CN106544437B - A kind of multiple fluorescence PCR kit detecting leukemia fusion gene - Google Patents
A kind of multiple fluorescence PCR kit detecting leukemia fusion gene Download PDFInfo
- Publication number
- CN106544437B CN106544437B CN201611055457.8A CN201611055457A CN106544437B CN 106544437 B CN106544437 B CN 106544437B CN 201611055457 A CN201611055457 A CN 201611055457A CN 106544437 B CN106544437 B CN 106544437B
- Authority
- CN
- China
- Prior art keywords
- sequence
- single strand
- strand dna
- dna shown
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of multiple fluorescence PCR kits and method for detecting leukemia fusion gene.The present invention provides a kind of primer sets for detecting leukemia fusion gene, are made of primer pair 1- primer pair 23；23 nucleotide sequence of primer pair 1- primer pair is respectively sequence 1- sequence 46.Kit provided by the invention and detection method, it is at low cost, easy to operate, detection sensitivity is high, result be easy interpretation, suitable for being detected daily physical examination to high-volume sample.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of multiple fluorescence PCR examination for detecting leukemia fusion gene
Agent box and method.
Background technique
Leukaemia is the Clonal malignant disease of a kind of candidate stem cell exception, is that children and young and middle-aged disease incidence are higher
One of malignant cancer.Leukaemia has a variety of hypotypes, and genetics characteristics are complicated and changeable.As Protocols in Molecular Biology is sent out
Exhibition, it was demonstrated that many leukaemics have the variation, including chromosome translocation, inversion and missing etc. of chromosome.Leukaemic
Chromosomal variation can generate new fusion, and these fusions are the key factors of leukaemia morbidity.Fusion can
Molecular marker as leukemia diagnosis and parting.If BCR-ABL p210 type fusion is the pass of chronic myelocytic leukemia
Key factor, 95% or more patients with chronic myeloid leukemia have BCR-ABL p210 type fusion；98% or more urgency
Property progranulocyte leukemia patient have PML-RARA fusion；50% or more children acute lymphoblastic leukaemia patient
With MLL-AF4 fusion；40% or more M2 type Patients with Acute Myeloid Leukemia has AML1-ETO fusion.White blood
Patient is mostly after abnormal symptom or quantity of leucocyte exception occurs in body, just to go whether diagnosis is leukaemia, is made a definite diagnosis in mostly
Advanced stage.Leukaemia is made a definite diagnosis in early detection, is conducive to improve therapeutic effect, extends patient survival.But lacks be applicable at present
In the kit and detection method of leukaemia early diagnosis.In view of fusion have become molecular marker that leukaemia is made a definite diagnosis it
One, the early diagnosis kit and method of exploitation detection leukemia fusion gene have important clinical meaning and market value.
Currently, screening leukemia fusion gene mainly uses multiplex PCR combination agarose gel electrophoresis to be detected.It is multiple
PCR working principle is the PCR reaction that multipair primer is added in same PCR reaction system, while expanding multiple target genes.
It is compared with regular-PCR, has the characteristics that efficient, economical and convenient.But by primer concentration, amplifying specific in same reaction system
Property etc. factors limitation, while there is cumbersome time-consuming, the general most multipotency of optimization reaction condition to detect 4 target genes simultaneously, examine
Survey the disadvantages of sensitivity and specificity are lower.At present clinically frequently with 29 kinds of leukemia fusion gene multiplex PCR detection sides
Method is that will test sample to make 8 pipe PCR reaction respectively, then carries out molecule electrophoresis, identified according to PCR product clip size.
This method detection program is cumbersome, and a patient needs multitube PCR to react, and gel electrophoresis is needed to be identified；Detection sensitivity
Low, price is not suitable for early diagnosing generally at 1000 yuan or more.Leukaemia is carried out to general population in daily physical examination
Fusion screening, detection kit must satisfy the features such as inexpensive, fast and convenient, sensitivity and specificity are high.
Fluorescent PCR is the probe that fluorescent dye or fluorescent marker are added in the reaction system, and inspection is marked to PCR product
It surveys, without carrying out molecule electrophoresis, has the characteristics that sensitivity and specificity are high.Common fluorescent dye is SYBR Green, it
It can be incorporated on double-stranded DNA.When the template in PCR reaction system is amplified, SYBR Green dyestuff can be combined effectively
On to newly synthesized DNA double chain and fluorescence is excited, quantitative detection is carried out to PCR product by the detection to fluorescence.
Summary of the invention
It is an object of the present invention to provide a kind of primer sets for detecting leukemia fusion gene.
