CN109554474A - A kind of method and kit of BCR-ABL fusion quantitative detection - Google Patents
A kind of method and kit of BCR-ABL fusion quantitative detection 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
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- 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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Abstract
The present invention provides the methods and kit of a kind of BCR-ABL fusion quantitative detection, specific technical solution of the present invention only needs two different upstream primers for two kinds of BCR-ABL fusion types, and common downstream primer is detected with the probe of fluorescent marker and a set of ABL reference gene detection system.Using specific primer provided by the invention and probe, 1 pipe is only needed to can be detected 2 kinds of fused types of BCR-ABL, and the specific and good sensibility with height, accuracy are strong.
Description
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to a kind of BCR-ABL fusion quantitative detections
Method and kit.
Background technique
Leukaemia, also referred to as leukemia are the malignant clone diseases of candidate stem cell.Leukaemia cell generates in marrow,
Spreading to blood and whole body.According to foreign statistic, leukaemia accounts for about 3% or so of tumour total incidence, is children and youth
Middle one of the most common type malignant tumour.The disease incidence of leukaemia is 4.8-7.1/10 ten thousand in male, is 3.2-4.6/ in women
100000；In countries in the world, Europe and North America disease incidence highest, the death rate are ten thousand population of 3.2-7.4/10.Asia and South America
Continent disease incidence is lower, and the death rate is ten thousand population of 2.8-4.5/10.
Typical Philadelphia (Ph) chromosome, i.e. the abl original cancer base of 9q34 is presented in chronic myelogenous leukemia (CML) malignant cell
Because forming t (9 with the bcr Gene Fusion of 22q11；22) it transcribes and translates into the bcr/ with high protein tyrosine kinase activity
Abl fusion protein.Its expression has activated tyrosine protein kinase, changes the protein-tyrosine level and actin of cell
Binding ability activates multi-signal pathway, makes cell hyperproliferation and cell regulation is made to get muddled.
Due to the breaking point of ABL and BCR gene and the difference of amalgamation mode, corresponding 3 kinds of differences may finally be generated
The isomers of BCR-ABL fusion, respectively main type (M), secondary type (m) and microminiature (μ), wherein main type and time
Type is wanted to reach 95% or more.Main type, that is, e13a2 or e14a2, expresses p210 fusion protein, sees the CML patient of 97-98%
In；Secondary type, that is, e1a2 expresses p190 fusion protein, sees in 75% ALL patient；Microminiature, that is, e6a2, e8a2 or e19a2
Deng, express p230 fusion protein, see in the CML patient of 2-3%.
BCR-ABL fusion is commonly detected including CYTOGENETIC ANALYSIS OF ONE, fluorescence in situ hybridization, immune point at present
The detection methods such as type, polymerase chain reaction.
(1) CYTOGENETIC ANALYSIS OF ONE, acquires marrow or peripheral blood carries out Short-term Culture, routinely harvests and prepare chromosome,
Marrow specimen adds simultaneously does direct method, and karyotyping is all made of RHG banding technique.But leukaemia cell's splitting index is low, dyeing
Volume morphing is short and small, is easy aobvious with unclear, and structure is complicated or subtle change is not easy to prepare identification.
(2) fluorescence in situ hybridization (FISH detection), acquires marrow or peripheral blood as research object, to metaphase and
Phase nucleus can detect, and synchronous can also test and analyze to concealment type with variability.Sensitivity is higher, and specificity is preferable, but often
It, can be because of bcr and abl gene signal random distribution or optics when the double-colored single fusion probe in detecting bcr/abl fusion seen
Overlapping, leads to the false positive rate of 4%-10%.
(3) immunophenotyping, using flow cytometer (FCM), hemolytic agent (optiplyse C) changes cell membrane permeability
The antibody of kit and FITC label, can quickly analyze a large amount of cells, accurate convenient.But FCM method needs specific apparatus,
Operating technology is more demanding, expensive, and result repeatability is bad.
(4) polymerase chain reaction, there are mainly two types of: one is RT-PCR, directly detect slow grain BCR-ABL mRNA.But
Since primer mispairing and non-specific return goods occur, it is easy to produce non-specific result；Another kind is that Taqman fluorescence probe is fixed
PCR is measured, Taqman probe 5 ' holds one fluorescent molecule of label, 3 ' end one fluorescence quencher molecule of label.When probe is complete, two
Fluorescence resonance energy transmitting (FRET) occurs for person.When PCR amplification, due to Taq enzyme 5 ' → 3 ' 5 prime excision enzyme activities, by probe hydrolysis,
Increase fluorescent molecule and quencher molecule spacing, fluorescence monitoring system detects fluorescence signal.But it relies on Ct value higher, standard
Exactness is inadequate, and sensitivity is bad.2.3 inventive solution
(5) digital pcr, digital pcr (dPCR) are able to achieve the absolute quantitation of nucleic acid molecules, melt in detection BCR-ABL gene
Sensitivity and specificity with higher when conjunction.It is distributed a PCR reaction system by way of physically or chemically dividing
In the reactor small to up to ten thousand, includes in each microreactor or the target nucleic acid not comprising one or more copies divides
Son carries out " unimolecule template PCR amplifications ".After amplification, calculated by the number and statistical method of positive reaction unit
The copy number of target gene in original sample.Therefore, dPCR can be directly determined down to single without establishing standard curve and be copied target to be checked
The absolute number of molecule.
