CN104372095A - Primer, probe and kit for detecting human BCR-ABL gene mutation - Google Patents
Primer, probe and kit for detecting human BCR-ABL gene mutation Download PDFInfo
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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
- C12Q1/6876—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
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention relates to gene mutation detection and particularly relates to a primer, probe and kit for detecting human BCR-ABL gene mutation. The primer and probe for detecting human BCR-ABL gene mutation are a combination of one or several groups selected from the following seven groups of mutation primer and probe sequences. By using the primer, the probe and the kit, seven kinds of point mutation in a BCR-ABL gene can be simultaneously detected; a sample to be detected is detected by using a mutant primer and probe which are designed according to different mutation ways; and only a mutant sample can be successfully amplified to obtain a double-stranded DNA product and is combined with the probe to give a fluorescence signal so as to be detected. The primer, the probe and the kit have the advantages of high speed, simplicity, convenience, safety, high sensitivity, high flux, low cost and the like, and can be used for screening BCR-ABL gene mutation of a great number of clinical samples.
Description
Technical field
The present invention relates to the detection of transgenation, relating to a kind of primer, probe and test kit thereof for detecting mankind BCR-ABL transgenation particularly.
Background technology
Chronic granulocyte is a kind of malignant proliferative disorders originating from hemopoietic stem cell from blood disease (CML), accounts for all leukemic 13%, with No. 9 and No. 22 chromosome translocation t (9:22) (q34; Ql1) Philadelphia chromosome (Ph) formed is feature.This transposition forms a kind of new fusion gene (BCR-ABL), this genes encoding pattern of fusion albumen p210, i.e. a kind of active tyrosine kinase, can cause the abnormal clonal expansion of medullary system hematopoiesis, this is the basic reason causing CML, is also the target site for the treatment of.
Imatinib is the first tyrosine kinase inhibitor going through to be applied to first-line treatment CML.Imatinib can targeting in BCR-ABL, show the clinical efficacy of its brilliance.But, still have 17%CML patient can not obtain complete cytogenetic response (CCyR), about 18% patient having obtained CCyR finally loses curative effect, 14% patient having obtained CCyR can not obtain main molecules reaction (MMR), and the patient of 6% does not tolerate imatinib.
Dasatinib (Dasatinib) is a kind of BCR-ABL and Src kinases double inhibitor of synthetic, is used for the treatment of the adult patient of imatinib mesylate resistance or all stadium of not tolerant chronic lymphocytic leukemia.FDA has ratified Dasatinib treatment to the acute lymphoblastic leukemia adult patient of other therapy resistances or not tolerant Philadelphia Chromosome Positive.Although Dasatinib can work to the mutational site of majority, sudden change can cause its resistance also to have moiety site to occur.Therefore gene test is carried out to its resistance site, for patient directions clinical application, there is important reference value.
Dasatinib is 2nd generation BCR-ABL tyrosine kinase, and it is stronger than imatinib 325 times to the restraining effect of BCR-ABL activity, stronger than AMN107 16 times.Majority can be suppressed to cause the BCR-ABL of imatinib-resistant to suddenly change, but still have some sudden changes that patient can be caused to produce resistance, such as: V299L, T315A, T315I, F317L/I/V/S etc.Therefore gene test is carried out to its resistance site, for patient directions clinical application, there is important reference value.
At present, mainly sample in tumor tissues for detection in Gene Mutation.But the cancer patient of the 70%-80% of first visit clinically all reaches an advanced stage, lose surgical engine meeting, tumor tissues obtains difficulty, so that loses machines meeting.Clinical in setting up easy and widely used monitoring index, the individual resistance (or susceptibility) to targeted drug of evaluate patient, reduces the financial loss that blindly medication brings to patient.