Primer set provided by the invention is made of primer pair 1- primer pair 23；
Single strand dna shown in the single strand dna shown in sequence 1 of primer pair 1 and sequence 2 forms；
Single strand dna shown in the single strand dna shown in sequence 3 of primer pair 2 and sequence 4 forms；
Single strand dna shown in the single strand dna shown in sequence 5 of primer pair 3 and sequence 6 forms；
Single strand dna shown in the single strand dna shown in sequence 7 of primer pair 4 and sequence 8 forms；
Single strand dna shown in the single strand dna shown in sequence 9 of primer pair 5 and sequence 10 forms；
Single strand dna shown in the single strand dna shown in sequence 11 of primer pair 6 and sequence 12 forms；
Single strand dna shown in the single strand dna shown in sequence 13 of primer pair 7 and sequence 14 forms；
Single strand dna shown in the single strand dna shown in sequence 15 of primer pair 8 and sequence 16 forms；
Single strand dna shown in the single strand dna shown in sequence 17 of primer pair 9 and sequence 18 forms；
Single strand dna shown in the single strand dna shown in sequence 19 of primer pair 10 and sequence 20 forms；
Single strand dna shown in the single strand dna shown in sequence 21 of primer pair 11 and sequence 22 forms；
Single strand dna shown in the single strand dna shown in sequence 23 of primer pair 12 and sequence 24 forms；
Single strand dna shown in the single strand dna shown in sequence 25 of primer pair 13 and sequence 26 forms；
Single strand dna shown in the single strand dna shown in sequence 27 of primer pair 14 and sequence 28 forms；
Single strand dna shown in the single strand dna shown in sequence 29 of primer pair 15 and sequence 30 forms；
Single strand dna shown in the single strand dna shown in sequence 31 of primer pair 16 and sequence 32 forms；
Single strand dna shown in the single strand dna shown in sequence 33 of primer pair 17 and sequence 34 forms；
Single strand dna shown in the single strand dna shown in sequence 35 of primer pair 18 and sequence 36 forms；
Single strand dna shown in the single strand dna shown in sequence 37 of primer pair 19 and sequence 38 forms；
Single strand dna shown in the single strand dna shown in sequence 39 of primer pair 20 and sequence 40 forms；
Single strand dna shown in the single strand dna shown in sequence 41 of primer pair 21 and sequence 42 forms；
Single strand dna shown in the single strand dna shown in sequence 43 of primer pair 22 and sequence 44 forms；
Single strand dna shown in the single strand dna shown in sequence 45 of primer pair 23 and sequence 46 forms.
In above-mentioned primer, it is characterised in that: the mole ratio of each primer is 1:1 in the primer pair 1-22；
The mole ratio of every primer is 0.2-1:0.01- in the every primer and the primer pair 1 of the primer pair 23
0.1；
Or the mole ratio of every primer is 0.2-1 in the every primer and the primer pair 1 of the primer pair 23:
0.06；
Or the mole ratio of every primer is 0.3:0.01- in the every primer and the primer pair 1 of the primer pair 23
0.1。
Another object of the present invention is to provide a kind of PCR reagent for detecting leukemia fusion gene.
PCR reagent provided by the invention comprising above-mentioned primer.
Above-mentioned PCR reagent further includes Taq archaeal dna polymerase, dNTP, SYBR Green, MgCl2And water；
Above-mentioned PCR reagent is by above-mentioned primer, Taq archaeal dna polymerase, dNTP, SYBR Green, MgCl2, PCR buffer and
Water composition.
In above-mentioned PCR reagent, concentration of each primer in the PCR reagent is 0.01- in the primer pair 1-22
0.1μM；
Concentration of each primer in the PCR reagent is 0.2-1 μM in the primer pair 23；
Or concentration of each primer in the PCR reagent is 0.06 μM in the primer pair 1-22；
Concentration of each primer in the PCR reagent is 0.3 μM in the primer pair 23.
Kit containing above-mentioned primer or containing above-mentioned PCR reagent is also the scope of protection of the invention.
Mentioned reagent box further includes the carrier for being described below detection method:
Fluorescent PCR amplification is carried out with above-mentioned primer pair sample to be tested, obtains amplified production；
The Ct value of amplified production is detected, if the Ct value of sample to be tested is greater than or equal to 35, sample to be tested is not contained or waited
Choosing does not contain leukemia fusion gene；If the Ct value of sample to be tested is less than 35, sample to be tested contains or candidate contains leukaemia
Fusion；
The template of the fluorescent PCR amplification is cDNA；
The fluorescent PCR amplification condition be first 94-98 DEG C holdings 30-60 seconds, 50-55 DEG C holding 30-60 seconds, 72 DEG C keep
30-90 seconds, react 5-15 circulation, then 94-98 DEG C holdings 30-60 seconds, 72 DEG C holding 60-120 seconds, reaction 5-10 recycle；
94-98 DEG C holding 30-60 seconds again, 55-62 DEG C holding 30-60 seconds, 72 DEG C holding 30-90 seconds, reaction 35-40 recycle.
Whether above-mentioned primer contains leukaemia in identification sample to be tested containing above-mentioned PCR reagent or above-mentioned kit
Application in fusion is also the scope of protection of the invention；
Above-mentioned primer or containing above-mentioned PCR reagent or above-mentioned kit in system of identification for whether containing in sample to be tested
Application in leukemia fusion gene product is also the scope of protection of the invention.