A large number of studies show that tyrosine kinase can be assessed by monitoring the BCR-ABL fusion transcriptional level of patient
The curative effect of inhibitor (TKI), thus EARLY RECOGNITION drug resistance or progression of disease, to instruct therapeutic intervention.To sum up, it needs at this stage
It is a kind of in high sensitivity can fast and reliable ground detect the expression of the main type of BCR-ABL fusion and secondary type mRNA simultaneously
Method and kit.
Therefore, those skilled in the art are dedicated to exploitation can detect a variety of leukemia fusion genes and mutated gene simultaneously
Detection method reduces testing cost to improve detection efficiency.
Summary of the invention
The purpose of the present invention is to provide the methods and kit of a kind of BCR-ABL fusion quantitative detection.
In the first aspect of the present invention, a kind of primer pair group of quantitative detection BCR-ABL fusion is provided, it is described to draw
Object includes the first upstream primer and the first downstream primer to group, shown in the first upstream primer sequence SEQ ID NO.:1；Institute
The first downstream primer sequence is stated as shown in SEQ ID NO.:3.
In another preferred example, the primer pair group further includes the second upstream primer, the second upstream primer sequence SEQ
Shown in ID NO.:2.
In another preferred example, the primer pair group further includes internal control primer pair, and the internal control primer pair includes:
Internal control upstream primer shown in SEQ ID NO:5；With internal control downstream primer shown in SEQ ID NO.:6.
The second aspect of the present invention provides a kind of probe groups of quantitative detection BCR-ABL fusion, the probe groups
Including probe sequence shown in SEQ ID NO.:4.
In another preferred example, the probe groups further include internal control probe sequence shown in SEQ ID NO.:7.
In another preferred example, 5 ' ends of SEQ ID NO:4 nucleotide sequence are marked with FAM, 3 ' ends are marked with BHQ1.
In another preferred example, 5 ' ends of SEQ ID NO:7 nucleotide sequence are marked with VIC, 3 ' ends are marked with BHQ1+
ZIP。
The third aspect of the present invention provides a kind of kit of quantitative detection BCR-ABL fusion, the kit
Including primer pair group described in first aspect present invention.
In another preferred example, the kit further includes probe groups described in second aspect of the present invention.
In another preferred example, the kit further includes RT- buffer, and the RT- buffer includes (NH4)2SO4、
KCl、Tris-HCl、MgCl2And DTT.
In another preferred example, the kit further includes RNA enzyme, the RNA enzyme include dNTPs, hot start Taq polymerase,
And reverse transcriptase.
In another preferred example, the kit further includes 7-deaza-dGTP.
The fifth aspect of the present invention provides a kind of method of quantitative detection BCR-ABL fusion, the method includes
Step:
(1) sample to be detected is provided, and extracts sample gene to be tested group nucleic acid；
(2) dPCR amplification reaction system is prepared, is carried out amplification reaction:
Wherein, the dPCR amplification reaction system includes the sample to be detected that step (1) provides；First aspect present invention institute
Probe groups described in the primer pair group and second aspect of the present invention stated.
In another preferred example, described to be detected as non-diagnostic purpose；For example it can be carried out using method of the invention public
The information analysis of health field altogether, or the sample to be tested for being originated from animal pattern is analyzed during new drug development.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Specific embodiment
The present inventor by extensive and in-depth research, obtain a kind of quantitative detection BCR-ABL fusion method and
Kit only needs two different upstream primers for two kinds of BCR-ABL fusion types, common downstream primer with one
The probe of fluorescent marker and a set of ABL reference gene detection system are detected.Using specific primer provided by the invention with
Probe only needs 1 pipe to can be detected 2 kinds of fused types of BCR-ABL, can calculate IS%, and then judges that molecules is reacted
(MMR).The present invention can detect that IS% is 0.0032%, that is, reach molecules reaction MR4.5 high sensitivity.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this
Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and
And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated
" about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method
And material, place enumerates preferred method and material herein.
Digital pcr, digital pcr (dPCR) are able to achieve the absolute quantitation of nucleic acid molecules, when detecting BCR-ABL Gene Fusion
Sensitivity and specificity with higher.One PCR reaction system is assigned to by it by way of physically or chemically dividing
In ten thousand small reactors, in each microreactor include or the target nucleic acid molecules not comprising one or more copies, into
Row " unimolecule template PCR amplifications ".After amplification, original sample is calculated by the number and statistical method of positive reaction unit
The copy number of target gene in this.Therefore, dPCR can be directly determined without establishing standard curve down to single copy target molecule to be checked
Absolute number.
The present inventor to it is existing it is leucocythemia related because variation, carry out go deep into compare analysis after, devise primer and spy
Needle, then the primer and probe of design is in optimized selection and is verified, it has finally determined and can be used for multiple digital PCR amplification
Primer and probe sequence provide the detection kit of quantitative detection BCR-ABL fusion on this basis.
Method of the invention can be based on STILLA digital pcr platform, quantitative detection chronic myelogenous leukemia (CML), urgency
The expression of the main type of BCR-ABL fusion and secondary type mRNA in the hematological system tumors such as property lymphocytic leukemia (ALL)
It is horizontal.
