CN107083419A - BCR/ABL Gene Fusions and ASS gene delection detection kits - Google Patents
BCR/ABL Gene Fusions and ASS gene delection detection kits Download PDFInfo
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Abstract
The invention discloses a kind of BCR/ABL Gene Fusions and ASS gene delection detection kits, the kit includes dye marker, for ASS and first group of probe of abl gene sequence, for second group of probe of BCR gene M BCR broken site upstream gene sequences, the color of the dyestuff of two groups of probe marks is all different；Two groups of probes are, respectively using human gene group DNA as template, obtained amplified production to be expanded by primer.The length of FISH probe of the present invention is that inventor passes through many experiments, experimental result is compared and statistical analysis after the optimization length that draws, the optimal balance between detection specificity and hybridization duration can be reached, both result specificity and sensitivity can guarantee that, hybridization time can be shortened again, so that detection can be completed within 78 hours, detection efficiency is improved.
Description
Technical field
The invention belongs to biology field, it is related to medical science and biotechnology, relates particularly to a kind of BCR/ABL bases
Because of fusion and ASS gene delection detection kits.
Background technology
Chronic myelocytic leukemia (Chronic Myelogenous Leukemia, CML) is that one kind betides Hematopoietic Stem
The hematological system malignant clone proliferative disease of cell.Can be found in the cell line of involvement Philadelphia chromosome (Ph) or (and)
BCR/ABL gene rearrangements.Due to t (9；22)(q34；Q11.2) and produce Philadelphia chromosome or (and) BCR/ABL gene rearrangements
There is important diagnosis and prognosis meaning in neoplastic hematologic disorder, CML, the 30% adult acute's lymph for coming across more than 90% are thin
The acute grain of born of the same parents' leukaemia (Acute lymphoblastic leukemia, ALL), 2%-10% children ALL and minority
Chronic myeloid leukemia (Acute Myelogenous Leukemia, AML) and Huppert's disease (multiple myeloma, MM)
Patient.
BCR genes positioned at 22q11 and the mutual transposition of abl gene positioned at 9q34, form BCR/ABL fusions.BCR
Gene totally 23 extrons, mainly there is two kinds of breakage hot spots：Nearly all (exons 1 2-16 is crossed in CML for M-BCR sites
58kb regions), and in ALL about 2/3 be m-BCR sites (the 55kb regions of First Intron).Abl gene has 1b, 1a
With 2~11 totally 12 extrons, upstream is connected with ASS genes, and it, which is broken, is located at the 1st or intron 2.Because breakaway poing differs,
BCR/ABL fusions and its mRNA and protein product are in various, and different fusion products and the type of disease and disease are entered
Exhibition is related, and with different carciongenic potencies.BCR/ABL fusions are a kind of genes of anti-apoptotic, with height junket
Histidine kinase activity, activates multi-signal pathway, suppresses Apoptosis, makes cell hyperproliferation and makes cell regulation and control generation
It is disorderly.Patient's poor prognosis with BCR/ABL fusions, clinically can be according to patient with the presence or absence of BCR/ABL fusion bases
Because optionally to use molecular targeted therapy.About 10-30% patient ABL/BCR on derivative No. 9 chromosome
Partially or completely lacking occurs in fusion, and absent region is located at ABL with place of BCR Gene Fusions, being several MB, typical body
It is now to include the region where ASS genes.The patient that lacking occurs in this region of clinical discovery means shorter DFS phase
And Overall survival, therapeutic effect and prognosis are worse.Therefore, BCR/ABL fusions are detected, can preferably instructs patient's molecule
Targeted therapy and Index for diagnosis.