Whether above-mentioned primer infects white blood in system of identification containing above-mentioned PCR reagent or above-mentioned kit for sample to be tested
Application in sick product is also the scope of protection of the invention.
Third purpose of the present invention be to provide in a kind of identification sample to be tested whether the method containing leukemia fusion gene.
Method provided by the invention, includes the following steps:
Fluorescent PCR amplification is carried out to sample to be tested with above-mentioned primer or containing above-mentioned PCR reagent or above-mentioned kit, is obtained
To amplified production；
The Ct value of amplified production is detected, if the Ct value of sample to be tested is greater than or equal to 35, sample to be tested is not contained or waited
Choosing does not contain leukemia fusion gene；If the Ct value of sample to be tested is less than 35, sample to be tested contains or candidate contains leukaemia
Fusion.
In the above method, the template of the fluorescent PCR amplification is cDNA；
Or, the fluorescent PCR amplification condition be first 94-98 DEG C holding 30-60 seconds, 50-55 DEG C holding 30-60 seconds, 72 DEG C
Kept for 30-90 second, react 5-15 circulation, then 94-98 DEG C holding 30-60 seconds, 72 DEG C holding 60-120 seconds, it is a to react 5-10
Circulation；94-98 DEG C holding 30-60 seconds again, 55-62 DEG C holding 30-60 seconds, 72 DEG C holding 30-90 seconds, reaction 35-40 is followed
Ring.
Among the above, the leukemia fusion gene be E2A-PBX1 gene, MLL-AF4 gene, TEL-AML1 gene,
BCR-ABL p210 gene, BCR-ABL p190 gene, SIL-TAL gene, PML-RARA L type gene, PML-RARA S
At least one of type gene, AML1-ETO gene, CBFB-MYH11A type gene and CBFB-MYH11DE type gene.
Compared with prior art, the present invention having the advantage that
(1) easy to operate, only need 1 pipe PCR reaction that can identify 11 kinds of fusions simultaneously.
(2) totally-enclosed reaction is not necessarily to gel electrophoresis, avoids the pollution of PCR product.
(3) it is reacted by three step PCR, the first step carries out the enrichment of purpose fusion using fusion specific primer,
Because the primer that step reaction is added is extremely low, can be interfered with each other to avoid between primer dimer, mispairing, non-specific amplification, primer
Phenomena such as generation, and each target gene has two pairs of primers, and the probability of amplifying target genes significantly improves, without harsh
Condition optimizing；Second step reaction improves annealing temperature, and only tailed primer can expand on the inside of fusion, by the mesh of enrichment
Mark gene is carried out plus universal primer sequence；Third step reaction carries out Fluorescence PCR, the universal primer using universal primer
It is high that specificity is detected without any homology to human source gene by optimization.
(4) at present detection method due to price it is high, cumbersome, can not be sieved suitable for the early stage daily physical examination
It looks into.
It is at low cost, easy to operate, detection sensitivity is high, knot in conclusion kit provided by the invention and detection method
Fruit is easy interpretation, suitable for detecting daily physical examination to high-volume sample.
Detailed description of the invention
Fig. 1 is the amplification curve for detecting Healthy People and leukaemic's clinical sample.
Fig. 2 is the amplification curve of the fusion positive sample of detectable concentration gradient dilution.
Specific embodiment
A specific embodiment of the invention is described further below with reference to embodiment.These embodiments are only exemplary
, it is not intended to limit the scope of the present invention in any way.It will be understood by those skilled in the art that without departing from of the invention
Can be with the details and forms of the technical scheme of the invention are modified or replaced under spirit and scope, but these modifications and replacement are equal
It falls within the scope of protection of the present invention.
Experimental method used in following embodiments is unless otherwise specified conventional method.
Material as used in the following examples, reagent commercially obtain unless otherwise specified.
The foundation of embodiment 1, the primer pair and its method of detection leukaemia
1, the primer pair of leukaemia is detected
According to 11 kinds of leukemia fusion genes: E2A-PBX1, MLL-AF4, TEL-AML1, BCR-ABL p210, BCR-ABL
p190、SIL-TAL、PML-RARA L type、PML-RARA S type、AML1-ETO、CBFB-MYH11A type、 CBFB-
MYH11DE type designs primer pair as shown in table 1 below.
Table 1 is the primer pair for detecting leukaemia
2, the kit or system of leukaemia are detected
Multiple fluorescence PCR reaction system, 23 pairs of primer pairs including table 1, Taq archaeal dna polymerase, dNTP, SYBR
Green、MgCl2, PCR buffer and ultrapure water.