Primer that BCR-ABL fusion immue quantitative detection reagent box of the invention includes, probe and including the primed probe
(especially addition 7-deaza-dGTP increases positive signal fluorescent value, reaches more highly sensitive for mixed liquor and dPCR reaction solution
Degree).Detection method and kit of the invention has high sensitivity, can achieve IS%=0.0032%, i.e. molecules is reacted
MR4.5。
The present invention provides one kind to be based on digital pcr platform, one-step method quantitative detection chronic myelogenous leukemia (CML), urgency
The expression of the main type of BCR-ABL fusion and secondary type mRNA in the hematological system tumors such as property lymphocytic leukemia (ALL)
Horizontal method, primer, probe and its kit has simple experimentation, absolute quantitation, draws and visit that dosage is low, sample easily obtains
The advantages that taking.The present invention reaches positive signal fluorescent value using addition 7-deaza-dGTP and increases, and improves sensitivity.
Technical scheme is as follows:
One kind being based on digital pcr platform, quantitative detection chronic myelogenous leukemia (CML), acute lymphoblastic leukemia
(ALL) etc. the method for the main type of BCR-ABL fusion and the expression of secondary type mRNA in hematological system tumors, primer,
Probe and kit including the primed probe mixed liquor.It provides and melts for detecting BCR-ABL P210 or BCR-ABL P190
Specific primer and probe needed for closing gene transcription level.Addition 7-deaza-dGTP increases positive signal fluorescent value,
Reach more highly sensitive.
The invention discloses a kind of quantitative detection chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) etc.
Primer, the probe of the main type of BCR-ABL fusion and the expression of secondary type mRNA in hematological system tumor:
The upstream primer nucleotide sequence of the detection BCR-ABL P210 fusion is as shown in SEQ ID NO:1, institute
The upstream primer nucleotide sequence of detection BCR-ABL P190 fusion is stated as shown in SEQ ID NO:2, the detection BCR-
The downstream primer sequence of ABL P210 fusion and BCR-ABL P190 fusion is as shown in SEQ ID NO:3, the inspection
The upstream primer nucleotide sequence of ABL internal control gene is surveyed as shown in SEQ ID NO:5, the downstream of the detection ABL internal control gene
Primer nucleotide sequences are as shown in SEQ ID NO:6；
The probe includes the probe and internal control Gene A BL probe for detecting BCR-ABL fusion transcriptional level；It is described
The nucleotide sequence of the probe of BCR-ABL fusion transcriptional level such as SEQ ID NO:4, the probe nucleotide sequence of internal control is such as
Shown in SEQ ID NO:7.
Further, 5 ' ends of the SEQ ID NO:4 nucleotide sequence are marked with FAM, 3 ' ends are marked with BHQ1, described
5 ' ends of SEQ ID NO:7 nucleotide sequence are marked with VIC, 3 ' ends are marked with BHQ1+ZIP.
Preferably, the final concentration of 0.8 μm of ol/L of the upstream primer in the reaction system, the downstream primer are being reacted
Final concentration of 0.8 μm of ol/L in system, the final concentration of 0.3 μm of ol/L of the fusion probe in the reaction system；Institute
State the final concentration of 1.5 μm of ol/L of internal control upstream primer in the reaction system, the internal control downstream primer is in the reaction system
Final concentration of 1.5 μm of ol/L, the final concentration of 0.2 μm of ol/L of the internal control probe in the reaction system；
The primer probe sequence such as following table of the BCR-ABL fusion and internal control,
Detect the primed probe nucleotide sequence information of BCR-ABL fusion:
Above-mentioned PCR primer and the probe for indicating different type fluorescence can be used for merging BCR-ABL the main type of situation
(BCR-ABL P210) and secondary type (BCR-ABL P190) type is detected, according to result present fluorescence signal whether there is or not,
Whether judgement sample DNA profiling merges；Above-mentioned primer and probe is according to 2 kinds of people BCR-ABL P210, BCR-ABL P190 types
Fusion and abl gene group DNA sequence dna design and synthesize, can detecte 2 kinds of BCR-ABL P210, BCR-ABL P190
Fused type；The common fused type of BCR-ABL gene is as follows:
BCR-ABL P210 is transcribed by b3a2 or b2a2 forms bcr/abl fusion mRNA, encodes the endochylema of 210Da molecular weight
Albumen P210；
BCR-ABL P190 encodes P190 fusion protein by the heterozygosis mRNA of ela2 connector；
The specific primer and probe can accurately detect whether BCR-ABL gene merges, in detection fusion
Meanwhile gene transcript levels can be immediately arrived at correctly to evaluate patient's curative effect.Using fusion probe in detecting BCR-ABL
Copy number and internal control probe in detecting ABL copy number, determine whether BCR-ABL occurs Gene Fusion and meter by copy number ratio
IS% is calculated, according to BCR-ABLIS and then judges that molecules reacts (MMR).Present invention combination dPCR system to detect body
System can achieve molecules reaction MR4.5.
Kit prepared by above-mentioned specific primer and probe can detect BCR-ABL fusion based on dPCR platform
Two kinds of fused types, provide reference for whether patient needs to carry out targeted therapy, it can also be used to which leukaemic's is highly sensitive
Spend early detection and curative effect monitoring.