At present, conventional BCR/ABL fusions detection method mainly includes following 3 kinds：Cytogenetics is detected, is based on
The detection of PCR method and FISH detections.Cytogenetics detection need to cultivate cell, time-consuming longer, can only detect the cell of mid-term,
And success rate and sensitivity are low, it is impossible to which patient that is negative to Ph but there is BCR/ABL fusions makes diagnosis and prognosis；It is based on
PCR detection such as RT-PCR, Q-PCR etc. easily pollute, and false positive rate is high, and can only detect known breakaway poing, to unknown or contain
The BCR/ABL fusions of a small amount of transcript can not be detected.FISH is detected merges base to the BCR/ABL of patient's peripheral blood or marrow
Because having the ability of high sensitivity and detection concealment type transposition, its false positive rate is detected well below PCR, can provide relative
In the more structurally sound molecular genetics evidence of karyotyping, available for Index for diagnosis, medication curative effect, minimal disease residual and transplanting
The monitoring of postoperative effect.Although current FISH detection methods are widely used and improved, but still with following defect：
(1) there is more repetitive sequence in the detection probe prepared using artificial chromosome, influence the specificity of hybridization, cause simultaneously
Higher background fluorescence；(2) detecting step is numerous and diverse, and probe sequence is long, and probe generally requires 12-16h ability with sample hybridization
Ensure fully hybridization, waste time and energy；(3) probe sequence of some optimizations is too short, causes to detect specificity reduction and weak output signal
It is difficult to be detected.Therefore, it is badly in need of a kind of FISH detection methods, its probe is free of repetitive sequence, suitable length, both can guarantee that hybridization
The specific and optimal fluoroscopic examination intensity of reaction, can shorten FISH detection durations to greatest extent, improve detection specificity again
And detection efficiency.
The content of the invention
Base is merged it is an object of the invention to provide a kind of high specificity, the BCR/ABL that sensitivity is high and detection efficiency is high
Because of detection kit.
Realize that the technical scheme of above-mentioned purpose is as follows.
A kind of BCR/ABL Gene Fusions and ASS gene delection detection kits, include dye marker, for ASS and
First group of probe of abl gene sequence, for second group of probe of BCR gene M-BCR broken site upstream gene sequences, two groups
Probe is marked with dyestuff, and the dye colour of same group of probe is identical, and the dye colour of difference group probe is different；Two groups of probes
For respectively using human gene group DNA as template, obtained amplified production is expanded by primer；
It is for the amplimer of first group of probe：Selected from SEQ ID NO.1 and SEQ ID NO.2, SEQ ID
NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID
NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ
ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20,
SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID
At least one pair of primer in NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30；
It is for the amplimer of second group of probe：Selected from SEQ ID NO.31 and SEQ ID NO.32, SEQ ID
NO.33 and SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQ
ID NO.39 and SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44,
SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID
NO.50, SEQ ID NO.51 and SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54, SEQ ID NO.55 and SEQ
At least one pair of in ID NO.56, SEQ ID NO.57 and SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60.
A kind of BCR/ABL gene mutation detection kits, include dye marker, for the first of abl gene sequence
Group probe, for the 3rd group of probe of BCR complete genome sequences；The color of the dyestuff of two groups of probe marks is different；Two groups of spies
Pin is, respectively using human gene group DNA as template, obtained amplified production to be expanded by primer；
It is for the amplimer of first group of probe：Selected from SEQ ID NO.1 and SEQ ID NO.2, SEQ ID
NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID
NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ
ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20,
SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID
At least one pair of primer in NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30；
It is for the amplimer of the 3rd group of probe：Selected from SEQ ID NO.61 and SEQ ID NO.62, SEQ ID
NO.63 and SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, SEQ ID NO.67 and SEQ ID NO.68, SEQ
ID NO.69 and SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72, SEQ ID NO.73 and SEQ ID NO.74,
SEQ ID NO.75 and SEQ ID NO.76, SEQ ID NO.77 and SEQ ID NO.78, SEQ ID NO.79 and SEQ ID
NO.80, SEQ ID NO.81 and SEQ ID NO.82, SEQ ID NO.83 and SEQ ID NO.84, SEQ ID NO.85 and SEQ
At least one pair of in ID NO.86, SEQ ID NO.87 and SEQ ID NO.88, SEQ ID NO.89 and SEQ ID NO.90.
In one of which embodiment, first group of probe is by being expanded selected from least the above corresponding three pairs of primers
The product arrived, the 3rd group of probe is the product by being obtained selected from least the above corresponding three pairs of primers amplification.
In one of which embodiment, first group of probe is by being expanded selected from least the above corresponding three pairs of primers
The product arrived, the 3rd group of probe is the product by being obtained selected from least the above corresponding three pairs of primers amplification.
In one of which embodiment, the size of obtained amplified production is 100-500bp.