Multiple fluorescence PCR reaction system of the present invention is 50 μ L, including 400 μM of final concentration of Taq archaeal dna polymerase 1U, dNTPs,
MgCl22 μM of final concentration of final concentration 3mM, SYBR Green, 2 μ L of sample to be tested cDNA, 11 kinds of fusion primers are in PCR
Final concentration in reaction system is respectively 0.01-0.1 μM, it is therefore preferable to 0.06 μM, final concentration of 0.2-1 μM of universal primer, excellent
Selection of land is 0.3 μM, and ultrapure water complements to 50 μ L.
Also all the present invention establishes the kit or multiple fluorescence PCR reaction system of 23 pairs of primer pairs containing table 1.
3, detection method
1) the RNA reverse transcription for extracting sample obtains cDNA as template；
2) PCR amplification
Using above-mentioned cDNA as template, 23 pairs of primer pairs shown in table 1 carry out multiple fluorescence PCR amplification, detection amplification
Product.
Above-mentioned multiple fluorescence PCR reaction system is 50 μ L, including 400 μM of final concentration of Taq archaeal dna polymerase 1U, dNTPs,
MgCl22 μM of final concentration of final concentration 3mM, SYBR Green (being purchased from Dalian treasured biotech firm, article No. PR820A), sample to be tested
2 μ L of cDNA, 11 kinds of fusion primer final concentrations are respectively 0.01-0.1 μM, it is therefore preferable to and 0.06 μM, universal primer final concentration
It is 0.2-1 μM, it is therefore preferable to which 0.3 μM, ultrapure water complements to 50 μ L.
The program of above-mentioned multiple fluorescence PCR amplification are as follows:
First step PCR reaction condition are as follows: 94-98 DEG C holding 30-60 seconds, 50-55 DEG C holding 30-60 seconds, 72 DEG C of holding 30-
90 seconds, 5-15 circulation is reacted, step reaction does not monitor SYBR Green fluorescence signal.
Second step PCR reaction condition are as follows: 94-98 DEG C holding 30-60 seconds, 72 DEG C holding 60-120 seconds, reaction 5-10 is followed
Ring, step reaction do not monitor SYBR Green fluorescence signal.
Third step PCR reaction condition are as follows: 94-98 DEG C holding 30-60 seconds, 55-62 DEG C holding 30-60 seconds, 72 DEG C of holding 30-
90 seconds, 35-40 circulation is reacted, step reaction monitors SYBR Green fluorescence signal at the end of 72 DEG C of reactions, recycles every time
Monitoring is primary.
If as a result the Ct value of institute's test sample is greater than or equal to 35 and is determined as that institute's test sample does not contain or candidate does not contain
Leukemia fusion gene or institute to test sample are not or candidate is not leukaemia sample；Less than 35, it is determined as that institute's test sample contains
Or candidate containing leukemia fusion gene or institute it is to test sample or candidate is leukaemia sample.
By be determined as containing or candidate institute's test sample containing leukemia fusion gene PCR product, use universal primer
Sequencing analysis is carried out, sequence alignment determines fusion parting.
The application of embodiment 2, the primer pair of detection leukaemia
Using kit and method the detection Healthy People and leukaemic's clinical sample of embodiment 1.
The present embodiment acquires 20, healthy human peripheral blood sample, identified fusion positive mutations in leukemia patients by peripheral blood
20, sample (patient knows).
1, the acquisition of sample cDNA
(1) periphery blood specimen wants EDTA anticoagulant
25ml erythrocyte cracked liquid is added in 50ml centrifuge tube.In vitro periphery blood specimen is mixed by inversion, 50ml is poured into
Centrifuge tube is placed in roller bearing oscillator and cracks 10 minutes, and 1500 turns are centrifuged 5 minutes.1ml phosphate buffer is added to blow afloat, turns
1.5ml centrifuge tube is moved to, 1600 turns are centrifuged for 3 minutes, and phosphate buffer is added, is in charge of according to cell quantity, every pipe
About 107A cell.1600 turns are centrifuged for 3 minutes, abandon supernatant.Add 1ml Trizol, abundant pressure-vaccum is mixed, frozen to -80 DEG C of ice
Case directly extracts RNA.
(2) extraction of RNA: to the sample for having added Trizol, adding 200 μ l chloroforms, be first mixed by inversion, then acutely oscillation is mixed
It is even, 3min is stood, is centrifuged 12000 turns, 10min, 4 DEG C.400 μ l of upper strata aqueous phase is inhaled to add in corresponding new 1.5ml pipe, then plus
Enter isometric isopropanol.It is mixed by inversion, stands 10min, be centrifuged 12000 turns later, 4 DEG C of centrifugation 10min.Supernatant is abandoned, is added
75% ethyl alcohol is pre-chilled in 1ml, is mixed by inversion, bounces RNA, is centrifuged 7500 turns, 4 DEG C of centrifugation 10min.Abandon supernatant, after drying, root
According to precipitation volume plus DEPC water, RNA is dissolved.RNA concentration is controlled in 500ng/ μ l or so.