Peripheral blood sampling has easy advantage of drawing materials, and is the first-selected Specimen origin carrying out state of illness monitoring clinically.Research shows, a large amount of in the circulation of blood of solid tumor patient exist free Tumour DNA, and its content is about more than 10 times of Healthy People.In recent years, along with continuing to optimize of nucleic acid amplification technologies, start report abroad and adopt the transgenation of peripheral blood Samples detection dissociative DNA, the result coincidence rate of sensitivity and specificity and tissue sample is higher, this greatly facilitates the progress of targeted drug detection method of gene mutation, is that target molecule inquires into clinical drug efficacy and prognosis molecule mark is just becoming current study hotspot with dissociative DNA.But in peripheral blood, the mutant DNA of cancer cells is the very rare mutator gene be present under a large amount of wild type gene background, and it exists ratio and is generally less than 0.2%.Therefore, challenge can be proposed to existing detection technique by this kind of rare mutation of accurate noninvasive detection.
Summary of the invention
For solving the problem, the invention provides a kind of primer, probe and test kit thereof for detecting mankind BCR-ABL transgenation.
The technical solution used in the present invention is for achieving the above object:
For detecting primer, the probe of mankind BCR-ABL transgenation, the combination by a group in following 7 groups of mutant primers and probe sequence or several groups:
(1)AGATCAAACACCCTAACCATT(SEQ ID NO：1)
GTCGGCAGAGCACAAATATTC(SEQ ID NO：2)
FAM--CAGCTCCTTGGTGAGTAAGCC--BHQ1(SEQ ID NO：3)
(2)GTCAGAATCCATCAGAAGGC(SEQ ID NO：4)
CCGTAGGTCATGAACTCTTC(SEQ ID NO：5)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：6)
(3)GTCAGAATCCATCAGAAGGC(SEQ ID NO：7)
TCCCGTAGGTCATGAACAGAA(SEQ ID NO：8)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：9)
(4)GTCAGAATCCTTCAGAACGC(SEQ ID NO：10)
GGAGGTTCCCGTAGGTCAAAAG(SEQ ID NO：11)
GGAGGTTCCCGTAGGTTGTT(SEQ ID NO：12)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：13)
(5)GTCAGAATCCATCAGAAGGC(SEQ ID NO：14)
AGGTTCCCGTAGGTCAAAAT(SEQ ID NO：15)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：16)
(6)CCGTTCTATATCATCACTCTGG(SEQ ID NO：17)
CCTCTACCTGTGGATGAAGT(SEQ ID NO：18)
FAM--CTTCTCCAGGTACTCCATGGC--BHQ1(SEQ ID NO：19)
(7)GTCAGAATCCTTCAGAACGC(SEQ ID NO：20)
GGAGGTTCCCGTAGGTCCGGG(SEQ ID NO：21)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：22)。
A kind of method detecting mankind BCR-ABL transgenation, to extract the DNA of testing sample as template, adopt primer and the probe of above-mentioned record, increase mutator gene to be measured sequence by fluorescent PCR, the fluorescence intensity of detection reaction system, the standard that during to reach the threshold value of setting, required cycle index Ct value judges as a result, Ct value is 0 or 45: negative; Ct value < 38: positive.
Described testing sample can be whole blood or sample of bone marrow.
The reaction system of described quantitative fluorescent PCR is:
The reaction conditions of described quantitative fluorescent PCR is:
For detecting a test kit for mankind BCR-ABL transgenation, test kit comprises primer described at least one group and probe.
Also comprise for reaction system in described test kit.
The present invention be directed to 7 sudden changes (see table 1) of BCR-ABL gene, design primer and probe divide 7 PCR pipe to detect to measuring samples, primer thalline is wild-type sequence according to mankind BCR-ABL gene 5,6 exon and corresponding mutant gene sequence, designs multipair high specific primer and multiple specific probe.
Described BCR-ABL transgenation specifically refers to table 1:
The 7 kinds of sudden changes of table 1:BCR-ABL gene
The invention has the beneficial effects as follows:
The present invention can detect 7 kinds of point mutation in BCR-ABL gene simultaneously, adopt respectively and for the mutant primers of its different mutational formats design and probe, testing sample is detected, only have saltant type sample could be amplified double stranded DNA product smoothly, and be combined with probe, send fluorescent signal thus be detected.
