CN110218774A - For detecting the primer, kit and method of SYT-SSX fusion - Google Patents
For detecting the primer, kit and method of SYT-SSX fusion Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses the primers, kit and detection method for detecting SYT-SSX fusion, including detecting the primer of SYT-SSX1 or SYT-SSX2 fusion and the primer and probe of probe and detection reference gene.Compared with the technologies such as direct Sequencing, the present invention detects high specificity, high sensitivity for SYT-SSX fusion, it is easy to operate quickly, high-throughput, interpretation result is reliable, the advantages such as simplicity.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of ARMS- for the detection of SYT-SSX fusion
QPCR primer, kit and method, can quick, sensitive, accurate detection SYT-SSX fusion.
Background technique
Synovial sarcoma is more typical soft tissue malignant tumour, accounts for the 5%-10% of soft tissue sarcoma.Histological type point 4
Kind: unipolar type, biphasic or bipolar type, single-phase epitheliated type and low differentiated [1].Synovial sarcoma metamorphosis is complicated, needs to take by many places
Material and find tumour two-phase differentiation feature can make a definite diagnosis.Such as between twenty and fifty patient of the typical case of synovial sarcoma, betides four limbs
Tumour near large joint, X-ray examination are shown in different degrees of calcification, the spy that in conjunction with slice discovery tumour there is two-phase to break up
Sign, makes a definite diagnosis not difficult.But some synovial sarcomas such as kidney, basis cranii, pectoralis major etc. for betiding gerontal patient and special-site,
If the Epithelial differentiation of tumour is unobvious, diagnose more difficult.Therefore, it is necessary to a kind of simple and reliable methods to diagnose
Synovial sarcoma.There are special chromosome abnormality t (x for the synovial sarcoma of 90% or more document report；18) (p11.2, q11.2),
SYT Gene Fusion on No. 18 chromosomes is caused to be formed SYT-SSX fusion [2] to X chromosome SSX gene.In non-synovial membrane meat
In tumor tissue, then it can't detect the transcription of the fusion.Therefore, SYT-SSX fusion has spy for synovial sarcoma
The opposite sex, the mRNA for detecting them can be used as the diagnosis index of synovial sarcoma.Since the breaking point of X dyeing is different, fusion
Main type is divided into SYT-SSX1 type and SYT-SSX2 type [3].Research shows that SYT-SSX fusion may with tumour growth and
Progress has a close ties, the poor prognosis of SYT-SSX1 type ratio SYT-SSX2, they can also be used for detection conditions of patients development and
Prognosis.
In practical applications, have much for the method for fusion, such as RT-PCR, northern blot.This project
SYT-SSX mRNA expression is detected using most popular real-time fluorescence quantitative PCR at present.Real-time fluorescence quantitative PCR have compared with
High sensitivity and specificity, and real-time online detection can be carried out to PCR, reaction fusion is the presence or absence of in the sample and just
A large amount of detection time has been saved in beginning content, test, it is thus also avoided that the generation of carryover contamination.Common method has SYBR
GreenI dye method, double probe hybrid methods and Taqman technology etc..Wherein SYBR GreenI is special due to being non-saturable dye
The opposite sex is not as good as double probe hybrid methods and Taqman method, it is necessary to its specificity is judged by observation solubility curve；And double probes
Method hybrid method cost again costly.Therefore this research is using real-time fluorescent PCR technology combination Taqman probe application in SYT-
The detection of SSX fusion.In addition, it is bent using double marks in calculation method, using this algorithm not only to each concentration of specimens without spy
It is different to require (the mRNA concentration of each sample adjust unanimously), and slope of a curve can also be corrected further caused by amplification accidentally
Difference.
Summary of the invention
In order to overcome the above-mentioned defects in the prior art, the present invention provides one kind for detecting SYT-SSX fusion
Primer, which is characterized in that the primer includes:
(1) primer and probe of SYT-SSX1 or SYT-SSX2 fusion, base sequence are detected are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAMRA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
(2) primer and probe of reference gene, base sequence are detected are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '.