(3) reverse transcription cDNA:RNA is denaturalized, and each PCR pipe adds 8 μ l DEPC water, 2 μ l RNA.65 degree of 5min, on ice
5min.Configure 2 × reverse transcription RT mix buffer (using ABI reverse transcription reagent box): DEPC water 4.2 μ l, 10 × RT
2 0.8 μ l, 10 × Random Primers of μ l, 25 × dNTP, 1 μ l, RNase inhibitor of buffer 1 μ l, Reverse
Transcriptase 1μl.It takes the configured 2 × RT mix buffer of 10 μ l to be added in denaturation RNA pipe, mixes.Reverse transcription
Condition: 25 DEG C, 10min；37 DEG C, 90min；85 DEG C, 5min.
2, PCR amplification
50 μ L:Taq enzyme premixed liquid of PCR reaction system (being purchased from ABI company) 25 μ L (include Taq enzyme, dNTPs, MgCl2、
SYBR Green), 2 μ L of sample cDNA, 5 μ L of primer mixed liquor (makes final concentration of the fusion primer in PCR reaction system
It is 0.06 μM, final concentration of 0.3 μM in PCR reaction system of universal primer), ultrapure water complements to 50 μ L.By what is prepared
PCR reaction system is added in 96 hole PCR plates, pad pasting, and closing, centrifugation mixes.
Multiple fluorescence PCR reaction condition are as follows: 95 DEG C are kept for 30 seconds, and 53 DEG C are kept for 30 seconds, and 72 DEG C are kept for 80 seconds, react 12
Circulation；95 DEG C are kept for 30 seconds, and 72 DEG C are kept for 90 seconds, react 6 circulations；95 DEG C are kept for 30 seconds, and 60 DEG C are kept for 30 seconds, 72 DEG C of holdings
60 seconds, 40 circulations are reacted, step reaction monitors SYBR Green fluorescence signal, each circulatory monitoring at the end of 72 DEG C of reactions
Once.
If the Ct value of institute's test sample is greater than or equal to 35 and is determined as that institute's test sample does not contain or candidate is without containing white blood
Sick fusion or institute to test sample are not or candidate is not leukaemia sample；Less than 35, it is determined as that institute's test sample contains or waits
Choosing containing leukemia fusion gene or institute to test sample is or candidate is leukaemia sample.
As a result as shown in Figure 1, Healthy People sample amplification curve does not play peak, Ct value is greater than 35；Fusion positive leukaemia
Clinical samples amplification curve plays peak, and Ct value is less than 35.Utilize kit of the present invention and method 20 fusions detected
Positive leukaemic's sample is containing leukemia fusion gene, is leukaemia sample, 20 Healthy People samples detected
It is feminine gender, does not contain leukemia fusion gene, be not leukaemia sample.
Commercial company is sent to carry out conventional sequencing the pcr amplification product of 20 fusion positive leukaemic's samples,
Sequencing primer is the universal primer in the present invention, determines fusion parting by sequence alignment.The leukaemia fusion detected
Gene is consistent with clinical diagnosis, illustrates that kit and method of the invention have high accuracy and specificity.
Embodiment 3, kit of the present invention and the detection of method sensitivity
1, the acquisition of template
(following document is documented in: " leukaemia lymph using the BCR-ABL p210 plasmid standard of building as template
Tumor ", 2007,16 (03): 161-164), plasmid is diluted to 105、104、103、102、101Copy/μ L.
2, PCR amplification
50 μ L:Taq enzyme premixed liquid of PCR reaction system (being purchased from ABI company) 25 μ L (include Taq enzyme, dNTPs, MgCl2、
SYBR Green), 1 μ L of plasmid standard, 5 μ L of primer mixed liquor (keeps end of the fusion primer in PCR reaction system dense
Degree is 0.06 μM, final concentration of 0.3 μM of final concentration of the universal primer in PCR reaction system), ultrapure water complements to 50 μ L.It will
The PCR reaction system prepared is added in 96 hole PCR plates, pad pasting, and closing, centrifugation mixes.
Multiple fluorescence PCR reaction condition are as follows: 95 DEG C are kept for 30 seconds, and 53 DEG C are kept for 30 seconds, and 72 DEG C are kept for 80 seconds, react 12
Circulation；95 DEG C are kept for 30 seconds, and 72 DEG C are kept for 90 seconds, react 6 circulations；95 DEG C are kept for 30 seconds, and 60 DEG C are kept for 30 seconds, 72 DEG C of holdings
60 seconds, 40 circulations are reacted, step reaction monitors SYBR Green fluorescence signal, each circulatory monitoring at the end of 72 DEG C of reactions
Once.
Testing result is shown in Fig. 2, and kit of the present invention and method are to 105、104、103、102、101Copy/μ L standard product examine
That surveys has amplification curve, illustrates that kit of the present invention and method detection sensitivity are higher, can reach 10 copies/50 μ L PCR
Reaction system.
