ 2 hour by 16 ~ 18 hours detection time, can detect TOP2A gene appearance rapidly, be conducive to formulating follow-up treatment plan more accurately.
Accompanying drawing explanation
What Fig. 1 represented is after sample passes through process, the microscopy result after hybridizing with TOP2A gene-specific probe and No. 17 chromosome-specific probes, as shown in the figure, after hybridization dyeing, nucleus profile is clear, in nucleus danger signal and green high-visible, may be selected to be counting cells.
Embodiment
Embodiment 1:
one,the fluorescence in situ hybridization probe of rapid detection TOP2A gene unconventionality, comprises TOP2A gene-specific probe and No. 17 chromosome-specific probes.
1. wherein, the sequence of probe is respectively:
TOP2A gene-specific probe sequence:
| Probe 1 | TGCTCCGCCCAGACACCTACATTG |
| Probe 2 | TAAAGGAAGGTTCAAGTGGAGCTCT |
| Probe 3 | CCTAACCGACGCGCGTCTGTGGAG |
| Probe 4 | GCGGCTTGGTCGGGGGTGGTCTCG |
| Probe 5 | GGGTCCTGCCTGTTTAGTCGCTTTC |
| Probe 6 | GGGTTCTTGAGCCCCTTCACGACCG |
| Probe 7 | TCACCATGGAAGTGTCACCATTGC |
| Probe 8 | GTGACGGGTGAAGCTCTGGGGCT |
| Probe 9 | GGTCAGGCCGGCGACCGGCTTG |
| Probe 10 | CATTCCCGGTTCCGGGGTGATCGT |
No. 17 chromosome-specific probe sequences:
| Probe 1 | ATTTCAGCTGACTAAACAGAAGC |
| Probe 2 | TGGAAAACGAATTATGGTCACAT |
| Probe 3 | TGGTGACGACTGAGTTTAACTCACAG |
| Probe 4 | TTTGGACCACTCTGTGGCCTTCG |
| Probe 5 | TGGAAACGGGATAACTGCACCT |
| Probe 6 | AGGGCTTTGTGGTTTGTGGTGGA |
| Probe 7 | CAGAACCTTCTTCGTGATGTCTGC |
| Probe 8 | CTCCGAAGATGTCTTTGGAAACGG |
| Probe 9 | AGGGGACATGTAGACCTCTTTGAA |
2. above-mentioned probe is for repeatability is high on homologue, the short data records that specificity is good.
3., in the probe of the use in above-mentioned detection kit, the fluorophor that TOP2A gene-specific probe connects is green fluorescence group; The fluorophor of No. 17 chromosome-specific probe connections is red fluorescence group.
4. probe damping fluid used is: 20mM ~ 50mMTris-Hcl(PH8.0 ~ 8.8), 10mMMgcl
2, 10% ~ 30% methane amide.
5. the hybridising region on probe slide is placed in hybridization instrument and carries out sex change hybridization, denaturation temperature is 95 DEG C, and denaturation time is 10min, and hybridization temperature is 42 DEG C, and hybridization time is 20min.
two,fISH detects
1. paraffin embedding sample slide is put into the roasting sheet of 65 ± 5 DEG C of thermostat containers to spend the night;
2. take out slide, to put it in room temperature dimethylbenzene 15 minutes, repeat once;
3. take out slide, then to put it in room temperature dehydrated alcohol 10 minutes;
4. take out slide, then it is put into successively room temperature 100% ethanol, 90% ethanol, each 3 minutes of 70% ethanol;
5. take out slide, then to put it in deionized water at room temperature 3 minutes;
6. take out slide, then boil sheet 10 minutes (section is placed horizontally in container, and sample faces up) in the sample treatment solution putting it into 95 DEG C-99 DEG C;
7. take out slide, wait it cool to room temperature, then to put it in sterile pure water 3 minutes, suck excessive moisture (can not tissue be touched) with lint-free paper handkerchief;
8. room temperature is dried, and is divided by slide and is placed on thermostat container or heating platform, drips the Proteinase K reaction solution of appropriate (covering whole sample area) in sample areas, and 37 ° digest 5 ~ 15 minutes;
9. remove the surplus liquid in glass surface, to put it in room temperature washing lotion 15 minutes, repeat once
10. take out slide, then it is put into room temperature 70% successively, 90%, 100% each 2 minutes of graded ethanol dehydration, room temperature is dried
The 11. centrifugal 5min of probe dry powder 12000r/min taking out 10nmol/ pipe from-20 DEG C of refrigerators; Add the damping fluid of 100ul, concussion mixing.