Further, SYT-SSX-F, SSX1-R SSX2-R and SYT-SSX-TaqMan concentration ratio be 1:1:1.
Further, the use concentration ratio of Actin-F, Actin-R and Actin-Probe are 1:1:1.
The present invention also provides a kind of for detecting the ARMS-QPCR kit of SYT-SSX fusion, and feature exists
In the kit includes qPCR reaction system, and qPCR reaction system includes that qPCR mixed reaction solution, reaction primer and the positive are right
Product in the same old way；
QPCR mixed reaction solution includes PCR buffer, dNTP, MgCl2, KOD enzyme, PCR upstream and downstream primer and TaqMan visit
Needle, the primer for detecting reference gene and probe and positive control sample；In qPCR reaction system, the final concentration of each component is distinguished
Are as follows: PCR buffer is 1X；DNTP is 1.5mM；MgCl2For 2mM；KOD enzyme is 0.6U/ μ l；PCR upstream and downstream primer is 0.1 μM；
TaqMan probe is 0.1 μM；Positive control sample is 1ng/ μ l；Wherein
The PCR upstream and downstream primer, the sequence of TaqMan probe are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAMRA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
The primer and probe of the detection reference gene can expand all difference cDNA templates, base sequence are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '.
The positive control sample is respectively the cDNA plasmid of SYT-SSX1 or SYT-SSX2 fusion.
The present invention finally provides a kind of ARMS-qPCR method for detecting SYT-SSX fusion, which is characterized in that packet
Include following steps:
(1) total serum IgE is extracted from cell or blood, reverse transcription is at cDNA as template；
(2) real-time fluorescence quantitative PCR amplification is carried out, 5 pipes of detection point of each sample carry out, and each pipe is added identical
QPCR mixed reaction solution, template cDNA, every pipe are separately added into primer, and one of probe and two kinds of ARMS primers carry out SYT-
The detection of SSX fusion；
Wherein, the primer and probe of SYT-SSX1 or SYT-SSX2 fusion, base sequence are detected are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAM RA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
Detect the primer and probe of reference gene, base sequence are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '；
(4) result interpretation:
A. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid
Pipe is positive, and SSX1-R pipe amplification curve feminine gender and SSX2-R pipe amplification curve are negative, which is determined as that SYT-SSX is merged
Gene negative；
B. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid
The pipe positive SSX1-R pipe amplification curve positive and SSX2-R pipe amplification curve are positive；The sample results are determined as that SYT-SSX is merged
Gene masculine；
C. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid
Pipe positive SSX1-R pipe amplification curve is positive or SSX2-R pipe amplification curve is positive, the sample results be determined as SYT-SSX1 or
SYT-SSX2 fusion is positive；
D. negative referring to primer Actin pipe amplification curve, it prompts the sample extraction to fail, needs to re-start extraction.
Further, in step (3), the condition for carrying out pcr amplification reaction is 95 DEG C of initial denaturations 60s, 95 DEG C of denaturation 15s,
60 DEG C of extension 35s, 40 circulations.
The Full Name in English of qPCR is Real-time Quantitative PCR Detecting System in the present invention,
Real time fluorescent quantitative nucleic acid amplification detection system is also real-time quantitative gene magnification fluorescence detecting system, abbreviation qPCR.
The present invention provides the ARMS-QPCR kits of SYT-SSX fusion detection, to solve existing fusion inspection
Time-consuming in survey technology, the problems such as program is cumbersome and easy to pollute.This kit can quickly, accurate, cheap, high throughput detects
SYT-SSX fusion, high sensitivity, high specificity are suitable for common sample such as blood, tissue, paraffin, mucous membrane of mouth
And hair etc..Compared with the technology of the Genotypings such as direct Sequencing, the present invention has special for the detection of SYT-SSX fusion
Property strong, high sensitivity, it is easy to operate quickly, high-throughput, interpretation result is reliable, the advantages such as simplicity.
Kit of the invention can be quick, cheap, accurate, detects blood of human body or other histocytes with high throughput
In SYT-SSX fusion situation be current cost performance the best way for screening detection technique, convenient for clinical or
Health detection is carried out on a large scale.
ARMS used in the present invention (amplification refractory mutation system) expands obstruction
Abruptly-changing system, for being detected to known mutations gene.By design, 3 ' end primers carry out two parallel PCR to the method respectively,
It only just may extend away with the primer of SSX1 or SSX2cDNA complete complementary and obtain pcr amplification product.If mispairing is located at primer
3 ' ends then cause PCR that cannot extend, then referred to as ARMS.
The sensitivity of ARMS detection dependent on ARMS primer specificity and MgCl2 in reaction condition such as reaction solution it is dense
Degree, adds the optimization that DMSO etc. is not added.In order to increase the specificity of primer, reduces primer and prolong with the mispairing for targeting the wrong timing of cDNA
It stretches, another or two base mismatch can be introduced by the 1st, 3 base held in primer 3 ', be allowed to the shape between template
Extended at multiple mispairing with IMPEDANCE WRONG.
Probe used in the present invention is the TaqMan probe of 5 ' end FAM labels, and probe both ends distinguish mark fluorescent and report base
Group (R) and fluorescent quenching group (Q).When probe is complete, i.e., in stochastic regime and when without PCR product hybridized state, report base
The fluorescence that group issues is quenched group absorptions, can't detect the presence of fluorescence.In ARMS-qPCR amplification procedure, when special
When hybridization reaction occurs for PCR product and TaqMan probe, 5 ' end 5 prime excision enzyme activities of KOD enzyme also the base of TaqMan probe by
A shearing, the fluorimeter that the fluorescence that reporter group is released can be built into PCR instrument detect.PCR passes through one
Circulation, fluorescence signal is also as target fragment, as soon as there is a process for sync index amplification, the power of fluorescence signal represents mould
The number of plate cDNA copy number.Therefore the present invention is a tool for good Genotyping.
The advantages of combining ARMS primer and real-time fluorescence quantitative PCR, compared with the technologies such as direct Sequencing, the present invention
For having the advantage that for SYT-SSX fusion detection
1, high specificity: the ARMS primer of design can specifically expand corresponding SYT-SSX fusion；In ARMS primer
The the 1st, 3 base of inverse at 3 ' ends introduces another or two base mismatch, increases the specificity of ARMS primer.
2, the sensitivity of the technology of the present invention is up to 1%, much higher than the sensitivity of DNA direct Sequencing 15%；
3, a possibility that detection process is stopped pipe reaction, greatly reduces pollution and result error；
4, operation is quick and easy, can complete within 3 hours from sample inspection to result is obtained.And direct sequencing detects
Complex steps: inspection sample → extraction DNA → PCR amplification → verifying PCR product (electrophoresis) → purified pcr product → direct Sequencing
→ interpretation of result, the electrophoresis process Jing Guo two PCR products, pollution probability is very big, is not suitable for carrying out on a large scale in hospital；
5, interpretation result is clearly objective, if desired when can to result carry out quantitative analysis；
6, high-throughput, it once can detecte 96 samples；
7, safety does not include poisonous and harmful substance in whole system, without the post-processing of PCR product, to operator and
Environment is all harmless.
In short, compared with the technologies such as direct Sequencing, the present invention is for SYT-SSX fusion detection high specificity, sensitive
Degree is high, it is easy to operate quickly, high-throughput, interpretation result is reliable, the advantages such as simplicity.
Detailed description of the invention
Fig. 1 is the curve graph that normal sample is detected with SYT-SSX fusion ARMS-qPCR method.Wherein positive plasmid
It is shown as initial line morning, Ct value is forward；And internal reference is later in plasmid initial line.Negative sample does not have initial line.
Specific embodiment
Combined with specific embodiments below and attached drawing, the present invention is further explained.It should be noted that not specified in embodiment
Normal condition and method, usually routinely use method according to fields experimenter: for example, Ao Sibai and James Kingston chief editor
" fine works molecular biology experiment guide " fourth edition, or according to step proposed by manufacturer and condition.
Embodiment 1
For detecting the ARMS-QPCR kit of SYT-SSX fusion, including qPCR reaction system, qPCR reactant
System includes qPCR mixed reaction solution, reaction primer and positive control sample；
QPCR mixed reaction solution includes PCR buffer, dNTP, MgCl2, KOD enzyme, PCR upstream and downstream primer and TaqMan visit
Needle, the primer for detecting reference gene and probe and positive control sample；In qPCR reaction system, the final concentration of each component is distinguished
Are as follows: PCR buffer is 1X；DNTP is 1.5mM；MgCl2For 2mM；KOD enzyme is 0.6U/ μ l；PCR upstream and downstream primer is 0.1 μM；
TaqMan probe is 0.1 μM；Positive control sample is 1ng/ μ l；Wherein
PCR upstream and downstream primer, the sequence of TaqMan probe are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAMRA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
The primer and probe for detecting reference gene can expand all difference cDNA templates, base sequence are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '.
Positive control sample is respectively the cDNA plasmid of SYT-SSX1 or SYT-SSX2 fusion.
Embodiment 2
Testing process:
(1) the tissue RNA in blood is extracted.
(2) reverse transcription: with reference to the Rever Tra Ace qPCR RT Kit kit specification of TOYOBO company, by (1)
In tissue RNA reverse transcription be cDNA.
(3) reagent configures: detecting qPCR by detection person-portion number configuration fusion detection qPCR reaction solution and reference gene
Reaction solution；
(4) it is loaded: the cDNA obtained in 2ul step (2) being taken to be added to the fusion detection qPCR configured in (3) respectively
In reaction solution and reference gene detection qPCR reaction solution；For positive reference substance experiment, directly add 2ul positive reference substance；It is right
For negative controls experiment, directly add 2ul negative controls；For the experiment of blank control product, add 2ul physiological saline or not
Add any substance.
(5) detect: detection carries out on real-time fluorescence PCR instrument, can include ABI7300, the 7500 (U.S. with instrument
Applied Biosystems company) etc..Reaction condition: 95 DEG C of initial denaturation 60s, 95 DEG C of denaturation 15s, 60 DEG C of extension 35s, 40
Circulation, fluorescence signal acquire when 58 DEG C of 35sec.
(6) result judges:
A. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid
Pipe is positive, and SSX1-R pipe amplification curve feminine gender and SSX2-R pipe amplification curve are negative, which is determined as that SYT-SSX is merged
Gene negative；
B. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid
The pipe positive SSX1-R pipe amplification curve positive and SSX2-R pipe amplification curve are positive；The sample results are determined as that SYT-SSX is merged
Gene masculine；
C. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid
Pipe positive SSX1-R pipe amplification curve is positive or SSX2-R pipe amplification curve is positive, the sample results be determined as SYT-SSX1 or
SYT-SSX2 fusion is positive；
D. negative referring to primer Actin pipe amplification curve, it prompts the sample extraction to fail, needs to re-start extraction.
Embodiment 3
Clinical blood sample is detected using kit for detecting nucleic acid of the present invention.
20, clinical blood sample to be checked is taken, extracts the tissue in blood sample by testing process described in embodiment 2
RNA, it is cDNA, preparation of reagents by the tissue RNA reverse transcription and detects.
For every part of sample, 2ul cDNA obtained is taken to be added to the detection qPCR reaction of SYT-SSX fusion respectively
In liquid and reference gene detection qPCR reaction solution.Carry out positive, negative and blank control product experiment simultaneously.
The optimization of reaction system:
(1) optimization of primer concentration, in the reaction system in the identical situation of other conditions, by primer concentration from 0.05 μ
M/L~1.5 μM/L makees multiple proportions serial dilution, determines that optium concentration is 0.1 μM/L by the analysis of experimental result.
(2) optimization of concentration and probe concentration, in the reaction system in the identical situation of other conditions, by primer concentration from 0.05 μ
M/L~0.5 μM/L makees multiple proportions serial dilution, determines that optium concentration is 0.1 μM/L by the analysis of experimental result.
(3) optimization of annealing temperature, in the reaction system in the identical situation of other conditions, progress grads PCR (55 DEG C~
65 DEG C), determine that optimum temperature is 60 DEG C by the analysis of experimental result.
(4) optimization of amplified reaction enzyme chooses KOD enzyme by comparing various PCR amplification enzymes in the market.
Reaction detects each sample on ABI7500 detector and carries out 5 tube reactions, and identical qPCR is added in every tube reaction
Mix (i.e. qPCR mixed reaction solution), upstream and downstream primer, TaqMan probe.QPCR reaction condition are as follows: 95 DEG C of initial denaturation 60s, 95 DEG C
15s, 60 DEG C of 35s, amplified reaction are 40 circulations.
As a result are as follows: 20 normal blood samples do not detect SYT-SSX fusion, and SYT-SSX1 and SYT-SSX2 is merged
Positive plasmid test positive, and reference gene test positive, so detection complies fully with requirement, false positive rate occurs very
Low or almost without generation, specificity is good, meets Clinical screening requirement.Negative sample detection curve figure is as shown in Figure 1.
The above is only a preferred embodiment of the present invention, for those of ordinary skill in the art, according to the present invention
Thought, there will be changes in the specific implementation manner and application range, and the content of the present specification should not be construed as to the present invention
Limitation.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<400> 2
accagatcat gcccaagaag ccagc 25
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cactcccttc gaatcatttt cg 22
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cacttcctcc gaatcatttc ct 22
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgagcgaggc tacagctt 18
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tccttgatgt cgcgcacgat tt 22
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accaccacgg ccgagccc 18
Claims (6)
1. for detecting the primer of SYT-SSX fusion, which is characterized in that the primer includes:
(1) primer and probe of SYT-SSX1 or SYT-SSX2 fusion, base sequence are detected are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAM RA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
(2) primer and probe of reference gene, base sequence are detected are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '.
2. primer as described in claim 1, which is characterized in that SYT-SSX-F, SSX1-R SSX2-R and SYT-SSX-
The concentration ratio of TaqMan is 1:1:1.
3. primer as described in claim 1, which is characterized in that Actin-F, Actin-R and Actin-Probe using dense
Degree is than being 1:1:1.
4. the ARMS-QPCR kit for detecting SYT-SSX fusion, which is characterized in that the kit includes qPCR
Reaction system, qPCR reaction system include qPCR mixed reaction solution, reaction primer and positive control sample；
QPCR mixed reaction solution includes PCR buffer, dNTP, MgCl2, KOD enzyme, PCR upstream and downstream primer and TaqMan probe, inspection
The primer and probe and positive control sample of survey reference gene；In qPCR reaction system, the final concentration of each component is respectively as follows: PCR
Buffer is 1X；DNTP is 1.5mM；MgCl2For 2mM；KOD enzyme is 0.6U/ μ l；PCR upstream and downstream primer is 0.1 μM；TaqMan
Probe is 0.1 μM；Positive control sample is 1ng/ μ l；Wherein
The PCR upstream and downstream primer, the sequence of TaqMan probe are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAM RA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
The primer and probe of the detection reference gene can expand all difference cDNA templates, base sequence are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '.
The positive control sample is respectively the cDNA plasmid of SYT-SSX1 or SYT-SSX2 fusion.
5. detecting the ARMS-qPCR method of SYT-SSX fusion, which comprises the following steps:
(1) total serum IgE is extracted from cell or blood, reverse transcription is at cDNA as template；
(2) real-time fluorescence quantitative PCR amplification is carried out, 5 pipes of detection point of each sample carry out, and it is mixed that identical qPCR is added in each pipe
Reaction solution, template cDNA are closed, every pipe is separately added into primer, and one of probe and two kinds of ARMS primers carry out SYT-SSX fusion
Genetic test；
Wherein, the primer and probe of SYT-SSX1 or SYT-SSX2 fusion, base sequence are detected are as follows:
SYT-SSX-F:5 '-CACCACAGCCACCCCAGC-3 '
SYT-SSX-TaqMan:5 '-FAM ACCAGATCATGCCCAAGAAGCCAGC-TAM RA-3 '；
SSX1-R:5 '-CACTCCCTTCGAATCATTTTCG-3 '；
SSX2-R:5 '-CACTTCCTCCGAATCATTTCCT-3 '；
Detect the primer and probe of reference gene, base sequence are as follows:
Actin-F:5 '-TGAGCGAGGCTACAGCTT-3 '；
Actin-R:5 '-TCCTTGATGTCGCGCACGATTT-3 '；
Actin-Probe:5 '-FAM-ACCACCACGGCCGAGCCC-TAMRA-3 '；
(4) result interpretation:
A. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid Guan Yang
Property, SSX1-R pipe amplification curve feminine gender and SSX2-R pipe amplification curve are negative, which is determined as SYT-SSX fusion
It is negative；
B. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid Guan Yang
Property the SSX1-R pipe amplification curve positive and SSX2-R pipe amplification curve it is positive；The sample results are determined as SYT-SSX fusion
It is positive；
C. positive referring to primer Actin pipe amplification curve, SYT-SSX1 positive plasmid pipe is positive, SYT-SSX2 positive plasmid Guan Yang
Property SSX1-R pipe amplification curve it is positive or SSX2-R pipe amplification curve is positive, which is determined as SYT-SSX1 or SYT-
SSX2 fusion is positive；
D. negative referring to primer Actin pipe amplification curve, it prompts the sample extraction to fail, needs to re-start extraction.
6. the method as described in claim 1, which is characterized in that in step (3), the condition for carrying out pcr amplification reaction is 95 DEG C
Initial denaturation 60s, 95 DEG C of denaturation 15s, 60 DEG C of extension 35s, 40 recycle.
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| CN1315000A (en) * | 1998-06-26 | 2001-09-26 | 路德维格癌症研究院 | Methods for determining presence of cancer in a sample by determining expression of an ssx gene, peptide derived from said ssx gene and NY-ESO-1 gene, and uses thereof |
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| JP2017175953A (en) * | 2016-03-28 | 2017-10-05 | アークレイ株式会社 | SYT-SSX fusion gene detection probe, SYT-SSX fusion gene detection probe set, SYT-SSX fusion gene detection method and SYT-SSX fusion gene detection kit |
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2019
- 2019-06-27 CN CN201910566697.1A patent/CN110218774A/en active Pending
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| CN1315000A (en) * | 1998-06-26 | 2001-09-26 | 路德维格癌症研究院 | Methods for determining presence of cancer in a sample by determining expression of an ssx gene, peptide derived from said ssx gene and NY-ESO-1 gene, and uses thereof |
| JP2017175953A (en) * | 2016-03-28 | 2017-10-05 | アークレイ株式会社 | SYT-SSX fusion gene detection probe, SYT-SSX fusion gene detection probe set, SYT-SSX fusion gene detection method and SYT-SSX fusion gene detection kit |
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| Title |
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| D.ASHLEY HILL: "Real-time Polymerase Chain Reaction as an Aid for the Detection of SYT-SSX1 and SYT-SSX2 Transcripts", 《PEDIATRIC AND DEVELOPMENTAL PATHOLOGY》 * |
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