Sequence table
<110>Xuzhou medical university
<120>a kind of multiple fluorescence PCR kit and method for detecting leukemia fusion gene
<160> 46
<170> PatentIn version 3.5
<210> 1
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
cagaagaacg gcatcaaggt gagcaccagc ctcatgcaca a 41
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
ctggttggag gcagccacc 19
<210> 3
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
cggactgggt gctcaggtag gttcctcctc ctgggctcct 40
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 4
tgtccagccg catcagctg 19
<210> 5
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 5
cagaagaacg gcatcaaggt gcccgcccaa gtatccctgt a 41
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 6
caggtccaga gcagagcaaa c 21
<210> 7
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 7
cggactgggt gctcaggtag gctgagagtc ctttgtaggg a 41
<210> 8
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 8
tcatattgtt tttggttttg gg 22
<210> 9
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 9
cagaagaacg gcatcaaggt gtcatggtct ctgtctcccc g 41
<210> 10
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 10
ctctctcatc gggaagacct g 21
<210> 11
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 11
cggactgggt gctcaggtag gtggaaggcg gcgtgaag 38
<210> 12
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 12
caacgcctcg ctcatcttg 19
<210> 13
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 13
cagaagaacg gcatcaaggt gccagactgt ccacagcatt cc 42
<210> 14
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 14
gctgcagatg ctgaccaact c 21
<210> 15
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 15
cggactgggt gctcaggtag acgagcggct tcactcagac 40
<210> 16
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 16
cactgggtcc agcgagaagg 20
<210> 17
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 17
cagaagaacg gcatcaaggt ggcagatctg gcccaacgat 40
<210> 18
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 18
cgtgtccgag gccaccat 18
<210> 19
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 19
cggactgggt gctcaggtag acgagcggct tcactcagac 40
<210> 20
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 20
cactgggtcc agcgagaagg 20
<210> 21
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 21
cagaagaacg gcatcaaggt gccgctccta ccctgcaaac 40
<210> 22
<211> 17
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 22
cggtggcgct ccttgga 17
<210> 23
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 23
cggactgggt gctcaggtag gcggaagccg aggaagag 38
<210> 24
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 24
ctctgaatag gatctccact cc 22
<210> 25
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 25
cagaagaacg gcatcaaggt gcccgtcata ggaagtgagg tc 42
<210> 26
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 26
cacctggatg gaccgcctag 20
<210> 27
<211> 42
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 27
cggactgggt gctcaggtag gacaaagcaa ggcttgtaga tg 42
<210> 28
<211> 20
<212> DNA
<213> 28
<400> 28
cgctgacccc atagtggtag 20
<210> 29
<211> 38
<212> DNA
<213> 29
<400> 29
cagaagaacg gcatcaaggt gagagcctgc aagctgcc 38
<210> 30
<211> 19
<212> DNA
<213> 30
<400> 30
gacatgcacg gtttcctgc 19
<210> 31
<211> 42
<212> DNA
<213> 31
<400> 31
cggactgggt gctcaggtag gacaaagcaa ggcttgtaga tg 42
<210> 32
<211> 20
<212> DNA
<213> 32
<400> 32
cgctgacccc atagtggtag 20
<210> 33
<211> 41
<212> DNA
<213> 33
<400> 33
cagaagaacg gcatcaaggt ggcaagtcgc cacctaccac a 41
<210> 34
<211> 23
<212> DNA
<213> 34
<400> 34
gcttcactct gaccatcact gtc 23
<210> 35
<211> 42
<212> DNA
<213> 35
<400> 35
cggactgggt gctcaggtag cgtcttcaca tccacaggtg ag 42
<210> 36
<211> 21
<212> DNA
<213> 36
<400> 36
ggtggcattg ttggaggagt c 21
<210> 37
<211> 41
<212> DNA
<213> 37
<400> 37
cagaagaacg gcatcaaggt ggcaggagga tgcattagca c 41
<210> 38
<211> 20
<212> DNA
<213> 38
<400> 38
gggctgtctg gagtttgatg 20
<210> 39
<211> 41
<212> DNA
<213> 39
<400> 39
cggactgggt gctcaggtag ctcctccatc tgggtctcca g 41
<210> 40
<211> 20
<212> DNA
<213> 40
<400> 40
cttgcagctc gtcctccagc 20
<210> 41
<211> 41
<212> DNA
<213> 41
<400> 41
cagaagaacg gcatcaaggt ggcaggagga tgcattagca c 41
<210> 42
<211> 20
<212> DNA
<213> 42
<400> 42
gggctgtctg gagtttgatg 20
<210> 43
<211> 39
<212> DNA
<213> 43
<400> 43
cggactgggt gctcaggtag cttcccctcg gcctcgtta 39
<210> 44
<211> 20
<212> DNA
<213> 44
<400> 44
gtcctggagc tgggaactga 20
<210> 45
<211> 21
<212> DNA
<213> 45
<400> 45
cagaagaacg gcatcaaggt g 21
<210> 46
<211> 20
<212> DNA
<213> 46
<400> 46
cggactgggt gctcaggtag 20
Claims (14)
1. a kind of primer set for detecting leukemia fusion gene, is made of primer pair 1- primer pair 23；
Single strand dna shown in the single strand dna shown in sequence 1 of primer pair 1 and sequence 2 forms；
Single strand dna shown in the single strand dna shown in sequence 3 of primer pair 2 and sequence 4 forms；