The invention also discloses a kind of kits of BCR-ABL fusion detection, including for preparing dPCR reaction solution
Primed probe mixed liquor, RT-buffer, RNA enzyme, Fluorescein sodium salt, 7-deaza-dGTP, control sample
This and RNase-free water, wherein primed probe mixed liquor includes following ingredient, such as table 1,
1 primed probe mixed liquor of table
Wherein, the primer for detecting BCR-ABL fusion and ABL internal control gene is distinguished as follows with probe,
The upstream primer nucleotide sequence of the detection BCR-ABL P210 fusion is as shown in SEQ ID NO:1, institute
The upstream primer nucleotide sequence of detection BCR-ABL P190 fusion is stated as shown in SEQ ID NO:2, the detection BCR-
The downstream primer sequence of ABL P210 fusion and BCR-ABL P190 fusion is as shown in SEQ ID NO:3, the inspection
The upstream primer nucleotide sequence of ABL internal control gene is surveyed as shown in SEQ ID NO:5, the downstream of the detection ABL internal control gene
Primer nucleotide sequences are as shown in SEQ ID NO:6；
The probe includes the probe and ABL internal control probe for detecting BCR-ABL Gene Fusion；The BCR-ABL gene melts
The nucleotide sequence such as SEQ ID NO:4 of probe is closed, 5 ' ends are marked with FAM fluorescent reporter group, the ABL internal control probe
For nucleotide sequence as shown in SEQ ID NO:7,5 ' ends are marked with VIC fluorescent reporter group, 3 ' end marks of all probes
Note has BHQ1 fluorescent quenching group, 3 ' end labels of ABL internal control probe plus Zip modification；
When BCR-ABL gene merges, fusion probe in conjunction with the target fragment that upstream and downstream primer is expanded,
Discharge FAM fluorescence signal；The internal control primer and probe are designed and synthesized according to people's abl gene conservative fragments, for examining
It surveys containing the abl gene that Gene Fusion does not occur；
The RT-buffer of the dPCR reaction solution includes following ingredient, such as table 2,
2 RT-buffer of table
| Number | Component | Main component in component |
| 1 | RT-buffer | (NH4)2SO4、KCl、Tris-HCl、MgCl2、DTT |
The 7-deaza-dGTP of the dPCR reaction solution can significantly improve positive signal fluorescent value, reach positive and background
Discrimination is more preferably.
The reference substance includes following ingredient, such as table 3,
3 check sample of table
Preferably, contain Caco2 cell strain nucleic acid in the cell strain DNA mixed liquor of the gDNA control；
Contain in the cell strain DNA mixed liquor of the positive control:
Detect the outer conjunction large fragment 1 of BCR-ABL P210 fusion；
Detect the outer conjunction large fragment 2 of BCR-ABL P190 fusion；
Detect the outer conjunction large fragment 3 of ABL reference gene.
It is peripheral blood DNA nucleic acid that kit of the present invention, which is applicable in sample,.
Kit of the present invention be used for determines detect validity standard are as follows: every time detect be respectively provided with negative control group,
GDNA control group and positive controls, when the positive controls of testing result are the positive, and negative control group and gDNA control group
When being feminine gender, show that experimental result is effective.The detection sensitivity of kit of the present invention can achieve 0.0032%, that is, divide
Son learns reaction MR4.5.
The invention also discloses a kind of method of BCR-ABL fusion quantitative detection, specific steps include:
1. processing sample to be tested simultaneously extracts sample DNA template；Preferably, sample to be tested is peripheral blood DNA nucleic acid；
2. dPCR reaction system is prepared, by sample to be tested DNA profiling, specific primer and probe, RT-buffer, RNA
Enzyme, Fluorescein sodium salt, the mixing of 7-deaza-dGTP and RNase-free water, are prepared dPCR reaction solution；
DPCR reaction solution constitutes such as table 4,
4 dPCR reaction solution of table
| Specific primer and probe | 1μL |
| DNA profiling | 5μL |
| RT-buffer | 5μL |
| RNA enzyme | 3μL |
| 7-deaza-dGTP | 1μL |
| Fluorescein sodium salt | 2.5μL |
| RNase-free water | Complement to 25 μ L |
3. Sapphire chip is smoothly placed on experimental bench, gently rotates white lid 1/4 and enclose, abandon white lid, pipette
The dPCR reaction solution that 25 μ L are prepared is added to the Kong Jingzhong of Sapphire chip, stands 2-3min and covers the long lid of white.
4. opening Naica Geode droplet generates amplification system and pulsometer switch, air pressure pump output pressure and micro- is confirmed
Drop generate amplification system input (input) pressure stablize in 1150+/- 50mbar, if not in this range, adjusted to
1150+/-50mbar。
5. according to " digital pcr tests protocol " setting response procedures: 40 DEG C of Partition at, " Sapphire
V1 ", the first stage, 50 DEG C reverse transcription 30 minutes；Second stage, 95 DEG C initial denaturation 10 minutes；Phase III, 95 DEG C of denaturation 30
Second, 56 DEG C extend 30 seconds, 45 circulations；Release P.
It is added in module 6. being lightly placed in Sapphire chip, covers Naica Geode machine cover, confirm journey
After sequence is errorless, " play " button is clicked, starts to generate droplet and PCR response procedures.
7. being scanned with " Crytal Reader " software to amplification, and " Cristal is opened in interface
Minder " analyzes scanning result, and the copy of target molecules in each sample is calculated based on Poisson distribution Statistics
Number；
8. determining whether sample to be tested merges according to the intensity and ratio of each fluorescence signal, and then calculate IS%
(IS%=BCR-ABL fusion copy number/ABL reference gene copy number × 100%).