The present invention, on the basis of Auele Specific Primer and specific probe technology, establishes 7 kinds of transgenations that Multiplex real-time PCR detects mankind BCR-ABL simultaneously, adopts the inventive method can detect 7 kinds of mutator genes under same reaction conditions simultaneously; Only have saltant type sample could be amplified double stranded DNA product smoothly, and be combined with probe, send fluorescent signal thus be detected.The present invention has quick, easy, safe, highly sensitive, the advantage such as high-throughput and low cost, can be used for the BCR-ABL gene mutation for screening of a large amount of clinical sample.Be specially highly sensitive, the sudden change of 5-10 copy can detect; High specificity, 20ng wild type gene group DNA does not have nonspecific signals; Detection speed is fast, and whole testing process needs 90 minutes.The present invention sports detected object with 7 of BCR-ABL gene kinds, by the optimum combination of specially primer and use specific probe, thus realizes quick and precisely, simply detecting 7 kinds of sudden changes, and detects the ability of suddenling change up to 0.1%.
Accompanying drawing explanation
The detection curve figure of real-time PCR detection 7 kinds of BCR-ABL transgenations that Fig. 1 provides for the embodiment of the present invention, wherein, a is that the present invention detects negative control graphic representation; B is wherein a kind of detection curve figure that the present invention detects 7 kinds of sudden change BCR-ABL transgenation;
The real-time PCR detection BCR-ABL detection in Gene Mutation graphic representation that Fig. 2 provides for the embodiment of the present invention, wherein: a is the fluorescence intensity curves that the fluorescent PCR amplification instrument do not suddenlyd change containing BCR-ABL gene T315A in sample measures; B is the fluorescence intensity curves that the fluorescent PCR amplification instrument suddenlyd change containing BCR-ABL gene T315A in sample measures; C is sensitivity analysis experimental result graphic representation; D is selectivity experimental result graphic representation.
The real-time PCR detection BCR-ABL detection in Gene Mutation graphic representation that Fig. 3 provides for the embodiment of the present invention, wherein: a is the fluorescence intensity curves that the fluorescent PCR amplification instrument do not suddenlyd change containing BCR-ABL gene T315I in sample measures; B is the fluorescence intensity curves that the fluorescent PCR amplification instrument suddenlyd change containing BCR-ABL gene T315I in sample measures; C is sensitivity analysis experimental result graphic representation; D is selectivity experimental result graphic representation.
The real-time PCR detection BCR-ABL gene V299L that Fig. 4 provides for the embodiment of the present invention and F317V abrupt climatic change graphic representation, wherein: a is the fluorescence intensity curves that the fluorescent PCR amplification instrument do not suddenlyd change containing BCR-ABL gene V299L and F317V in sample measures; B is the fluorescence intensity curves that the fluorescent PCR amplification instrument suddenlyd change containing BCR-ABL gene V299L and F317V in sample measures.
Embodiment
Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiments be for illustration of
The present invention and be not limited to limit the scope of the invention.The implementation condition adopted in embodiment can do further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in normal experiment.The present invention with the leukocytic genomic dna of Healthy People for wild-type template, the mutant plasmid that has in table 1 and record mutational site is constructed as mutagenesis template using existing genetic engineering technique, set up the reaction system of real-time PCR detection BCR-ABL gene 7 kinds sudden change, and this system is used for the rapid detection of BCR-ABL.This method mainly comprises the following steps:
(1) according to wild-type sequence and 7 kinds of mutant gene sequence of BCR-ABL gene 5,6 exon of Cosmic data announcement, multipair high specific primer and high specific probe is designed.
(2) testing sample process and template extraction.
(3) fluorescent PCR amplification
(4) fluorescent signal is detected, the standard that during to reach the threshold value of setting, required cycle index Ct value judges as a result.
According to the wild-type sequence of BCR-ABL gene 5,6 exon of Cosmic data announcement and 7 kinds of mutant gene sequence in step (1), design multipair high specific primer and high specific probe, refer to table 2.
Wherein step (2) sample preparation and template extraction, the sample scope of application comprises whole blood and marrow.For the operation instructions of corresponding genome DNA extracting reagent kit on the process reference market of sample.