Single strand dna shown in the single strand dna shown in sequence 5 of primer pair 3 and sequence 6 forms；
Single strand dna shown in the single strand dna shown in sequence 7 of primer pair 4 and sequence 8 forms；
Single strand dna shown in the single strand dna shown in sequence 9 of primer pair 5 and sequence 10 forms；
Single strand dna shown in the single strand dna shown in sequence 11 of primer pair 6 and sequence 12 forms；
Single strand dna shown in the single strand dna shown in sequence 13 of primer pair 7 and sequence 14 forms；
Single strand dna shown in the single strand dna shown in sequence 15 of primer pair 8 and sequence 16 forms；
Single strand dna shown in the single strand dna shown in sequence 17 of primer pair 9 and sequence 18 forms；
Single strand dna shown in the single strand dna shown in sequence 19 of primer pair 10 and sequence 20 forms；
Single strand dna shown in the single strand dna shown in sequence 21 of primer pair 11 and sequence 22 forms；
Single strand dna shown in the single strand dna shown in sequence 23 of primer pair 12 and sequence 24 forms；
Single strand dna shown in the single strand dna shown in sequence 25 of primer pair 13 and sequence 26 forms；
Single strand dna shown in the single strand dna shown in sequence 27 of primer pair 14 and sequence 28 forms；
Single strand dna shown in the single strand dna shown in sequence 29 of primer pair 15 and sequence 30 forms；
Single strand dna shown in the single strand dna shown in sequence 31 of primer pair 16 and sequence 32 forms；
Single strand dna shown in the single strand dna shown in sequence 33 of primer pair 17 and sequence 34 forms；
Single strand dna shown in the single strand dna shown in sequence 35 of primer pair 18 and sequence 36 forms；
Single strand dna shown in the single strand dna shown in sequence 37 of primer pair 19 and sequence 38 forms；
Single strand dna shown in the single strand dna shown in sequence 39 of primer pair 20 and sequence 40 forms；
Single strand dna shown in the single strand dna shown in sequence 41 of primer pair 21 and sequence 42 forms；
Single strand dna shown in the single strand dna shown in sequence 43 of primer pair 22 and sequence 44 forms；
Single strand dna shown in the single strand dna shown in sequence 45 of primer pair 23 and sequence 46 forms.
2. primer according to claim 1, it is characterised in that: the mole ratio phase of each primer in the primer pair 1-22
Together；
The mole ratio of every primer is 0.2-1:0.01-0.1 in the every primer and the primer pair 1 of the primer pair 23.
3. primer according to claim 2, it is characterised in that:
The mole ratio of every primer is 0.2-1:0.06 in the every primer and the primer pair 1 of the primer pair 23.
4. primer according to claim 2, it is characterised in that:
The mole ratio of every primer is 0.3:0.01-0.1 in the every primer and the primer pair 1 of the primer pair 23.
5. a kind of PCR reagent for detecting leukemia fusion gene comprising primer as claimed in claim 1 or 2.
6. PCR reagent according to claim 5, it is characterised in that: each primer is in the PCR in the primer pair 1-22
Concentration in reagent is 0.01-0.1 μM；
Concentration of each primer in the PCR reagent is 0.2-1 μM in the primer pair 23.
7. PCR reagent according to claim 6, it is characterised in that:
Concentration of each primer in the PCR reagent is 0.06 μM in the primer pair 1- primer pair 22；
Concentration of each primer in the PCR reagent is 0.3 μM in the primer pair 23.
8. the kit containing primer described in claim 1-4 any claim.
9. the kit containing PCR reagent described in claim 5-7 any claim.
10. kit according to claim 8 or claim 9, it is characterised in that: the kit further includes being described below detection side
The carrier of method:
Fluorescent PCR amplification is carried out with primer pair sample to be tested as claimed in claim 1 or 2, obtains amplified production；
Detect the Ct value of amplified production, if the Ct value of sample to be tested is greater than or equal to 35, sample to be tested do not contain or it is candidate not
Contain leukemia fusion gene；If the Ct value of sample to be tested, less than 35, sample to be tested contains or the candidate leukaemia that contains merges
Gene；
The template of the fluorescent PCR amplification is cDNA；
The fluorescent PCR amplification condition be first 94-98 DEG C holding 30-60 seconds, 50-55 DEG C holding 30-60 seconds, 72 DEG C of holding 30-
90 seconds, react 5-15 circulation, then 94-98 DEG C holdings 30-60 seconds, 72 DEG C holding 60-120 seconds, reaction 5-10 recycle；Again
94-98 DEG C holding 30-60 seconds, 55-62 DEG C holding 30-60 seconds, 72 DEG C holding 30-90 seconds, reaction 35-40 recycle.