In one of the embodiments, the fluorescent dye is selected from：FAM、TET、JOE、HEX、Cy3、TAMRA、ROX、
Texas Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488 and Alexa Fluor 750, and for difference
The fluorescent dye of probe groups is different.
Main advantages of the present invention are：
(1) FISH probe of the present invention is that inventor passes through after sequence alignment, according to non-duplicate and highly conserved sequence
Design primer amplification is prepared into, and with very strong specificity, background noise is lower.
(2) length of FISH probe of the present invention is that inventor passes through many experiments, and experimental result is compared and counted
The optimization length drawn after analysis, can reach the optimal balance between detection specificity and hybridization duration, both can guarantee that result was special
The opposite sex and sensitivity, can shorten hybridization time again so that detection can be completed within 7-8 hour, improve detection efficiency.
(3) conventional FISH probe labeling method can not realize the amplification of fluorescence signal, it is therefore desirable to longer probe
The sensitivity of detection is just can guarantee that, so as to cause hybridization time long.The present invention uses new probes label methods, can be by probe
On fluorescence signal be amplified, greatly improve detection sensitivity.Probes label methods and spy that the present invention is amplified by signal
The optimization of pin length, greatly shortens probe and fully hybridizes required duration with target gene, improve detection sensitivity and detection
Efficiency.
(4) present invention includes three groups of probe types, and the combination of different probe type has different Detection results：First group
Probe and second group of probe are used in combination, and can distinguish BCR gene breaks site, judge fracture mode, while detectable ASS genes
Deletion condition, be that patient's molecular targeted therapy and Index for diagnosis provide more accurately foundation；First group of probe and the 3rd group of spy
Pin is used in combination, and can detect the whole variation type of ABL and BCR genes, is suitable for the less sample of those mutant ratios
This, uses during such as clinical observation residual.
Brief description of the drawings
Fig. 1 is that kit a-signal of the present invention counts guide-typical positive cell signal pattern；
Fig. 2 is kit signal-count guide of the present invention-typical case's negative cells signal mode；
Fig. 3 is that kit B signal of the present invention counts guide-typical positive cell signal pattern.
Embodiment
For the ease of understanding the present invention, the present invention will be described more fully below.The present invention can be with many not
With form realize, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is made to this
The understanding of the disclosure of invention is more thorough comprehensive.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook
People, molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) in institute
The condition stated, or according to the condition proposed by manufacturer.Used various conventional chemical reagent, are commercially available in embodiment
Product.
Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention
The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose of example is applied, the limitation present invention is not used in.Term "and/or" used in the present invention includes one or more related listed
The arbitrary and all combination of project.
The kit forms of embodiment 1
BCR/ABL fusion detection kits described in the present embodiment, are mainly included：
First, the probe of fluorochrome label
The probe includes first group of probe for ASS and abl gene, on BCR gene M-BCR broken sites
Swim the second group of probe and the 3rd group of probe for BCR complete genome sequences of gene order.The probe using human gene group DNA as
Template, is obtained, its preparation method is as follows using 90 pairs of specific primers to carrying out pcr amplification reaction：
1st, the design of amplimer
In order to detect the variation situation of ASS, ABL and BCR gene on No. 9 chromosomes and No. 22 chromosomes, 3 have been separately designed
Group amplimer：First group of amplimer for ASS and abl gene, for BCR gene M-BCR broken site upstream genes
Second group of amplimer of sequence and the 3rd group of amplimer for BCR complete genome sequences.The amplification of the amplimer group
Region is respectively non-duplicate and highly conserved fragment in object detection area, and fragment length is more than 1000bp.Amplimer phase
The amplified production length answered has respectively constituted first group, second group and the 3rd group probe library between 100-500bp.Amplification is drawn
Thing sequence information and its corresponding amplified production are shown in Table 1 (note：1F/1R is pair of primers, and forward primer is represented respectively and is reversely drawn
Thing；Other are by that analogy).
The amplimer of table 1 and amplified production
Primer sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and every sequence after synthesis is used respectively
10mmol/L Tris Buffer are configured to 100pmol/mL storage liquid, and carry out mark.
2nd, probe library is built
Using the primer pair of above-mentioned design, performing PCR amplification, corresponding amplified production are entered by template of human genome DNA
First group, second group and the 3rd group probe library is respectively constituted.
(1) human gene group DNA extracts：According to state of the art, refer to《Molecular Cloning: A Laboratory guidance-the third edition》
(Science Press) or operated according to commercially available peripheral blood cells DNA extraction kit product description.
(2) PCR primer working solution is configured：For first group of probe library, by corresponding amplimer (1F/1R-15F/15R)
It is divided into 3 amplimer groups, 3 non-duplicate and highly conserved region progress to ASS and abl gene are expanded：Take respectively corresponding
Amplimer (1F/1R-5F/5R, 6F/6R-10F/10R and 11F/11R-15F/15R) storing liquid 50ul be placed in 3 1.5ml
In centrifuge tube, it is configured to final concentration using 10mmol/L Tris Buffer and is respectively worked for 10pmol/mL 3 amplimers
Liquid, accordingly carries out mark；For second group of probe library, corresponding amplimer (16F/16R-30F/30R) is divided into 3 amplifications
3 non-duplicate and highly conserved regions of BCR gene M-BCR broken site upstream genes are expanded by primer sets：Take respectively
Corresponding amplimer (16F/16R-20F/20R, 21F/21R-25F/25R and 26F/26R-30F/30R) storing liquid 50ul is put
In 3 1.5ml centrifuge tubes, 3 amplifications for being configured to final concentration respectively for 10pmol/mL using 10mmol/L Tris Buffer
Primer working solution, accordingly carries out mark；For the 3rd group of probe library, by corresponding amplimer (31F/31R-45F/45R) point
For 3 amplimer groups, 3 non-duplicate and highly conserved regions of BCR full genomes are expanded：Corresponding amplification is taken respectively
Primer (31F/31R-35F/35R, 36F/36R-40F/40R, 41F/41R-45F/45R) storing liquid 50ul be placed in 3 1.5ml from
In heart pipe, 3 amplimer working solutions that final concentration is respectively 10pmol/mL are configured to using 10mmol/L Tris Buffer,
Accordingly carry out mark.
(3) PCR amplification system is configured：The amplification of above-mentioned 9 systems is carried out respectively, and amplification system reagent composition is as follows：
(4) PCR is expanded：Configure and be gently mixed after system uniformly, brief centrifugation 5-10s is expanded by following program：
95 DEG C of 5min, 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations since second step, 72 DEG C of 5min, 4 DEG C of holdings.
(5) product identification and purifying：By for ASS and the non-duplicate and highly conserved region of abl gene 3 amplified production
It is well mixed, obtain first group of probe library；And height non-duplicate for BCR gene M-BCR broken sites upstream gene 3 is protected
The amplified production in defending zone domain is well mixed, and obtains second group of probe library；By for the 3 of BCR full genomes non-duplicate and height guarantors
The amplified production in defending zone domain is well mixed, and obtains the 3rd group of probe library；Two groups of probe libraries are entered by 2% agarose gel electrophoresis
Row identification, product of the cutting size between 100-500bp, and product is reclaimed, that is, obtain first group and second group of spy
Pin storehouse, accordingly carries out mark.
3rd, dye marker probe
The present embodiment preferred Cy3 (red fluorescence) first group of probe of mark, preferably Alexa Fluor 488 (green fluorescence)
Second group of probe of mark, preferably the 3rd group of probe of Alexa Fluor 488 (green fluorescence) mark.Artificial synthesized Cy3 mark and
The polyA sequences that Alexa Fluor 488 are marked, the polyA sequences include 1-30 base, preferably comprising 10-20
Individual base, more preferably comprising 2-8 base, polyA sequences are end modified Streptavidin.By Cy3 marks and Alexa
The polyA sequences that Fluor 488 is marked are mixed with being modified with first group, second group and the 3rd group probe library of biotin respectively
(the polyA sequences that 20uL fluorescence labelings are added per 100ug probe libraries), 30min is incubated in 37 DEG C of slow shake, and obtains corresponding glimmering
The probe of signal.Probe is purified and precipitation, is dissolved in TE buffer solutions, that is, obtains first group of probe of red-label and green
Second group of color marker and the 3rd group of probe, probe is kept in dark place in -20 DEG C.