The reaction system of the fluorescent PCR amplification of step (3) is:
Above reagent constituents: 10 × PCR buffer (Free Mg
2+), MgCl
2, dNTP MIX and Taq enzyme be purchased from TaKaRa biotech firm.
Reaction conditions is:
Step (4) detects the Ct value of fluorescent signal, is to adopt StepOne
tMreal-time fluorescence quantitative PCR instrument annealing stage detects fluorescence, once can detect 96 increment product (comprising yin and yang attribute Quality Control).Ct value judged result according to the display of fluorescent PCR amplification instrument: Ct value is that 0 or 45 expressions are negative; Ct value < 38 expression is positive; When Ct value is between 38 ~ 45, sample need detect again.Testing process required time is about 90 minutes.
Embodiment 1
The whole blood sample BCR-ABL gene T315A detecting clinical acquisitions using the reagent in test kit sports example as sample.Record T315A sudden change in employing table 2, designed primer pair and probe are respectively M2-F, M2-R, M2-P; Primer pair and probe are that the wildtype gene sequence of BCR-ABL gene extron 6 announced according to Cosmic data and mutant gene sequence comparative analysis obtained, simultaneously according to the wildtype gene sequence analysis of the HAK gene of GeneBank data announcement, design pair of primers and probe are respectively: HAK-F, HAK-R, HAK-P (detailed sequence table 3).According to the wildtype gene sequence analysis of the mankind BCR-ABL gene extron 11 that GeneBank data are announced, design pair of primers and probe are respectively W-F, W-R, W-P (detailed sequence table 3).
Table 3 primer and probe
The method utilizing above-mentioned fluorescent PCR system to detect the whole blood sample BCR-ABL gene T315A abrupt climatic change of clinical acquisitions comprises the following steps:
(1) DNA in TIANGEN company poba gene group DNA extraction kit extraction whole blood is used, step is as follows: sample thief 200 μ L adds the cell pyrolysis liquid CL of 400 μ L, 200 μ L damping fluid GS are added wherein after centrifugal collecting cell core precipitation, add 20 μ L Proteinase K solution again, 200 μ L damping fluid GB are added after mixing, fully put upside down mixing, place 10min for 56 DEG C.Transfer to after adding 200 μ L dehydrated alcohols in an adsorption column, the centrifugal 1min of 12,000rpm, abandons waste liquid.In adsorption column, add 500 μ L damping fluid GD, the centrifugal 1min of 12,000rpm, abandons waste liquid.In adsorption column, add 600 μ L rinsing liquid PW12, the centrifugal 1min of 000rpm, abandons waste liquid.In adsorption column, add 600 μ L rinsing liquid PW, the centrifugal 1min of 12,000rpm, outwells waste liquid.Drip 50 μ L elution buffer TB to adsorption film mid-way, room temperature places the centrifugal 2min of 2min, 12,000rpm, by solution collection in centrifuge tube.Get 2 μ L and survey concentration, the sample DNA of extraction is diluted to 20ng/ μ L, gets 2 μ L and carry out PCR reaction.
(2) fluorescent PCR amplification, its reaction system is:
Real-time PCR reactions condition is:
(3) detect: adopt StepOne TM real-time fluorescence quantitative PCR instrument (ABI company) annealing stage to detect fluorescence, the PCR pipe containing reaction solution is put into fluorescent PCR amplification instrument one by one and detects.Ct value judged result according to the display of fluorescent PCR amplification instrument: Ct value is that 0 or 45 expressions are negative; Ct value < 38 expression is positive; When Ct value is between 38 ~ 45, sample need detect again.Testing process required time is about 90 minutes.
From Fig. 2 a and 2b, the fluorescence intensity curves display result that the fluorescent PCR amplification instrument do not suddenlyd change containing BCR-ABL gene T315A in sample measures is negative; When suddenling change containing BCR-ABL gene T315A in sample, it is 17.25 that fluorescence intensity curves shows its Ct value, and result is positive.