12. add the hybridization probe of 10 μ l to hybridising region on slide, cover rapidly 22 × 22mm cover glass, and light pressure makes hybridization solution be uniformly distributed, and avoid producing bubble;
13. use rubber glue along cover glass edge mounting, cover the position that cover glass contacts with slide glass completely;
Slide is put into hybridization instrument by 14., moistening in situ hybridization instrument humidity bar, inserts wet bar, covers hybridization instrument upper cover, arrange " Denat & Hyb " program, hybridization (sex change 95 DEG C of 10min hybridize 42 DEG C of 20min).
Slide takes out by 15., tears rubber glue gently off, removes cover glass (if cover glass is difficult to remove, can puts it in washing lotion 2 and slightly rock, be beneficial to it and come off);
16. slides put into washing lotion 3 incubated at room 5 minutes;
17. take out slides, then to put it in washing lotion 4 incubated at room 2 minutes;
18. take out slide, natural drying at room temperature slide;
19. room temperatures, drip the cover glass of 10ulDAPI counterstain to 22 × 22mm, slide glass target area down, is put down gently on cover glass, is gently pressed, and avoids producing bubble, in the dark deposits, to be seen.
Precaution: FISH detects 15-19 step needs lucifuge to operate.
three,detected result:
Microscopy result as shown in Figure 1.As can be seen from the figure, the probe used in detection kit of the present invention, can the combination stable with aim sequence produce fluorescent signal, and detected result accurately and reliably, substantially reduce the fluoroscopic examination time of TOP2A gene simultaneously, be beneficial to early diagnosis and the treatment of disease.
SEQUENCELISTING
<110> Guangzhou two kinds of substance synthesis into another Technology Co., Ltd.
The fluorescence in situ hybridization probe of a <120> rapid detection TOP2A gene unconventionality
<130>
<160>19
<170>PatentInversion3.5
<210>1
<211>24
<212>DNA
<213> artificial sequence
<400>1
tgctccgcccagacacctacattg24
<210>2
<211>25
<212>DNA
<213> artificial sequence
<400>2
taaaggaaggttcaagtggagctct25
<210>3
<211>24
<212>DNA
<213> artificial sequence
<400>3
cctaaccgacgcgcgtctgtggag24
<210>4
<211>24
<212>DNA
<213> artificial sequence
<400>4
gcggcttggtcgggggtggtctcg24
<210>5
<211>25
<212>DNA
<213> artificial sequence
<400>5
gggtcctgcctgtttagtcgctttc25
<210>6
<211>25
<212>DNA
<213> artificial sequence
<400>6
gggttcttgagccccttcacgaccg25
<210>7
<211>24
<212>DNA
<213> artificial sequence
<400>7
tcaccatggaagtgtcaccattgc24
<210>8
<211>23
<212>DNA
<213> artificial sequence
<400>8
gtgacgggtgaagctctggggct23
<210>9
<211>22
<212>DNA
<213> artificial sequence
<400>9
ggtcaggccggcgaccggcttg22
<210>10
<211>24
<212>DNA
<213> artificial sequence
<400>10
cattcccggttccggggtgatcgt24
<210>11
<211>23
<212>DNA
<213> artificial sequence
<400>11
atttcagctgactaaacagaagc23
<210>12
<211>23
<212>DNA
<213> artificial sequence
<400>12
tggaaaacgaattatggtcacat23
<210>13
<211>26
<212>DNA
<213> artificial sequence
<400>13
tggtgacgactgagtttaactcacag26
<210>14
<211>23
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<400>14
tttggaccactctgtggccttcg23
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<400>15
tggaaacgggataactgcacct22
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<211>23
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<400>16
agggctttgtggtttgtggtgga23
<210>17
<211>24
<212>DNA
<213> artificial sequence
<400>17
cagaaccttcttcgtgatgtctgc24
<210>18
<211>24
<212>DNA
<213> artificial sequence
<400>18
ctccgaagatgtctttggaaacgg24
<210>19
<211>24
<212>DNA
<213> artificial sequence
<400>19
aggggacatgtagacctctttgaa24
Claims (6)
1. a fluorescence in situ hybridization probe for rapid detection TOP2A gene unconventionality, comprises TOP2A gene-specific probe, No. 17 chromosome-specific probes, damping fluid and staining fluid, and wherein, the sequence of probe is respectively:
TOP2A gene-specific probe sequence:
No. 17 chromosome-specific probe sequences:
。
2. the fluorescence in situ hybridization probe of rapid detection TOP2A gene unconventionality according to claim 1, is characterized in that: the fluorophor that TOP2A gene-specific probe connects is green fluorescence group; The fluorophor of No. 17 chromosome-specific probe connections is red fluorescence group.