The application is as follows using dPCR testing principle: dPCR technology is that the reaction reagent in single PCR pipe is divided into about
Micro- reactions up to ten thousand in each micro- reaction or are free of nucleic acid target molecule to be checked, or contain 1 to several separate target nucleic acids to be checked
Son, and each micro- reaction is used as an independent PCR reaction member.After PCR process, fluorescence is carried out one by one to micro- reaction
Detection identifies that Yin/Yang is reacted.Micro- reaction containing different DNA profilings releases different fluorescence signals, and template is not micro-
Reaction does not generate fluorescence signal then.Finally, calculating target to be checked according to the ratio of Poisson distribution Statistics and positive micro- reaction
Mark molecule copy number.Since the interpretation of dPCR result only determines whether amplification, Ct value is not depended on, to the resistance to of PCR reaction suppressor
Greatly improved by ability, without necessarily referring to product and standard curve can accurate quantification, provided for this patent a kind of completely new
Technical thought and means.
It is preferably carried out in mode at of the invention one, the method for the present invention includes steps:
Step 1: sample to be tested DNA profiling extracts
Extract mutations in leukemia patients by peripheral blood 8-10mL be placed in EDTA or citron receive anti-coagulants heparin tube in, mark really
After guarantor's label information is errorless, 4 DEG C of preservations.Use the nucleic acid extraction or purified reagent of Da'an Gene Company, Zhongshan University
(Guangdong fringe tool is for No. 20170666), are carried out according to kit specification nucleic acid extraction；It is proposed with spectrophotometer (such as
NanoDrop2000 ultramicrospectrophotometer or other spectrophotometer instruments) nucleic acid after extraction is detected, nucleic acid
Purity should meet A260/A280 ratio range between 1.8-2.2, concentration should be not less than 10ng/ μ L, template nucleic acid can be direct
It for subsequent experimental or is placed in -80 DEG C and saves backup, avoid multigelation.
Step 2: the preparation of dPCR system
DPCR system prepare before prepare: take out kit in specific primer and probe, RT-buffer, RNA enzyme,
Fluorescein sodium salt, 7-deaza-dGTP, RNase-free water etc., room temperature melt, be vortexed concussion mix after from
The heart 10 seconds, prepare dPCR system；DPCR system constitutes such as table 5:
5 dPCR system of table
| Specific primer and probe | 1μL |
| RT-buffer | 5μL |
| RNA enzyme | 3μL |
| Fluorescein sodium salt | 2.5μL |
| 7-deaza-dGTP | 1μL |
| RNase-free water | Complement to 20 μ L |
The nucleotide sequence information difference of the primed probe is as follows:
Detect the primed probe nucleotide sequence information of BCR-ABL fusion:
Table 6
Step 3: sample-adding
Take the 5 μ L of each check sample in 5 μ L of sample DNA template and kit prepared by step 1, sample-adding to step 2
In eight connecting legs of the dPCR reaction system of preparation, make the 25 μ L of total volume of every pipe dPCR reaction solution；Eight connecting leg pipe lids are covered tightly, are filled
Divide and mix, high speed centrifugation 10 seconds, is used to prepare micro- reaction；The check sample of the kit such as table 7:
The check sample of 7 kit of table
The gDNA check sample is the wild-type nucleic acid sample containing abl gene, derives from Caco2 cell strain DNA；Sun
Property control be to mix 2 kinds with ABL reference gene template DNA by a certain percentage respectively containing BCR-ABL fusion template DNA
It closes；Wherein, the DNA profiling containing the BCR-ABL P210 fusion positive derives from outer conjunction large fragment 1, contains BCR-ABL
The DNA profiling of the P190 fusion positive derives from outer conjunction large fragment 2, and the DNA profiling containing ABL reference gene derives from outer conjunction
Large fragment 3；Negative control sample is RNase-free water.
Step 4: preparing micro- reaction and PCR amplification
The dPCR reaction solution for taking 25mL to prepare, generating amplification system machine by pulsometer and Naica Geode droplet will
It is automatic, being equably subdivided into the reaction member of 35 000 nanoliter levels, simultaneously Direct PCR reaction expands.The reaction item of PCR amplification
Part: 40 DEG C of Partition at, " Sapphire V1 ", the first stage, 50 DEG C reverse transcription 30 minutes；Second stage, 95 DEG C pre-
Denaturation 10 minutes；Phase III, 95 DEG C are denaturalized 30 seconds, and 56 DEG C extend 30 seconds, 45 circulations；Release P.
Step 5: result is read and analysis
Sapphire chip is scanned amplification with " Crytal Reader " software, experiment is named, is glimmering
The information such as photoinitiator dye and Gene Name, LED exposure time, sample ID and chip ID bar code are configured.And in interface
It opens " Cristal Minder " to analyze scanning result, checks the value in the channel FAM and VIC channel fluorescence signal；Pass through
Negative control sample, the reference of positive control and no template control sample and the distribution of fluorescence signal, are adjusted micro- reaction
Signal threshold value.