(4) specificity analyses
Specific detection: to build the mutant plasmid V299L of acquisition in contrast through existing genetic engineering technique, according to above-mentioned reaction system and reaction conditions application fluorescence quantitative PCR method, BCR-ABL gene T315A abrupt climatic change is carried out to sample, specificity analyses result shows, the sample suddenlyd change containing BCR-ABL gene T315A is only had to have fluorescent signal, V299L plasmid unstressed configuration signal in contrast.
(5) sensitivity analysis
Carry out quantitative analysis with the plasmid DNA containing BCR-ABL gene T315A sudden change that existing genetic engineering technique synthesizes, concentration quantitatively of learning from else's experience is 1 × 10
4the sample DNA of individual copy number, carries out 10 times of dilutions, and work 4 extent of dilution, then gets 5 dilution 2 μ L successively, and 8 parallel group is carried out Fluorescence PCR (see Fig. 2 c).Highly sensitive by the visible fluorescence PCR method of the present invention of Fig. 2 c, 1-10copies/ μ L can detect.
(6) Choice tests
1000,100,10,1 copy numbers of the plasmid DNA that the BCR-ABL gene T315A synthesized with existing genetic engineering technique suddenlys change carry out fluorescent PCR detection (see Fig. 2 d) under the background of 20ng.Good by the visible fluorescence PCR method selectivity of the present invention of Fig. 2 d.
Embodiment 2
The whole blood sample BCR-ABL gene T315I detecting clinical acquisitions using the reagent in test kit sports example as sample.Record T315I sudden change in employing table 2, designed primer pair and probe are respectively M3-F, M3-R, M3-P; Primer pair and probe are that the wildtype gene sequence of BCR-ABL gene extron 6 announced according to Cosmic data and mutant gene sequence comparative analysis obtained, simultaneously according to the wildtype gene sequence analysis of the HAK gene of GeneBank data announcement, design pair of primers and probe are respectively: HAK-F, HAK-R, HAK-P (detailed sequence table 3).According to the wildtype gene sequence analysis of the mankind BCR-ABL gene extron 11 that GeneBank data are announced, design pair of primers and probe are respectively W-F, W-R, W-P (detailed sequence table 3).
The method utilizing above-mentioned fluorescent PCR system to detect the whole blood sample BCR-ABL gene T315I abrupt climatic change of clinical acquisitions comprises the following steps:
1) DNA in TIANGEN company poba gene group DNA extraction kit extraction whole blood is used, step is as follows: sample thief 200 μ L adds the cell pyrolysis liquid CL of 400 μ L, 200 μ L damping fluid GS are added wherein after centrifugal collecting cell core precipitation, add 20 μ L Proteinase K solution again, 200 μ L damping fluid GB are added after mixing, fully put upside down mixing, place 10min for 56 DEG C.Transfer to after adding 200 μ L dehydrated alcohols in an adsorption column, the centrifugal 1min of 12,000rpm, abandons waste liquid.In adsorption column, add 500 μ L damping fluid GD, the centrifugal 1min of 12,000rpm, abandons waste liquid.In adsorption column, add 600 μ L rinsing liquid PW12, the centrifugal 1min of 000rpm, abandons waste liquid.In adsorption column, add 600 μ L rinsing liquid PW, the centrifugal 1min of 12,000rpm, outwells waste liquid.Drip 50 μ L elution buffer TB to adsorption film mid-way, room temperature places the centrifugal 2min of 2min, 12,000rpm, by solution collection in centrifuge tube.Get 2 μ L and survey concentration, the sample DNA of extraction is diluted to 20ng/ μ L, gets 2 μ L and carry out PCR reaction.
(2) fluorescent PCR amplification, its reaction system is:
Real-time PCR reactions condition is:
(3) detect: adopt StepOne
tMreal-time fluorescence quantitative PCR instrument (ABI company) annealing stage detects fluorescence, the PCR pipe containing reaction solution is put into fluorescent PCR amplification instrument one by one and detects.Ct value judged result according to the display of fluorescent PCR amplification instrument: Ct value is that 0 or 45 expressions are negative; Ct value < 38 expression is positive; When Ct value is between 38 ~ 45, sample need detect again.Testing process required time is about 90 minutes.