3. according to claim 1, the fluorescence in situ hybridization probe of the rapid detection TOP2A gene unconventionality described in 2, is characterized in that: probe is for repeatability is high on homologue, the short data records that specificity is good.
4. according to claim 1, the fluorescence in situ hybridization probe of the rapid detection TOP2A gene unconventionality described in 2, is characterized in that: mixed according to 1:1 with No. 17 chromosome-specific probes by TOP2A gene-specific probe.
5. the fluorescence in situ hybridization probe of rapid detection TOP2A gene unconventionality according to claim 1, is characterized in that: probe damping fluid used is: 20mM ~ 50mMTris-Hcl(PH8.0 ~ 8.8), 10mMMgcl
2, 10% ~ 30% methane amide.
6. according to claim 1,2,3,4, the fluorescence in situ hybridization probe of the rapid detection TOP2A gene unconventionality described in 5, it is characterized in that: be added to by probe in the hybridising region on sample slide and carry out sex change hybridization in hybridization instrument, denaturation temperature is 95 DEG C of denaturation time is 10min, and hybridization temperature is 42 DEG C of hybridization time is 20min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628875A (en) * | 2019-09-25 | 2019-12-31 | 广州简册生物技术有限公司 | Probe, kit and detection method for detecting Top2A gene |
| WO2023098582A1 (en) * | 2021-11-30 | 2023-06-08 | 广州康立明生物科技股份有限公司 | Chromosome centromere probe |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399772A (en) * | 2010-09-15 | 2012-04-04 | 中山大学达安基因股份有限公司 | Preparation method for probes related to breast cancer molecular markers and application of same |
| CN104561360A (en) * | 2015-01-30 | 2015-04-29 | 益善生物技术股份有限公司 | TOP2A gene abnormality detection probe, kit and method |
-
2015
- 2015-08-13 CN CN201510496464.0A patent/CN105177120A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399772A (en) * | 2010-09-15 | 2012-04-04 | 中山大学达安基因股份有限公司 | Preparation method for probes related to breast cancer molecular markers and application of same |
| CN104561360A (en) * | 2015-01-30 | 2015-04-29 | 益善生物技术股份有限公司 | TOP2A gene abnormality detection probe, kit and method |
Non-Patent Citations (3)
| Title |
|---|
| 《METHODS IN MOLECULAR BIOLOGY》 * |
| ASLI REHBER BESER ET AL: "HER-2, TOP2A and Chromosome 17 Alterations in Breast Cancer", 《PATHOLOGY ONCOLOGY RESEARCH》 * |
| 邵飞飞: "乳腺癌HER2、TOP2A和17号染色体的研究", 《吉林大学硕士学位论文》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628875A (en) * | 2019-09-25 | 2019-12-31 | 广州简册生物技术有限公司 | Probe, kit and detection method for detecting Top2A gene |
| WO2023098582A1 (en) * | 2021-11-30 | 2023-06-08 | 广州康立明生物科技股份有限公司 | Chromosome centromere probe |
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