Compared with prior art, the beneficial effects of the present invention are:
The present invention provides method, primer, probe and the kits of a kind of BCR-ABL fusion quantitative detection.This examination
Agent box optimizes specific primer probe, only needs two different upstream primers for two kinds of BCR-ABL fusion types, altogether
With downstream primer detected with the probe of fluorescent marker and a set of ABL reference gene detection system.Use the present invention
The specific primer and probe of offer only need 1 pipe to can be detected 2 kinds of fused types of BCR-ABL, can calculate IS%, and then sentence
Disconnected molecules reaction (MMR) out.The present invention can detect that IS% is 0.0032%, that is, reach molecules reaction MR4.5 sensitivity
It is high.Have many advantages, such as that process optimization is simple, required sample is few, performance stability and high efficiency, high accuracy, can tell individually copying
Difference realizes absolute quantitation truly, and data are analysis automated, as a result can observe in real time.Detection process drop formation
Stopped pipe completely integrated with PCR amplification is completed, and the risk of PCR product pollution and operation error is effectively reduced.The present invention is applicable in
It is monitored in BCR-ABL fusion transcriptional level to Leukemia Patients, and assesses tyrosine kinase inhibitor (TKI)
Curative effect.Realize the dynamic tracing of therapeutic effect, thus EARLY RECOGNITION drug resistance or progression of disease, to instruct therapeutic intervention.This is
Leukaemia early diagnosis and the feasible way efficiently treated are explored, is worthy of popularization.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002)
Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment 1: kit
A kind of constituent of the kit of BCR-ABL fusion quantitative detection, packaging and quantity (48 reactions/box),
Such as table 8,
Constituent, packaging and the quantity of 8 kit of table
Embodiment 2: sensitivity technique and the experiment of minimum recall rate
GDNA check sample is the nucleic acid source containing BCR-ABL fusion wild type in Caco2 cell strain DNA；Spirit
Sensitivity reference material is by 2 kinds of BCR-ABL P210 fusions and BCR-ABL P190 fusion template DNA and ABL reference gene
Template DNA (reference gene copy number >=104) be mixed in a certain ratio respectively, the IS% of mixed liquor is respectively 0.1%,
0.01%, 0.0032%；Wherein, BCR-ABL P210 and BCR-ABL P190 template DNA from artificial synthesized large fragment 1,
2；Negative control is RNase-free water；
Negative control, gDNA control, each 5 μ L of sensitivity reference material are taken, the dPCR reaction system prepared to step 2 is loaded
Eight connecting legs in, make every 25 μ L of pipe dPCR reaction solution total volume；Eight connecting leg pipe lids are covered tightly, are mixed well, high speed centrifugation 10 seconds,
It is prepared for micro- reaction；
The dPCR reaction solution for taking 25 μ L to prepare, generating amplification system machine by pulsometer and Naica Geode droplet will
It is automatic, being equably subdivided into the reaction member of 35 000 nanoliter levels, simultaneously Direct PCR reaction expands.The reaction item of PCR amplification
Part: 40 DEG C of Partition at, " Sapphire V1 ", the first stage, 50 DEG C reverse transcription 30 minutes；Second stage, 95 DEG C pre-
Denaturation 10 minutes；Phase III, 95 DEG C are denaturalized 30 seconds, and 56 DEG C extend 30 seconds, 45 circulations；Release P；
Sapphire chip is scanned amplification with " Crytal Reader " software, experiment is named, is glimmering
The information such as photoinitiator dye and Gene Name, LED exposure time, sample ID and chip ID bar code are configured.And in interface
It opens " Cristal Minder " to analyze scanning result, checks the value in the channel FAM and VIC channel fluorescence signal；Pass through
Negative control sample, the reference of positive control and no template control sample and the distribution of fluorescence signal, are adjusted micro- reaction
Signal threshold value；
Sensitivity of the invention and minimum recall rate are detected with dPCR system, when above-mentioned positive control mixes
When the value of liquid theory IS% is respectively 0.1%, 0.01%, 0.0032%, actually measured IS% is as shown in table 9,
9 sensitivity technique result of table
The sensitivity technique result of kit is consistent with theoretical value, shows that primer and probe have preferable specificity, spirit
Sensitivity detection is good；When the positive sample IS% that BCR-ABL copy number is mixed with ABL copy number is 0.0032%, dPCR system
System can stable detection go out corresponding fusion type, actually detected IS% distinguishes average out to 0.0032%, 0.0031%, 0.0031%,
Therefore, the IS% that detects of the invention is 0.0032%.
Embodiment 3: accuracy detection
According to the copy number of measured check sample, accuracy reference material is prepared
By 2 kinds of BCR-ABL P210 fusions and BCR-ABL P190 fusion template DNA and ABL reference gene mould
(reference gene copy number >=10 plate DNA4) be mixed in a certain ratio respectively, the mixed liquor that IS% is 0.01% is made.