From Fig. 3 a and 3b, the fluorescence intensity curves display result that the fluorescent PCR amplification instrument do not suddenlyd change containing BCR-ABL gene T315I in sample measures is negative; When suddenling change containing BCR-ABL gene T315I in sample, it is 17.25 that fluorescence intensity curves shows its Ct value, and result is positive.
(4) specificity analyses
Specific detection: to build the mutant plasmid V299L of acquisition in contrast through existing genetic engineering technique, according to above-mentioned reaction system and reaction conditions application fluorescence quantitative PCR method, BCR-ABL gene T315I abrupt climatic change is carried out to sample, specificity analyses result shows, the sample suddenlyd change containing BCR-ABL gene T315I is only had to have fluorescent signal, V299L plasmid unstressed configuration signal in contrast.
(5) sensitivity analysis
Carry out quantitative analysis with the plasmid DNA containing BCR-ABL gene T315I sudden change that existing genetic engineering technique synthesizes, concentration quantitatively of learning from else's experience is 1 × 10
4the sample DNA of individual copy number, carries out 10 times of dilutions, and work 4 extent of dilution, then gets 5 dilution 2 μ L successively, and 8 parallel group is carried out Fluorescence PCR (see Fig. 3 c).Highly sensitive by the visible fluorescence PCR method of the present invention of Fig. 3 c, 1-10copies/ μ L can detect.
(6) Choice tests
1000,100,10,1 copy numbers of the plasmid DNA that the BCR-ABL gene T315I synthesized with existing genetic engineering technique suddenlys change carry out fluorescent PCR detection (see Fig. 3 d) under the background of 20ng.Good by the visible fluorescence PCR method selectivity of the present invention of Fig. 3 d.
Embodiment 3
Detect simulation whole blood sample BCR-ABL gene V299L and F317V using the reagent in test kit and sport example as sample.Record V299L and F317V sudden change in employing table 2, designed primer pair and probe are respectively M1-F1, M1-R, M1-P and M6-F, M6-R, M6-P; Primer pair and probe are that the wildtype gene sequence of BCR-ABL gene extron 5,6 announced according to Cosmic data and mutant gene sequence comparative analysis obtained, simultaneously according to the wildtype gene sequence analysis of the HAK gene of GeneBank data announcement, design pair of primers and probe are respectively: HAK-F, HAK-R, HAK-P (detailed sequence table 3).According to the wildtype gene sequence analysis of the mankind BCR-ABL gene extron 11 that GeneBank data are announced, design pair of primers and probe are respectively W-F, W-R, W-P (detailed sequence table 3).
The method utilizing above-mentioned fluorescent PCR system to detect simulation whole blood sample BCR-ABL gene V299L and F317V abrupt climatic change comprises the following steps:
(1) be 1 × 10 by concentration
3the V299L 859G>T saltant type of copy/μ L and the F317V saltant type of same concentrations are drawn 10 μ L respectively and are added in the Whole Blood of Healthy of 80 μ L, mixing.The mixing sample of 80 μ L Whole Blood of Healthies is added for contrast with 20 μ LPBS damping fluids.
(2) above-mentioned biased sample TIANGEN company poba gene group DNA extraction kit is extracted DNA sample, step is as follows: sample thief 200 μ L adds the cell pyrolysis liquid CL of 400 μ L, 200 μ L damping fluid GS are added wherein after centrifugal collecting cell core precipitation, add 20 μ L Proteinase K solution again, 200 μ L damping fluid GB are added after mixing, fully put upside down mixing, place 10min for 56 DEG C.Transfer to after adding 200 μ L dehydrated alcohols in an adsorption column, the centrifugal 1min of 12,000rpm, abandons waste liquid.In adsorption column, add 500 μ L damping fluid GD, the centrifugal 1min of 12,000rpm, abandons waste liquid.In adsorption column, add 600 μ L rinsing liquid PW12, the centrifugal 1min of 000rpm, abandons waste liquid.In adsorption column, add 600 μ L rinsing liquid PW, the centrifugal 1min of 12,000rpm, outwells waste liquid.Drip 50 μ L elution buffer TB to adsorption film mid-way, room temperature places the centrifugal 2min of 2min, 12,000rpm, by solution collection in centrifuge tube.Get 2 μ L and survey concentration, the sample DNA of extraction is diluted to 20ng/ μ L, gets 2 μ L and carry out PCR reaction.
(2) fluorescent PCR amplification, its reaction system is:
Real-time PCR reactions condition is:
(3) detect: adopt StepOne
tMreal-time fluorescence quantitative PCR instrument (ABI company) annealing stage detects fluorescence, the PCR pipe containing reaction solution is put into fluorescent PCR amplification instrument one by one and detects.Ct value judged result according to the display of fluorescent PCR amplification instrument: Ct value is that 0 or 45 expressions are negative; Ct value < 38 expression is positive; When Ct value is between 38 ~ 45, sample need detect again.Testing process required time is about 90 minutes.
From Fig. 4 a and 4b, the fluorescence intensity curves display result that the fluorescent PCR amplification instrument do not suddenlyd change containing BCR-ABL gene V299L and F317V in sample measures is negative; When suddenling change containing BCR-ABL gene V299L and F317V in sample, fluorescence intensity curves shows its Ct value and is respectively 31.72 and 31.84, and result is positive.
Claims (7)
1. for detecting primer, the probe of mankind BCR-ABL transgenation, it is characterized in that: the combination by a group in following 7 groups of mutant primers and probe sequence or several groups:
(1)AGATCAAACACCCTAACCATT(SEQ ID NO：1)
GTCGGCAGAGCACAAATATTC(SEQ ID NO：2)
FAM--CAGCTCCTTGGTGAGTAAGCC--BHQ1(SEQ ID NO：3)
(2)GTCAGAATCCATCAGAAGGC(SEQ ID NO：4)
CCGTAGGTCATGAACTCTTC(SEQ ID NO：5)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：6)
(3)GTCAGAATCCATCAGAAGGC(SEQ ID NO：7)
TCCCGTAGGTCATGAACAGAA(SEQ ID NO：8)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：9)
(4)GTCAGAATCCTTCAGAACGC(SEQ ID NO：10)
GGAGGTTCCCGTAGGTCAAAAG(SEQ ID NO：11)
GGAGGTTCCCGTAGGTTGTT(SEQ ID NO：12)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：13)
(5)GTCAGAATCCATCAGAAGGC(SEQ ID NO：14)
AGGTTCCCGTAGGTCAAAAT(SEQ ID NO：15)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：16)
(6)CCGTTCTATATCATCACTCTGG(SEQ ID NO：17)
CCTCTACCTGTGGATGAAGT(SEQ ID NO：18)
FAM--CTTCTCCAGGTACTCCATGGC--BHQ1(SEQ ID NO：19)
(7)GTCAGAATCCTTCAGAACGC(SEQ ID NO：20)
GGAGGTTCCCGTAGGTCCGGG(SEQ ID NO：21)
FAM--CCACGTGTTGAAGTCCTCGTTG--BHQ1(SEQ ID NO：22)。
2. detect a method for mankind BCR-ABL transgenation, it is characterized in that:
To extract the DNA of testing sample as template, adopt primer and the probe of above-mentioned record, increase mutator gene to be measured sequence by fluorescent PCR, the fluorescence intensity of detection reaction system, the standard that during to reach the threshold value of setting, required cycle index Ct value judges as a result, Ct value is 0 or 45: negative; Ct value < 38: positive.
3., by the method for detection mankind BCR-ABL according to claim 2 transgenation, it is characterized in that: described testing sample can be whole blood or sample of bone marrow.
4., by the method for detection mankind BCR-ABL according to claim 2 transgenation, it is characterized in that: the reaction system of described quantitative fluorescent PCR is:
5., by the method for detection mankind BCR-ABL according to claim 2 transgenation, it is characterized in that: the reaction conditions of described quantitative fluorescent PCR is:
6. for detecting a test kit for mankind BCR-ABL transgenation, it is characterized in that: test kit comprises primer described at least one group of claim 1 and probe.
7. by the test kit for detecting mankind BCR-ABL transgenation according to claim 6, it is characterized in that: described test kit is reaction system.
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