11. whether primer described in claim 1 contains answering in leukemia fusion gene product in preparation identification sample to be tested
With.
12. primer described in claim 1 preparation identification sample to be tested institute source main body whether the application in cases with leukemia product.
13. whether kit as claimed in claim 9 is in preparation identification sample to be tested containing in leukemia fusion gene product
Application.
14. whether kit as claimed in claim 9 is in preparation identification sample to be tested institute source main body in cases with leukemia product
Using.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611055457.8A CN106544437B (en) | 2016-11-25 | 2016-11-25 | A kind of multiple fluorescence PCR kit detecting leukemia fusion gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611055457.8A CN106544437B (en) | 2016-11-25 | 2016-11-25 | A kind of multiple fluorescence PCR kit detecting leukemia fusion gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106544437A CN106544437A (en) | 2017-03-29 |
| CN106544437B true CN106544437B (en) | 2019-11-19 |
Family
ID=58395277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611055457.8A Active CN106544437B (en) | 2016-11-25 | 2016-11-25 | A kind of multiple fluorescence PCR kit detecting leukemia fusion gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106544437B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107385080A (en) * | 2017-08-31 | 2017-11-24 | 南京美宁康诚生物科技有限公司 | A kind of leukemia of children correlation fusion gene multiple PCR detection kit and method |
| CN115992228A (en) * | 2022-07-19 | 2023-04-21 | 领航基因科技（杭州）有限公司 | Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
| CN103937891A (en) * | 2014-04-16 | 2014-07-23 | 苏州大学 | Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes |
-
2016
- 2016-11-25 CN CN201611055457.8A patent/CN106544437B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
| CN103937891A (en) * | 2014-04-16 | 2014-07-23 | 苏州大学 | Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes |
Non-Patent Citations (1)
| Title |
|---|
| 《应用多重巢式PCR方法检测急性淋巴细胞白血病相关融合基因及其临床特点分析》;刘芳;《中国博士学位论文全文数据库（电子期刊）医药卫生科技辑》;20130915;第9页摘要，第12页表2，第18页2.5.3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106544437A (en) | 2017-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Tumor heterogeneity and circulating tumor cells | |
| US20120244531A1 (en) | Method for providing information for diagnosing cancer using quantitative real-time pcr and kit for diagnosing cancer for the same | |
| Nezos et al. | Detection of circulating tumor cells in bladder cancer patients | |
| CN106544437B (en) | A kind of multiple fluorescence PCR kit detecting leukemia fusion gene | |
| CN106480201A (en) | Metastasis in Breast Cancer assesses test kit | |
| CN103074431B (en) | Special primer, kit and method for testing minRNA-128 in colorectal cancer serum | |
| CN102925444A (en) | Serum micro ribonucleic acid (miRNA) biomarker of bladder cancer and detection method of expression quantity thereof | |
| CN106282387A (en) | Gastric cancer detection primer probe and test kit thereof | |
| CN106350596A (en) | Prognosis detection primer probe and kit thereof for colorectal cancer | |
| CN103173449B (en) | Esophagus cancer postoperative early-stage recurrence and prognosis related miRNA marker and its application | |
| CN103173450B (en) | Esophagus cancer postoperative early-stage recurrence and prognosis related miRNA marker and its application | |
| US10889855B2 (en) | Detection of nucleic acid molecules | |
| CN107326092A (en) | Applications and colorectal cancer detection kit of the related miRNA of colorectal cancer as biomarker | |
| CN104630377B (en) | A kind of application of breast cancer miRNA detection kits and its miRNA | |
| CN102899390A (en) | Small cell lung cancer markers and their detection | |
| CN103114092B (en) | MiRNA marker associated with post-esophageal-cancer-operation early recurrence and prognosis and applications thereof | |
| US10131956B2 (en) | Marker used for detection of colon cancer, and application thereof | |
| CN109929921A (en) | MicroRNA 21 (MIR21) nucleic acid quantitative determination reagent kit (PCR- fluorescence probe method) | |
| JP2013085542A (en) | Stomach cancer detection method, blood marker for stomach cancer, and use thereof | |
| US11643690B2 (en) | Early detection of preliminary stages of testicular germ cell tumors | |
| CN108085386A (en) | The identification of the reference gene of osteosarcoma miRNA detections | |
| WO2016018047A1 (en) | Apparatus and method for diagnosing cancer | |
| CN103160509A (en) | miRNA marker relative to esophagus cancer postoperative early relapse and prognosis and application | |
| CN106520928A (en) | Esophagus cancer detection primer probe and kit thereof | |
| CN103667281A (en) | Molecular marker miR-301a for prostatic cancer and application of molecular marker miR-301a |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |