CN104278093B - Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion primer to and kit - Google Patents
Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion primer to and kit Download PDFInfo
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Abstract
The invention discloses the primer of a kind of Manganic pyrophosphate complex initiation method detection SIL TAL1 fusion to and the kit that comprises described primer pair, this kit can disposably detect SIL TAL1 (type II) and two kinds of fusions of SIL TAL1 (type III).By the mRNA design primer to SIL TAL1 fusion, carrying out sequencing analysis to product after reverse transcription PCR, this kit can realize the detection quick, accurate, high-throughout of SIL TAL1 fusion.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion
Primer pair, comprise the kit of described primer pair and the application of this kit.
Background technology
SIL-TAL1 fusion includes the polytype such as SIL-TAL1 (type II), SIL-TAL1 (type III), often
See in T cell ALL (T-ALL).
In the method for the outer widely used molecular Biological Detection SIL-TAL1 fusion of Present Domestic, fluorescent in situ is miscellaneous
Friendship technology (FISH) can only carry out qualitative detection, operation complexity；Quantitative fluorescent PCR also exists the limitation of detection flux, so
All can't really meet the needs of clinical diagnosis detection.Traditional solid phase biological chip (Biochip) or biochip technology
Also exist repeatable poor, insufficient sensitivity good and the prominent weakness of complex operation.
Pyrosequencing techniques (Pyrosequencing) is a new generation's DNA sequence analysis technology, possesses simultaneously in a large number
Sample carries out the ability of sequencing analysis, is high flux, low cost, carries out nucleotide analysis and clinical examination carries quickly and intuitively
Supply ideal technical operation platform.
Content of the invention
Goal of the invention: for technical problem present in prior art, the present invention proposes a kind of Manganic pyrophosphate complex initiation method simultaneously
The kit introducing to and containing described primer pair of detection SIL-TAL1 fusion, can detect SIL-TAL1 simultaneously
(type II) and two kinds of fusions of SIL-TAL1 (type III), have high sensitivity, high specific, high accuracy, detection
The advantage such as rapidly.
Technical scheme: for realizing above-mentioned technical purpose, the present invention proposes a kind of Manganic pyrophosphate complex initiation method and detects SIL-simultaneously
The primer pair of TAL1 fusion, described SIL-TAL1 is SIL-TAL1 (type II) and in SIL-TAL1 (type III)
Any one or two kinds, it is characterised in that described primer is to including:
(1) for SIL-TAL1 (type II) gene,
Amplimer is:
Upstream primer: 5 '-TACCCTGCAAACAGACCTCAGCT-3 ',
Downstream primer: 5 '-TTCCCCCTTTTTCGCTGAGA-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer is:
5′-AGAGGCCTGCAGTTAC-3′；
(2) for SIL-TAL1 (type III) gene:
Amplimer:
Upstream primer: 5 '-CCTCCCAAAATGCTGATCG-3 ',
Downstream primer: 5 '-GTCTTTGCTTTCCCCCTTTTTC-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer:
5′-GGCCTGCAGTTACGCT-3′。
Present invention further proposes the kit of a kind of Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion, this reagent
Box comprises above-mentioned primer, and specifically, described kit comprises following primer:
(1) for SIL-TAL1 (type II):
Amplimer:
Upstream primer: 5 '-TACCCTGCAAACAGACCTCAGCT-3 ',
Downstream primer: 5 '-TTCCCCCTTTTTCGCTGAGA-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer:
5′-AGAGGCCTGCAGTTAC-3′；
(2) for SIL-TAL1 (type III):
Amplimer:
Upstream primer: 5 '-CCTCCCAAAATGCTGATCG-3 ',
Downstream primer: 5 '-GTCTTTGCTTTCCCCCTTTTTC-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer:
5′-GGCCTGCAGTTACGCT-3′。
More specifically, described kit also includes quality-control product.Described quality-control product includes positive reference substance and negative controls,
Wherein, described positive reference substance is the mixing containing SIL-TAL1 (type II) and the plasmid of SIL-TAL1 (type III)
Liquid, described negative controls, as comparison, is in a ratio of without SIL-TAL1 (type II) and SIL-TAL1 with positive reference substance
The plasmid solution of (type III).
The invention allows for above-mentioned primer to and the kit that comprises above-mentioned primer pair melt at preparation detection SIL-TAL1
Close the application in the reagent of gene.
Beneficial effect: compared with prior art, the examination of the Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion of the present invention
Agent box be capable of quick, easy, efficient, accurately detect SIL-TAL1 (type II) and SIL-TAL1 (type III) melt
Close gene, be used for detecting SIL-TAL1 (type II) for preparation and the reagent of SIL-TAL1 (type III) fusion provides
Theoretical foundation.
Detailed description of the invention
Experiment material:
Primer is synthesized by invitrogen company；Trizol is purchased from invitrogen company；Reverse transcription cDNA synthetic agent
Box is purchased in Fermentas company；Multiple PCR reagent kit is purchased in QIAGEN company；Manganic pyrophosphate complex initiation reagent purchase in
QIAGEN company.
The kit of a kind of Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion, described SIL-TAL1 is SIL-TAL1
Any one or two kinds in (type II) and SIL-TAL1 (type III), including quality-control product and following primer:
(1) SIL-TAL1 (type II):
Amplimer:
Upstream primer: 5 '-TACCCTGCAAACAGACCTCAGCT-3 ',
Downstream primer: 5 '-TTCCCCCTTTTTCGCTGAGA-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer:
5 '-AGAGGCCTGCAGTTAC-3 ',
(2) SIL-TAL1 (type III):
Amplimer:
Upstream primer: 5 '-CCTCCCAAAATGCTGATCG-3 ',
Downstream primer: 5 '-GTCTTTGCTTTCCCCCTTTTTC-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer:
5′-GGCCTGCAGTTACGCT-3′。
Wherein, quality-control product (quality-control product DNA) includes positive reference substance and negative controls, described positive reference substance for containing
There is the mixed liquor of the plasmid of SIL-TAL1 (type II) and SIL-TAL1 (type III)；Described negative controls is as the moon
Property comparison, compared with positive reference substance, negative controls is for without SIL-TAL1 (type II) and SIL-TAL1 (type III)
Plasmid solution.
Detection method comprises the steps:
(1) RNA extracts:
Take the clinical sample of 20 T-ALL patients；The clinical sample of 1-20 patient extracts respectively according to the following steps
RNA:
(1) lymphocyte extract: take fresh whole blood 2ml and add 1ml 3wt% sodium citrate, after mixing at 4 DEG C 3000r/
Min centrifuges 10min, takes pale yellow chromatograph on blood cell layer and is lymphocyte, obtains lymphocyte liquid；
(2) take 1 centrifuge tube (through DEPC water process) and add the lymphocyte liquid 150 μ l in (1), then add 1ml
Trizol, room temperature places 5min so that it is fully crack；
(3) 12,000r/min centrifuges 5min, takes supernatant；
(4) adding 200 μ l chloroforms in supernatant, after acute vibration mixes, room temperature places 15min；
15min is centrifuged under (5) 4 DEG C of 12,000g；
(6) draw upper strata aqueous phase, aqueous phase is transferred in another centrifuge tube；
(7) adding 500 μ l isopropanols to mix in aqueous phase, room temperature places 5～10min；
(8) 4 DEG C 12, centrifuging 10min, abandon supernatant under 000g, RNA is sunken at the bottom of pipe；
(9) adding 1ml 75% (v/v) ethanol in the pipe of step (8), gentle vibration centrifuge tube, suspend precipitation；
(10) 4 DEG C 8,000g centrifuges 5min, abandons supernatant as far as possible；
(11) room temperature is dried or is vacuum dried 5～10min；
(12) with 50ul RNase-free dH2O dissolves RNA sample, 55～60 DEG C, 5～10min；
(13) OD value quantitative RNA concentration is surveyed.
According to above-mentioned steps, obtain the mRNA of 1-20 sample respectively.
(2) multiple reverse transcription PCR amplification:
Carry out multiple reverse transcription PCR amplification as follows to the mRNA of above-mentioned 1-20 sample:
(1) synthesis of cDNA the first chain
1. take the centrifuge tube of a RNase-free, be placed on ice, set up following reaction system；
Total RNA 5～10 μ g/3 μ l
Oligo(dT)18 Primer(0.5μg/μl) 1μl
RNase-free dH2O adds to 12 μ l
Mix reactant liquor gently, somewhat centrifuge the reactant liquor collected on tube wall with refrigerated centrifuge at the bottom of pipe；
2. centrifuge tube incubates 5 minutes in 70 DEG C, takes out rapidly afterwards and is placed in ice cooling, somewhat centrifugal with refrigerated centrifuge
Collect the reactant liquor on tube wall at the bottom of pipe；
3. centrifuge tube is positioned on ice, is sequentially added into following reactant liquor；
5×reaction buffer 4μl
RNase Inhibitor(20U/μl) 1μl
10mM dNTPs Mix 2μl
Mix reactant liquor gently, somewhat centrifuge the reactant liquor collected on tube wall with refrigerated centrifuge at the bottom of pipe；
4. centrifuge tube incubates 5 minutes in 37 DEG C；
5. adding 1 μ l RevertAidTM Reverse Transcriptase (200U/ μ l), final volume is 20 μ l；
6. centrifuge tube reacts 60 minutes in 42 DEG C；
7. centrifuge tube terminates reaction for 10 minutes in 70 DEG C of heating, takes out rapidly afterwards and is placed in ice cooling.
So, cDNA the first chain, i.e. template cDNA (Template cDNA) are synthesized.
(2) multiplex PCR
Using the Multiplex PCR kit of QIAGEN company, system is as follows
Template cDNA or quality-control product DNA 10 μ l
2×QIAGEN Multiplex PCR Master Mix 25μl
10×Primer mix 5μl
RNase-free dH2O 10μl
Final volume is 50 μ l
Wherein, the enzyme of TaqDNA containing thermal starting, MgCl in 2 × QIAGEN Multiplex PCR Master Mix2And dNTP
Mix；10 × Primer mix is the mixture comprising above-mentioned three kinds of fusion primers pair.
PCR amplification program: 95 DEG C of 15min；94 DEG C of 30s, 55 DEG C of 90s, 72 DEG C of 90s, 35 circulations；72℃10min；4℃
Insulation.
Pass through above-mentioned steps, it is thus achieved that PCR primer.
(3) Manganic pyrophosphate complex initiation
First, fixing PCR primer with microballon, biotin labeled PCR primer is mainly fixed to Streptavidin by it
On coated Agarose microbead (Streptavidin Sepharose High Performance), comprise the steps:
(1) jog is coated the Agarose microbead of Streptavidin, until obtaining homogeneous solution；
(2) in a test tube, mix Streptavidin coated Agarose microbead total amount (2 μ l/ sample) and combine buffering
Liquid (40 μ l/ sample), adds ultra-pure water to certain volume, and this volume to add, with follow-up, optimized biotin labeled
The summation of the volume of PCR primer is 80 μ l/ holes；
(3) add the solution of the certain volume obtaining in (2) to 24 hole PCR orifice plates；
(4) arrange according to orifice plate, each of the biotin labeled PCR primer that interpolation 5～20 μ l have optimized to PCR orifice plate
Hole slot so that it is in solution contained by every hole be 80 μ l；
(5) orifice plate bar lid is used to seal PCR orifice plate, it is ensured that not leakage between hole slot；
(6) shaker mixer (1400rpm) constantly vibration PCR orifice plate at least 5～10 minutes are used.
Through above-mentioned steps, biotin labeled PCR primer is fixed on the coated Agarose microbead of Streptavidin.
Then, start vacuum work station preparation, comprise the steps:
(1) following reagent is prepared: about 50ml 70% (v/v) ethanol；About 40ml denaturing soln (QIAGEN company)；
About 50ml 1 × lavation buffer solution (QIAGEN company))；About 50m1 ultra-pure water；About 70ml ultra-pure water；
(2) open vavuum pump: open vacuum switch, carry out testing experiment, to determine whether filter probe normally works；
(3) detect the permeability of filter probe before using vavuum pump every time with ultra-pure water, preprepared is installed
The centrifuge tube of ultra-pure water is inserted in PCR device, falls into filter probe in ultra-pure water, and ultra-pure water is as being evacuated then table in 20 seconds
Bright filter probe is normal, it is possible to use, on the contrary then need to change filter probe；
(4) take off the PCR orifice plate having vibrated, sample is put in PCR device, carefully fall filter probe to PCR orifice plate
In, stop 15 seconds；Guarantee that solution is all sucked away, with the microballon containing fixed form for the capture；
(5) vacuum plant is moved to the first reagent trough containing 70% (v/v) ethanol, washing and filtering probe 5 seconds；
(6) vacuum plant is moved to the second reagent trough containing denaturing soln, washing and filtering probe 5 seconds；
(7) vacuum plant is moved to the 3rd reagent trough containing lavation buffer solution, washing and filtering probe 10 seconds；
(8) vacuum plant is raised more than 90 ° of vertical lines 5 seconds, discharge opeing from filter probe；
(9) vacuum plant is gripped on Q24 orifice plate, the vacuum switch on shutoff device unsettled stop 5 seconds, allow negative pressure dissipate
Go；
(10) left and right jog vacuum plant is passed through, in release pearl to the orifice plate containing 3 kinds of sequencing primers；
(11) vacuum plant is transferred to the 4th reagent trough containing ultra-pure water in the case that vacuum plant cuts out, vibration
10 seconds；
(12) fall probe in another the 5th reagent trough containing ultra-pure water and apply vacuum, clean probe, use 70ml
Ultrapure water filter probe；
(13) vacuum plant is raised more than 90 ° of vertical lines 5 seconds, discharge opeing from filter probe；
(14) close the switch on vacuum plant, and be placed on static (P) position.
So become the preparation completing vacuum station, be then initially separated DNA strand and sample is discharged into
In PyroMark Q24 orifice plate, specifically comprise the steps:
(1) PyroMark Q24 orifice plate is placed on the orifice plate seat of preheating 80 DEG C of accurate heating 2 minutes；
(2) from orifice plate seat, take off orifice plate, make sample at room temperature at least cool down 5 minutes.
Through above-mentioned steps, sample is discharged in PyroMark Q24 orifice plate by cup.It is then ready for PyroMark Q24 examination
Agent, is carried out in the steps below:
(1) open PyroMark Q24 kit and take out the bottle containing enzyme and substrate freeze-dried powder, and containing nucleosides
The test tube of acid；
(2) dissolving and dispense enzyme and substrate according to kit specification ultra-pure water, all reagent need to recover after room temperature again
Use；
(3) volume going out according to computer calculation, adds enzyme, substrate and nucleotides A, T, G, C (reagent in agent bin
Storehouse at most uses 30 times, and agent bin must be dry before use).
After getting out PyroMark Q24 reagent, PyroMark Q24 instrument is tested in the steps below:
(1) open PyroMark Q24 software, click on new assay (newly test) → new AQ assay (new AQ test)
→ in sequence to analyze (sequence to be analyzed), input catastrophe point sequence, click on Generate Dispensation
Order (generation allocation order), clicks on and preserves；
(2) click on new run (newly running) → Instrument method (instrumental method) → 007, then click on and to survey
The grid of sequence, clicks on and preserves to USB flash disk；
(3) by the USB port before the USB flash disk inserting instrument containing operating file；
(4) orifice plate heating is put on instrument；
(5) label surface of agent bin (enzyme, substrate and nucleotides) is put into instrument towards oneself, open orifice plate support saddle frame and put
Enter orifice plate, close orifice plate support saddle frame and instrument lid；
(6) select Run (operation), and press OK；
(7) after entering Run (operation), file to be run is selected by select (selection)；
(8) instrument end of run confirm that operating file has preserved to USB flash disk, by close, takes out USB flash disk；
(9) open instrument, take out agent bin, and it is cleaned repeatedly, discarded orifice plate；
(10) when instrument does not runs, select shutdown (shutdown) from main menu and press OK；
(11) when It is now safe to turn offthe instrument (now can be with safety shutdown) occurs
When, instrument can be closed, power switch is positioned at after instrument.
(4) interpretation of result:
Open Pyromark Q24 software, analyze SIL-TAL1 (type II) and SIL-TAL1 (type III) and merge base
The type of cause, analysis result such as following table:
| Patient's fusion type | SIL-TAL1(type II) | SIL-TAL1(type III) |
| 1 | Negative | Positive |
| 2 | Positive | Negative |
| 3 | Positive | Negative |
| 4 | Negative | Positive |
| 5 | Positive | Negative |
| 6 | Negative | Positive |
| 7 | Positive | Negative |
| 8 | Positive | Negative |
| 9 | Negative | Positive |
| 10 | Positive | Negative |
| 11 | Negative | Positive |
| 12 | Positive | Negative |
| 13 | Negative | Positive |
| 14 | Positive | Negative |
| 15 | Positive | Negative |
| 16 | Positive | Negative |
| 17 | Negative | Positive |
| 18 | Positive | Negative |
| 19 | Positive | Negative |
| 20 | Negative | Positive |
| Positive control | Positive | Positive |
| Negative control | Negative | Negative |
Result shows, the 2nd, the 3rd, the 5th, the 7th, the 8th, the 10th, the 12nd, the 14th, the 15th, the 16th, the 18th, No. 19 patients merge for SIL-TAL1 (type II)
Gene masculine；1st, the 4th, the 6th, the 9th, the 11st, the 13rd, the 17th, No. 20 patients positive for SIL-TAL1 (type III) fusion.
To sum up, apply the present invention kit be capable of quick, easy, efficient, accurately detect SIL-TAL1 (type
And SIL-TAL1 (type III) fusion II).
After having read above with respect to the those set forth of the present invention, the present invention can be made respectively by those skilled in the art
Planting modification or change, including the various changes of primer, these equivalent form of values belong to the application claims equally
Scope defined in book.
Claims (6)
1. the primer pair of a Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion, described SIL-TAL1 is SIL-TAL1
Type II and SIL-TAL1 type III, it is characterised in that described primer is to including:
(1) for SIL-TAL1 type II gene,
Amplimer is:
Upstream primer: 5 '-TACCCTGCAAACAGACCTCAGCT-3 ',
Downstream primer: 5 '-TTCCCCCTTTTTCGCTGAGA-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer is:
5′-AGAGGCCTGCAGTTAC-3′；
(2) for SIL-TAL1 type III gene:
Amplimer is:
Upstream primer: 5 '-CCTCCCAAAATGCTGATCG-3 ',
Downstream primer: 5 '-GTCTTTGCTTTCCCCCTTTTTC-3 ',
Wherein 5 ' ends of downstream primer carry out biotin labeling；
Sequencing primer:
5′-GGCCTGCAGTTACGCT-3′。
2. the kit of a Manganic pyrophosphate complex initiation method detection SIL-TAL1 fusion, it is characterised in that described kit comprises
Primer pair as claimed in claim 1.
3. kit according to claim 2, it is characterised in that described kit includes quality-control product, described quality-control product
Including positive reference substance and negative controls.
4. kit according to claim 3, it is characterised in that described positive reference substance is for containing SIL-TAL1
Type II, the mixed liquor of plasmid of SIL-TAL1 type III, described negative controls is for without SIL-TALl type
The plasmid solution of II, SIL-TAL1 type III.
5. the primer described in claim 1 is to the application in the reagent of preparation detection SIL-TAL1 fusion.
6. application in the reagent of preparation detection SIL-TAL1 fusion for the kit described in Claims 2 or 3.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955991A (en) * | 2010-03-31 | 2011-01-26 | 江苏迈迪基因生物科技有限公司 | Joint detection method of fusion genes related to leukemia and diagnosis kit |
| CN102251031A (en) * | 2011-06-30 | 2011-11-23 | 北京思尔成生物技术有限公司 | TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by same |
| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
-
2014
- 2014-09-28 CN CN201410510911.9A patent/CN104278093B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955991A (en) * | 2010-03-31 | 2011-01-26 | 江苏迈迪基因生物科技有限公司 | Joint detection method of fusion genes related to leukemia and diagnosis kit |
| CN102251031A (en) * | 2011-06-30 | 2011-11-23 | 北京思尔成生物技术有限公司 | TaqMan MGB probe real-time fluorescence PCR detection method for leukemia fusion genes, and special primers, probes and kit used by same |
| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
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