Every kind of accuracy reference material is done 2 repetitions and is tested, totally 4 pipe；BCR-ABL P210 fusion accuracy is taken to refer to
5 μ L, BCR-ABL P190 fusion accuracy reference material of product, 5 μ L is loaded to the eight of the prepared dPCR reaction system of step 2
In connecting leg, so that the total volume of dPCR reaction solution is 25 μ L；Eight connecting leg pipe lids are covered tightly, are mixed well, high speed centrifugation 10 seconds, are used for
Micro- reaction preparation；
The dPCR reaction solution for taking 25 μ L to prepare, generating amplification system machine by pulsometer and Naica Geode droplet will
It is automatic, being equably subdivided into the reaction member of 35 000 nanoliter levels, simultaneously Direct PCR reaction expands.The reaction item of PCR amplification
Part: 40 DEG C of Partition at, " Sapphire V1 ", the first stage, 50 DEG C reverse transcription 30 minutes；Second stage, 95 DEG C pre-
Denaturation 10 minutes；Phase III, 95 DEG C are denaturalized 30 seconds, and 56 DEG C extend 30 seconds, 45 circulations；Release P；
Sapphire chip is scanned amplification with " Crytal Reader " software, experiment is named, is glimmering
The information such as photoinitiator dye and Gene Name, LED exposure time, sample ID and chip ID bar code are configured.And in interface
It opens " Cristal Minder " to analyze scanning result, checks the value in the channel FAM and VIC channel fluorescence signal；Pass through
Negative control sample, the reference of positive control and no template control sample and the distribution of fluorescence signal, are adjusted micro- reaction
Signal threshold value；
It is detected with accuracy of the dPCR system to kit of the present invention, obtains result such as table 10,
Table 10: accuracy testing result
As a result, the testing result positive rate of each quality-control product accuracy is 100% according to upper table, meet theoretical qualitative mark
Standard shows that the accuracy detection of kit of the present invention meets the requirements.
Embodiment 4: clinical application experiment
Extract 15 mutations in leukemia patients by peripheral blood 8-10mL be placed in EDTA or citron receive anti-coagulants heparin tube in, blood is provided
15 patients of sample have carried out fluorescence quantitative PCR detection, and are clearly a kind of BCR-ABL Gene Fusion type kind, or be free of
BCR-ABL Gene Fusion；It carries out sample labeling and ensures that label information is errorless, 4 DEG C of preservations.Peace gene stock is reached using Zhongshan University
The nucleic acid extraction or purified reagent (Guangdong fringe tool is for No. 20170666) of part Co., Ltd, are carried out according to kit specification nucleic acid and mention
It takes；Spectrophotometer (such as NanoDrop2000 ultramicrospectrophotometer or other spectrophotometer instruments) is proposed with to mentioning
Nucleic acid after taking is detected, and the purity of nucleic acid should meet A260/A280 ratio range between 1.8-2.2, and concentration should not be low
In 10ng/ μ L, template nucleic acid can be directly used for subsequent experimental or be placed in -80 DEG C saving backup, and avoid multigelation.
Each 5 μ L of check sample in the 5 μ L of DNA profiling and kit of each sample is taken, the dPCR prepared to step 2 is loaded
In eight connecting legs of reaction system, make the 25 μ L of total volume of every pipe dPCR reaction solution；Eight connecting leg pipe lids are covered tightly, are mixed well, it is high
Speed centrifugation 10 seconds, prepares for micro- reaction；
The dPCR reaction solution for taking 25 μ L to prepare, generating amplification system machine by pulsometer and Naica Geode droplet will
It is automatic, being equably subdivided into the reaction member of 35 000 nanoliter levels, simultaneously Direct PCR reaction expands.The reaction item of PCR amplification
Part: 40 DEG C of Partition at, " Sapphire V1 ", the first stage, 50 DEG C reverse transcription 30 minutes；Second stage, 95 DEG C pre-
Denaturation 10 minutes；Phase III, 95 DEG C are denaturalized 30 seconds, and 56 DEG C extend 30 seconds, 45 circulations；Release P；
Sapphire chip is scanned amplification with " Crytal Reader " software, experiment is named, is glimmering
The information such as photoinitiator dye and Gene Name, LED exposure time, sample ID and chip ID bar code are configured.And in interface
It opens " Cristal Minder " to analyze scanning result, checks the value in the channel FAM and VIC channel fluorescence signal；Pass through
Negative control sample, the reference of positive control and no template control sample and the distribution of fluorescence signal, are adjusted micro- reaction
Signal threshold value；
Testing result are as follows: in 15 samples, 5 samples are positive in BCR-ABL fusion, remaining sample is feminine gender, examine knot
The result consistency of fruit and fluorescence quantitative PCR detection is 100%.
Comparative example 1
The present inventor carries out after going deep into comparing analysis, sets for target sequence to the gene order of BCR-ABL fusion
Tens of pairs of primers and tens of kinds of probes are counted, it is expected that obtaining the primer sets and detection probe that can detect 2 kinds of fused types simultaneously.
Primer specific sex differernce, due to annealing temperature is inconsistent and primer dimer etc., it is difficult to obtain effective with multiple number
PCR amplification primer and probe sequence.The present inventor through a large number of experiments, is in optimized selection the primer and probe of design
And verify, primer, the probe sequence that can be used for detecting the multiple digital PCR amplification of 2 kinds of fused types simultaneously has finally been determined
And combinations thereof.
It was found that, even if the case where determining the primer pair and probe sequence that are directed to each target nucleic acid substantially
Under, there is also significant ground differences for effect of the different primers to combination progress multiplex amplification.
For example, being detected using following control primer pair, other detecting steps and the same above-described embodiment of condition:
Compare primer pair 1:
CCGTGGAGCTGCAGATGCT (SEQ ID NO.:8),
CAGTGCCATAAGCGGCACC(SEQ ID NO.:9)；
Compare primer pair 2:
TCTCCCTGACATCCGTGGA (SEQ ID NO.:10),
GCAGATCTGGCCCAACGAT(SEQ ID NO.:11)。
Sensitivity technique is carried out according to the method for embodiment 2, testing result shows that control primer pair 1 can detect IS% and be
0.01%；It is 0.1% that control primer pair 2, which can detect IS%,；The sensitivity for compareing primer pair 1 and control primer pair 2 is poor.It presses
Accuracy detection is carried out according to the method for embodiment 3, the results showed that control primer pair 1 and the accuracy for compareing primer pair 2 are poor, nothing
Method carries out theoretical qualitative.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Da'an Gene Company, Zhongshan University
<120>a kind of method and kit of BCR-ABL fusion quantitative detection
<130> 00020
<160> 11
<170> PatentIn version 3.5
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<213>artificial sequence (Artificial sequence)
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<213>artificial sequence (Artificial sequence)
<400> 2
ttggttgtcg tgtccgagg 19
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<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
gcgagaaggt tttccttgga g 21
<210> 4
<211> 24
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<213>artificial sequence (Artificial sequence)
<400> 4
cggcttcact cagaccctga ggct 24
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
cttggcgcaa aatgttgg 18
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
gagcggcttc actcagacc 19
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 7
cctgaagctg gtgggctgca a 21
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<213>artificial sequence (Artificial sequence)
<400> 8
ccgtggagct gcagatgct 19
<210> 9
<211> 19
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<213>artificial sequence (Artificial sequence)
<400> 9
cagtgccata agcggcacc 19
<210> 10
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<213>artificial sequence (Artificial sequence)
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tctccctgac atccgtgga 19
<210> 11
<211> 19
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gcagatctgg cccaacgat 19
Claims (10)
1. a kind of primer pair group of quantitative detection BCR-ABL fusion, which is characterized in that the primer pair group includes on first
Primer and the first downstream primer are swum, shown in the first upstream primer sequence SEQ ID NO.:1；The first downstream primer sequence
Column are as shown in SEQ ID NO.:3.
2. primer pair group as described in claim 1, which is characterized in that the primer pair group further includes the second upstream primer, institute
It states shown in the second upstream primer sequence SEQ ID NO.:2.
3. primer pair group as described in claim 1, which is characterized in that the primer pair group further includes internal control primer pair, described
Internal control primer pair includes:
Internal control upstream primer shown in SEQ ID NO:5；With internal control downstream primer shown in SEQ ID NO.:6.
4. a kind of probe groups of quantitative detection BCR-ABL fusion, which is characterized in that the probe groups include SEQ ID
Probe sequence shown in NO.:4.
5. probe groups as claimed in claim 4, which is characterized in that the probe groups further include interior shown in SEQ ID NO.:7
Control probe sequence.
6. probe groups as claimed in claim 4, which is characterized in that 5 ' ends of SEQ ID NO:4 nucleotide sequence are marked with
FAM, 3 ' ends are marked with BHQ1；And/or
5 ' ends of SEQ ID NO:7 nucleotide sequence are marked with VIC, 3 ' ends are marked with BHQ1+ZIP.
7. a kind of kit of quantitative detection BCR-ABL fusion, which is characterized in that the kit includes claim 1
The primer pair group.
8. kit as claimed in claim 7, which is characterized in that the kit further includes probe as claimed in claim 4
Group.
9. kit as claimed in claim 7, which is characterized in that the kit further includes RT- buffer, and the RT- is slow
Fliud flushing includes (NH4)2SO4、KCl、Tris-HCl、MgCl2And DTT.
10. a kind of method of quantitative detection BCR-ABL fusion, which is characterized in that the method includes the steps:
(1) sample to be detected is provided, and extracts sample gene to be tested group nucleic acid；
(2) dPCR amplification reaction system is prepared, is carried out amplification reaction:
Wherein, the dPCR amplification reaction system includes the sample to be detected that step (1) provides；Primer described in claim 1
To group and probe groups as claimed in claim 4.
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| CN110358813A (en) * | 2019-07-24 | 2019-10-22 | 艾普拜生物科技(苏州)有限公司 | A kind of detection method of the genome structure variation mediated from control |
| CN110724741A (en) * | 2019-07-24 | 2020-01-24 | 艾普拜生物科技(苏州)有限公司 | Primer, probe and kit for detecting minimal residual leukemia related fusion gene |
| CN110724741B (en) * | 2019-07-24 | 2020-05-19 | 艾普拜生物科技(苏州)有限公司 | Primer, probe and kit for detecting minimal residual leukemia related fusion gene |
| CN112501301A (en) * | 2020-12-08 | 2021-03-16 | 凯杰生物工程（深圳）有限公司 | Primer and probe combination for quantitatively detecting BCR-ABL fusion gene, kit and using method thereof |
| CN112501301B (en) * | 2020-12-08 | 2023-07-18 | 凯杰生物工程（深圳）有限公司 | Primer and probe combination for quantitatively detecting BCR-ABL fusion gene, kit and use method thereof |
| CN113789389A (en) * | 2021-11-18 | 2021-12-14 | 广东永诺医疗科技有限公司 | Human leukemia BCR-ABL fusion mutation one-step detection kit |
| CN116769921A (en) * | 2023-08-15 | 2023-09-19 | 思纳福(苏州)生命科技有限公司 | Primer probe combination for detecting BCR-ABL1 fusion gene and application thereof |
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