CN104561356A - ALK gene rearrangement detection probe, kit and method - Google Patents
ALK gene rearrangement detection probe, kit and method Download PDFInfo
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Abstract
The invention discloses an ALK gene rearrangement detection probe, a kit and a method. The detection probe comprises a first group of probes for labeling fluorescent dye and targeted to the upstream side of a breaking point, and a second group of probes targeted to the downstream side of the breaking point, wherein the first group of the probes are selected from at least one of probes as shown in SEQ ID NO.41-SEQ ID NO.50; the second group of the probes are selected from at least one of probes as shown in SEQ ID NO.51-SEQ ID NO.60; and the fluorescent dyes labeled by the two groups of the probes are different in color. A FISH probe in the ALK gene rearrangement detection kit provided by the invention is free from a repeated sequence, and capable of avoiding a cross reaction and undergoes hybridization recognition with an ALK target detection fragment in a chromosome; and the probe has the advantages of high accuracy, good specificity and low false positive rate.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, particularly relate to ALK gene and reset detection probes, test kit and method.
Background technology
First the Nucleophosmin-anaplastic lymphoma kinase (anaplasticlymphoma kinase, ALK) of ALK gene coding is found in primary cutaneous type, is one of important target of targeted therapy.ALK gene is positioned at (position is 2p23) on No. 2 karyomit(e)s, length is 728kb, full-length cDNA contains 29 exons, total length alk protein contains 1620 amino acid, molecular weight is 177 kilodaltons (kDa), 254 kinase amino acid structural domains are made up of 1123 to 1376 amino acids residues, are a short cross-film district be made up of amino acid before this.The breaking point that ALK gene is reset is between exons 19 and 20, ALK gene has biological function after there is rearrangement more, its expression product is chimeric Tyrosylprotein kinase, polymer is formed by Coiled Coil structural domain, the ALK of composition is caused to activate, ALK signal promotes cell proliferation constantly by activating the downstream signaling pathway such as RAS-MEK-ERK, JAK3-STAT3 and PI3K-AKT, leads oncogenic generation and transfer.
Clinical study shows, the targeted drug gram azoles for ALK gene exploitation replaces Buddhist nun, resets positive NSCLC patient, show significant therapeutic activity, and can extend the lifetime of patient for ALK gene.At present, gram azoles has obtained the approval of state food pharmaceuticals administration general bureau (CFDA) for Buddhist nun, is used for the treatment of the positive NSCLC patient of Nucleophosmin-anaplastic lymphoma kinase (ALK) in China.Therefore, with gram azoles for Buddhist nun treat supporting ALK gene resets detects have clear and definite clinical meaning and detection necessity.
Conventional ALK gene is reset detection method and is divided into following three kinds: the method (comprising qRT-PCR, RACE-PCR or specificity RT-PCR associating sequencing technologies) of fluorescence in situ hybridization (FISH) technology, immunohistochemistry (IHC) and PCR-based.But above-mentioned technology still comes with some shortcomings, as fluorescence in situ hybridization probe mainly uses artificial chromosome to prepare, because tumor-necrosis factor glycoproteins constantly occurs in whole genome in artificial chromosome, thus cause nonspecific fluorescent signal, greatly reduce specificity and the sensitivity of FISH detection; The positive criteria of conventional IHC judges not yet unification; Although RT-PCR method detect ALK fusion gene quick, simple and easy to do, can the type of the simultaneously fusion variant that clear and definite ALK is known, cannot detect unknown pattern of fusion is its maximum weak point.
Summary of the invention
An object of the present invention is that ALK gene resets detection kit, and this detection kit can be used for the situation of high specific and the rearrangement of high accuracy detection ALK gene.
The technical scheme realizing above-mentioned purpose is as follows:
ALK gene resets detection kit, include mark fluorescent dyestuff, first group of probe for breaking point upstream and second group of probe for breaking point downstream, described first group of probe is for being selected from least one probe in SEQ IDNO.41-SEQ ID NO.50, and described second group of probe is for being selected from least one probe in SEQ IDNO.51-SEQ ID NO.60; The fluorescence dye color that above-mentioned two groups of probes mark is different.
Wherein in an embodiment, described first group of probe is be selected from least five probes in SEQ ID NO.41-SEQ IDNO.50, and described second group of probe is be selected from least five probes in SEQ ID NO.51-SEQ IDNO.60.
Wherein in an embodiment, described first group of probe is be selected from least eight probes in SEQ ID NO.41-SEQ IDNO.50, and described second group of probe is be selected from least eight probes in SEQ ID NO.51-SEQ IDNO.60.
Wherein in an embodiment, described first group of probe is SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is SEQ ID NO.51-SEQ ID NO.60.
Wherein in an embodiment, described SEQ ID NO.41-SEQ ID NO.50 is prepared by following primer amplification successively respectively: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20.
Wherein in an embodiment, described SEQ ID NO.51-SEQ ID NO.60 is prepared by following primer amplification successively respectively: SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ IDNO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ IDNO.29 and SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.33 and SEQID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQID NO.39 and SEQ ID NO.40.
Another object of the present invention is to provide a kind of ALK gene and resets detection probes.
Realize above-mentioned purpose technical scheme as follows:
ALK gene resets detection probes, first group of probe for breaking point upstream and second group of probe for breaking point downstream, described first group of probe is for being selected from least one probe in SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is for being selected from least one probe in SEQ ID NO.51-SEQ ID NO.60.
Wherein in an embodiment, described first group of probe is be selected from least five probes in SEQ ID NO.41-SEQ IDNO.50, and described second group of probe is be selected from least five probes in SEQ ID NO.51-SEQ IDNO.60.
Another object of the present invention is to provide a kind of ALK gene and resets detection method.
The technical scheme realizing above-mentioned purpose is as follows.
A kind of ALK gene resets detection method, comprises
Detected sample section pre-treatment;
Utilize above-mentioned ALK gene to reset detection kit and carry out hybridization check;
Fluorescent signal is examined under a microscope after redying.
Major advantage of the present invention is:
1. the detected result of ALK gene rearrangement detection kit provided by the present invention and the identical rate of Cytocell Products ALK testing product are up to 100%.
2. the FISH probe that the detection ALK gene that prepared by the present invention is reset, not containing tumor-necrosis factor glycoproteins, can avoid cross reaction, carry out identification and hybridize, have accuracy high, the advantage that specificity is good and false positive is low with ALK target detect fragment in karyomit(e).
3. simple to operate, the safety of test kit of the present invention, decrease the use of the expensive reagent such as Cot-1DNA reagent, the anti-DigiTAb of rhodamine, economical and efficient simultaneously;
4. compared with prior art, the detection method of test kit of the present invention, without the need to using Cot-1DNA to close sample gene, easy to operate, detection speed is fast, can complete hybridization check experiment in 12-24 lab scale.
Accompanying drawing explanation
Fig. 1 is signal-count guide-Typical signal pattern.
Fig. 2 is signal-count guide-typical positive cell signal pattern.
Fig. 3 signal-count guide-typical negative cells signal mode.
Embodiment
In order to understand the present invention better, illustrate content of the present invention further below in conjunction with accompanying drawing, embodiment, but protection scope of the present invention is not limited to the following examples.
Embodiment 1
Detection ALK gene described in the present embodiment resets detection probes and test kit, and its design is as follows:
One, design of amplification primers
ALK gene is positioned at (position is 2p23) on No. 2 karyomit(e)s, the breaking point that ALK gene is reset is between exons 19 and 20, according to the feature of ALK gene, design two groups of amplimers respectively, be respectively: for first group of amplimer of breaking point upstream design, for second group of amplimer of breaking point downstream design, corresponding amplified production constitutes first group of probe library and second group of probe library respectively.Often organize amplimer, the amplified production obtained as the FISH probe of resetting, not containing tumor-necrosis factor glycoproteins, can be avoided cross reaction, carry out specific recognition hybridization with ALK target detect fragment in karyomit(e).
The following primer of synthetic, specifically in table 1, (note: 1F/1R is pair of primers, represents forward primer and reverse primer respectively for amplimer and corresponding amplified production thereof; Other are by that analogy).Every bar amplimer after synthesis is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Table 1 amplimer and amplified production
Two, the structure of probe library
Extract human genome DNA, technology described by the document in this area and condition, as with reference to " Molecular Cloning: A Laboratory instructs the third edition " (Science Press) or carry out according to commercially available genome DNA extracting reagent kit product description, obtain human genome STb gene.
Preparation PCR primer working fluid: respectively for the target amplification product in breaking point upstream and breaking point downstream, the stock solution 50ul respectively getting above-mentioned amplimer (1R/1F-20R/20F) is positioned in 1.5ml Eppendorf tube respectively, using 10mmol/L Tris Buffe r to be mixed with final concentration is respectively 20 amplimer working fluids of 10pmol/mL, for nucleotide sequence fragment corresponding in the above-mentioned human genome STb gene that increases.Carry out the amplification of 20 PCR system respectively, PCR reaction system is as follows:
Pcr amplification program is: 95 DEG C of 3min; 94 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 10min; 4 DEG C save backup.
Pcr amplification product, through 2% agarose gel electrophoresis, reclaims fragment, and carries out mark.
Checked order by the amplified production of above-mentioned recovery, the series of sequence amplified production corresponding to amplimer in table 1 forms unanimously.
Above-mentioned recovery through the correct amplified production that checks order can be directly used in FISH probe mark, or respectively by products therefrom in carrier T, to preserve, wherein, in the present embodiment, pcr amplification product through check order errorless after, be cloned in carrier T, comprise the steps:
Get the 200ul Eppendorf tube of 20 sterilizings, reaction system 10 μ l:
After above-mentioned mixed solution shakes gently, 12000rpm, 30 seconds centrifugal, is then placed in 14 DEG C of incubated overnight (12-16h).Product conversion after connection enters TOP10 competence, is placed in Liquid nitrogen storage, comprises the following steps:
1, competent cell is placed in thawed on ice, for 100ul competent cell.
2, in competent cell suspension, add the carrier T that need transform and connect product, notice that the volume of carrier T connection product does not exceed 1/10th of competent cell suspension volume, rotating centrifugal pipe is to mix content gently, and ice bath places 30 minutes.
3, centrifuge tube is placed in 42 DEG C of water-bath 60-90 seconds, is then quickly transferred in ice bath and places 2-3 minute, note not shaking centrifuge tube.
4, in centrifuge tube, the LB substratum of the aseptic nonreactive of 500ul is added, 37 DEG C of 180rpm shaking culture 1 hour.Object is that resistant maker gene relevant on plasmid is expressed, and thalline is recovered.
5, get the competent cell coating transformed in right amount dull and stereotyped containing the antibiotic LB of ammonia benzyl, be inverted for 37 DEG C and cultivate 12-16 hour.
6, blue hickie screening, select positive bacterium colony (should select positive bacterium colony in a large number, to guarantee that selected bacterium colony contains all object fragments), selected bacterium colony is placed in LB liquid nutrient medium and cultivates.
7, get the bacterium liquid of step 6, add glycerine (glycerine volume: bacteria liquid amasss=1:4), be then placed in Liquid nitrogen storage.
When again needing target fragment, bacterium liquid described in step 7 can be got respectively cultivate in the LB liquid nutrient medium of ammonia benzyl resistance, the extraction thalline plasmid kit of sampling commercially available extracts plasmid, then with the plasmid that the method process that enzyme is cut is extracted, a large amount of object fragments not containing tumor-necrosis factor glycoproteins can just be obtained.It is as follows that enzyme cuts system:
Reaction conditions: temperature 37 DEG C, enzyme cuts 1 hour 30 minutes.
According to actual needs, enzyme is cut the object fragment mixing obtained, respectively obtained first group of probe library and second group of probe library, for follow-up probe mark.
Three, probe mark
Alexa Fluor 594NHS ester (ruddiness) is selected to mark first group of probe library; Alexa Fluor488NHS ester (green glow) is selected to mark second group of probe library.Concrete steps are as follows:
Preparation boric acid mark damping fluid, gets sodium borate decahydrate 1.9g and is dissolved in 50mL ddH
2o, pH value is adjusted to 8.5, and 0.45 μm of frit, is stored in-25 DEG C to-18 DEG C, for subsequent use.
Get DMSO, NF water and prepare boric acid mark damping fluid balance to room temperature, abundant vortex oscillation mixing.
Get Green fluorescent dye and red fluorescence dyestuff (specification is 1mg), 10000g, centrifugal 2min.Carefully uncap, often pipe adds 56 μ L DMSO dissolving dye, keeps in Dark Place.
Dye labeling schemes sees the following form 2:
Table 2 FISH probe marks
Probe after mark is purified and precipitate, and natural air drying, is dissolved in DNA in TE damping fluid.Obtain FISH ruddiness probe and green probe, lucifuge ,-20 DEG C of storages.
Four, other component of test kit
1) compound method of SSC buffer reservoir liquid (20 × SSC, pH5.3) is in table 3.
The compound method of table 3 SSC buffer reservoir liquid (20 × SSC, pH5.3)
2) compound method of stomach en-damping fluid (0.1N HCl) is in table 4.
The compound method of table 4 stomach en-damping fluid (0.1N HCl)
3) compound method of ethanolic soln (70% and 85%) is in table 5.
The compound method of table 5 ethanolic soln (70% and 85%)
4) compound method of lavation buffer solution I is in table 6.
The compound method of table 6 lavation buffer solution I
5) compound method of lavation buffer solution II is in table 7.
The compound method of table 7 lavation buffer solution II
Embodiment 2 uses ALK gene in embodiment 1 to reset detection kit to the detection of clinical sample
Utilize the ALK gene of embodiment one to reset detection kit (comprising SEQ NO.41-50 and SEQNO.51-60 totally two ten probes) to carry out the 20 parts of paraffin-embedded tissues provided by hospital, step is as follows.One, sample preprocessing journey:
Preparation work: open roasting sheet machine (45 DEG C-60 DEG C); Open water bath (37 ± 1 DEG C); Stomach en-damping fluid is placed in 37 ± 1 DEG C of water-bath preheatings, and use the stomach en-working fluid that the stomach en-buffer concentration after stomach en-and preheating is 1.0mg/mL, working fluid is placed in 37 ± 1 DEG C of water-bath preheatings (preheating is 1h at least).
Section dewaxing: be immersed in dimethylbenzene by section under room temperature, dewaxing 10min, changes fresh dimethylbenzene and repeats dewaxing twice again.The section completing dimethylbenzene process is immersed in 5min in dehydrated alcohol, changes fresh dehydrated alcohol and repeats once.Air-dry section or to be placed on roasting sheet machine 2-5min and to dry section after dehydrated alcohol process.
Section pre-treatment: the beaker filling ultrapure water is placed in microwave oven and heats, the ultrapure water that boiling is put in section is processed 25min, and taking-up is dried.
Pepsin: pretreated section is placed in stomach en-working fluid and digests 5-60min, period every 5min microscopy 1 time (using 10 × object lens to observe under light field), with most tissues cell dispersal for individual cells is for having digested standard; After having digested, section is immersed in ultrapure water and washs 3min.Be placed in 2-5min on roasting sheet machine and dry section.
Two, FISH detects
FISH detection comprises dehydration, hybridization, post-hybridization washing and DAPI and redyes four steps.Because the fluorophor on probe is shown in that high light easily cancellation occurs, all need in the dark to operate (lucifuge operation) so hybridization, post-hybridization washing and DAPI redye three steps.
Preparation work: probe balance is to room temperature, and vortex mixing is placed on centrifugal 2-3s in Eppendorf centrifuge; Open and preheating hybridization instrument, the wet bar of hybridization instrument is immersed in distilled water (at least soaking 2h) for subsequent use, during hybridization, puts into hybridization instrument; Glass cutter is used to iris out sample hybridising region at section reverse side.
Dehydration: section is placed in successively 70%, 85%, 100% ethanol and dewaters each 1min.Be placed in 2-5min on roasting sheet machine and dry section.
Hybridization (lucifuge operation): add 10 μ L ALK in section sample hybridising region and reset probe solution (each standard person-portion probe amount, the corresponding sample area detected is about 20mm × 20mm, the sample that sample areas is larger can increase consumption according to practical situation), covered, guarantees bubble-free under cover glass, probe is uniformly distributed.Use mounting glue to cover cover glass surrounding position, form closed sealing-ring.The hybridization instrument installing wet bar is put in section, hybridization program is set: 85 DEG C of sex change 8min, 42 DEG C of hybridized overnight (14h ~ 18h).
Post-hybridization washing (lucifuge operation): open water bath (76 ± 1 DEG C), is placed in water-bath preheating by lavation buffer solution II.Section after hybridization taken out from hybridization instrument, remove mounting glue, be placed in lavation buffer solution I 5min of room temperature, wash cover glass off, then be placed in lavation buffer solution II 5min of 76 ± 1 DEG C, period can rock section 1-3s gently;
Repeated washing is distinguished once with lavation buffer solution I and lavation buffer solution II; Finally with washing rush liquid I again repeated washing once, time during often kind of lavation buffer solution repeated washing with first time identical with time during this buffer solution.To cut into slices after washing uprightly air-dry.
DAPI redyes (lucifuge operation): add 10 μ L in sample hybridising region and redye liquid, covered, can basis of microscopic observation fluorescent signal after room temperature lucifuge 5-10min.Section after DAPI redyes also can be placed in-25 DEG C to-18 DEG C keep in Dark Place (shelf time is no more than 72h), section is put and observe to room temperature when needing to detect.
Three, result judges
The result judging criterion that a kind of ALK gene resets detection kit is:
1), every routine sample counting 50 cells, if positive cell number is less than 5 (5/50 or < 10%), this sample is judged to be feminine gender.
2), every routine sample counting 50 cells, if positive cell number is greater than 25 (25/50 or > 50%), this sample is judged to be the positive.
3), every routine sample counting 50 cells, if positive cell number is between 5-25 (10-50%), need another diagosis people to count 50 cells in addition.Gather two diagosis people counting cells numbers and positive cell number, namely amount in 100 cells, if positive cell sum is less than 15 (< 15%), this sample is judged to be feminine gender, if positive cell sum is more than or equal to 15 (>=15%), this sample is judged to be the positive.
The fluorescent signal of basis of microscopic observation may manifest variform, according to Fig. 1 signal-count guide-Typical signal pattern) Quality estimation and counting are carried out to signal.
The positive of cell judges with negative:
Move up and down field of microscope, search and be allly present in endonuclear signal.
(1) following signal mode can be judged to be that ALK resets positive cell (details are shown in Fig. 2: signal-count guide-typical positive cell signal pattern):
A. have redness and green that one group is separated at least, and spacing is more than or equal to 2 times of signaling point diameter lengths (no signal disappearance);
B. there is separately a danger signal and there is in addition a pair red/green signal (merge, all can be separated) without corresponding green.
(2) following signal mode can be judged to be that ALK resets negative cells (details are shown in Fig. 3):
A. red overlapping with green (presenting orange-yellow), tightly adjacent or to separate but distance is less than 2 times of signaling point diameter lengths (no signal disappearance);
B. there is separately green and there is in addition a pair red/green signal (merge, all can be separated) without corresponding danger signal.
In note: Fig. 1,2,3, black color dots represents danger signal point; White point represents green point; Grey Point represents orange-yellow signal.
Four, result detects and data analysis
Utilize probe of the present invention to detect 20 increment product, utilize Cytocell company kit test method to detect sample, detected result is in table 8 simultaneously.Compare with the detection of Cytocell company and detected result of the present invention, the detected result calculating detection method provided by the present invention is coincide rate.The Product checking rate of coincideing of ALK gene rearrangement detected result and Cytocell company that present method detects 20 increments bases reaches 100%.Visible FISH probe provided by the present invention can detect that ALK gene is reset exactly, and result is reliable and stable.In addition, of the present invention provided FISH also has high specificity, the feature that signal is strong.
Table 8 clinical sample detected result (" N " represents negative findings, and " P " represents positive findings)
Table 9 pattern detection result (" N " represents negative findings, and " P " represents positive findings)
The selection of embodiment 3 primer pair quantity is on the impact of sample detection result
The upstream and downstream of breaking point is reset for ALK gene, build probe library, select first group of different quantities and second group of amplimer to design 4 groups test respectively, to study the amplified production of different quantities (namely, probe) whether it to form corresponding probe library Detection results consistent, and wherein test group 1 is each selection 1 pair of amplimer pair; Test 2 is each selection 3 pairs of amplimers pair; Test 3 is each selection 5 pairs of amplimers pair; Test 4 is each selection 10 pairs of amplimers pair.Concrete test arrangement is in table 10, and the synthesis of probe, the structure of probe library, probe mark and experimental procedure etc. are as implemented shown in 1 and embodiment 2.
Table 10 amplimer to and amplified production
Use the probe library that above-mentioned test group prepares respectively, detect sample (sample number into spectrum 21 ~ 40), and with the detected result of Cytocell Products in contrast.Detected result following (referring to table 11,12):
Table 11 sample detection result
Table 12 sample detection result
Can find out according to table 11,12, the upstream and downstream of breaking point is reset for ALK gene, select first group of different quantities and second group of amplimer to structure probe library respectively, the amplified production of different quantities (namely, probe) to form corresponding probe library Detection results consistent for it, wherein use the probe library that 5 pairs of amplimers build respectively, its sensitivity detected, specificity and stability are all fine, and use 8 to 10 pairs of amplimers pair, reach best to make the sensitivity of probe library, specificity and stability.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1.ALK gene rearrangement detection kit, it is characterized in that, include mark fluorescent dyestuff, first group of probe for breaking point upstream and second group of probe for breaking point downstream, described first group of probe is for being selected from least one probe in SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is for being selected from least one probe in SEQ ID NO.51-SEQ ID NO.60; The fluorescence dye color that described first group of probe and second group of probe mark is different.
2. ALK gene according to claim 1 resets detection kit, it is characterized in that, described first group of probe is be selected from least five probes in SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is be selected from least five probes in SEQ ID NO.51-SEQ ID NO.60.
3. ALK gene according to claim 2 resets detection kit, it is characterized in that, described first group of probe is be selected from least eight probes in SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is be selected from least eight probes in SEQ ID NO.51-SEQ ID NO.60.
4. ALK gene according to claim 3 resets detection kit, and it is characterized in that, described first group of probe is SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is SEQ IDNO.51-SEQ ID NO.60.
5. the ALK gene according to any one of claim 1-4 resets detection kit, it is characterized in that, described SEQ ID NO.41-SEQ ID NO.50 is prepared by following primer amplification successively respectively: SEQ IDNO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ IDNO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ IDNO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20.
6. the ALK gene according to any one of claim 1-4 resets detection kit, it is characterized in that, described SEQ ID NO.51-SEQ ID NO.60 is prepared by following primer amplification successively respectively: SEQ IDNO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.39 and SEQ IDNO.40.
7.ALK gene rearrangement detection probes, it is characterized in that, first group of probe for breaking point upstream and second group of probe for breaking point downstream, described first group of probe is for being selected from least one probe in SEQ ID NO.41-SEQ IDNO.50, and described second group of probe is for being selected from least one probe in SEQ ID NO.51-SEQ IDNO.60.
8. ALK gene according to claim 7 resets detection probes, it is characterized in that, described first group of probe is be selected from least five probes in SEQ ID NO.41-SEQ ID NO.50, and described second group of probe is be selected from least five probes in SEQ ID NO.51-SEQ ID NO.60.
9. ALK gene resets a detection method, it is characterized in that, comprises the following steps:
Detected sample section pre-treatment;
Utilize ALK gene described in any one of claim 1-6 to reset detection kit and carry out hybridization check;
Fluorescent signal is examined under a microscope after redying.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227350A (en) * | 2017-06-09 | 2017-10-03 | 苏州达麦迪生物医学科技有限公司 | A kind of probe groups, kit and the method for the fracture of quick detection ALK gene |
| CN109504738A (en) * | 2018-11-09 | 2019-03-22 | 益善生物技术股份有限公司 | A kind of Probe labelling kit and its application |
| CN110541032A (en) * | 2019-09-16 | 2019-12-06 | 重庆威斯腾生物医药科技有限责任公司 | Probe, FISH kit and method for rapidly detecting ALK gene rearrangement |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227350A (en) * | 2017-06-09 | 2017-10-03 | 苏州达麦迪生物医学科技有限公司 | A kind of probe groups, kit and the method for the fracture of quick detection ALK gene |
| CN109504738A (en) * | 2018-11-09 | 2019-03-22 | 益善生物技术股份有限公司 | A kind of Probe labelling kit and its application |
| CN110541032A (en) * | 2019-09-16 | 2019-12-06 | 重庆威斯腾生物医药科技有限责任公司 | Probe, FISH kit and method for rapidly detecting ALK gene rearrangement |
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