2nd, kit other components
1st, SSC buffer reservoirs liquid (20 × SSC, pH5.3)：Sodium chloride 88g, sodium citrate 44g, ultra-pure water 400mL, fully
Dissolving is mixed, and solution ph is adjusted into 5.3 at room temperature, and solution is settled into 500mL, 0.45 μm of filter filtering with ultra-pure water.
2nd, ethanol solution (70% and 85%)：Absolute ethyl alcohol 700mL/850mL, ultra-pure water 300mL/150mL, it is fully mixed
It is even.
3rd, lavation buffer solution I：20 × SSC (pH5.3) 35mL, Nonidet P40 (NP-40) 3mL, ultra-pure water
912mL, fully mixes, solution ph is adjusted into 7.0 at room temperature, solution is settled into 1000mL, 0.45 μm of filter with ultra-pure water
Filtering.
4th, lavation buffer solution II：20 × SSC (pH5.3) 100mL, Nonidet P40 (NP-40) 1mL, ultra-pure water
849mL, fully mixes, solution ph is adjusted into 7.0 at room temperature, solution is settled into 1000mL, 0.45 μm of filter with ultra-pure water
Filtering.
Preferably first group probe of the present embodiment and second group of probe are used in combination, and kit A are constituted, for detecting BCR/
ABL fusions fracture mode and ASS gene delection situations；
Preferably first group probe of the present embodiment and the 3rd group of probe are used in combination, constitute kit B, for detect ABL and
The whole variation type of BCR genes.
Embodiment 2 is detected using the kit A of embodiment 1 to clinical sample
First, sample preprocessing
1st, peripheral blood in patients 1-1.5ml, 2000rpm centrifugation 5min is gathered with liquaemin anticoagulant tube；
2nd, supernatant is abandoned, 5-10ml 0.075M KCl solution is added, piping and druming is uniform, 37 DEG C of Hypotonic treatment 30min；
3rd, 1ml Fresh fixative (methanol/glacial acetic acid 3 is added:1) uniform, standing 5min, is blown and beaten；
4th, 2000rpm centrifuges 5min, abandons supernatant；
5th, 10ml Fresh fixative (methanol/glacial acetic acid 3 is added:1) uniform, standing 10min, is blown and beaten；
6th, 2000rpm centrifuges 5min, abandons supernatant；
7th, 10ml Fresh fixative (methanol/glacial acetic acid 3 is added:1) uniform, 2000rpm centrifugation 5min, are blown and beaten, supernatant is abandoned；
8th, repeat step 7 twice, finally adds 10ul Fresh fixatives (methanol/glacial acetic acid 3:1) cell is resuspended；
9th, the cell suspension prepared is added drop-wise on slide.
2nd, FISH is detected
FISH detections mainly include pre-degeneration, are denatured, hybridize and redye four steps, and the operating procedure since hybridization is equal
Need to carry out under the conditions of lucifuge：
1st, preparation：Probe is balanced to room temperature, and being vortexed to be placed in microcentrifuge after mixing centrifuges 2-3s；Open and pre-
Hot hybridization instrument, wet bar is immersed in distilled water standby (at least soaking 2h), hybridization instrument is put into during hybridization；Using glass cutter in section
Reverse side irises out sample hybridising region.
2nd, pre-degeneration：Slide is soaked into 2min in room temperature 2 × SSC liquid, slide is then sequentially placed into 70%, 85%,
Each 2min is dehydrated in 100% ethanol.2-5min on roasting piece machine is placed in dry.
3rd, (lucifuge operation) is hybridized：Added in slide sample hybridising region 10ul probe solutions (concentration and probe concentration 15ng/ul,
Each standard person-portion probe amount, the sample area of correspondence detection is about 20mm × 20mm, and the larger sample of sample areas can basis
Actual conditions increase consumption), covered, it is ensured that bubble-free, probe are uniformly distributed under cover glass.Covered and covered using mounting glue
Slide surrounding position, forms the sealing ring of closure.Section is put into the hybridization instrument for having installed wet bar, hybridization reaction journey is set
Sequence：85 DEG C of denaturation 8min, 42 DEG C of hybridization 4h.
4th, post-hybridization washing (lucifuge operation)：Water-bath (76 ± 1 DEG C) is opened, lavation buffer solution II water-bath is placed in pre-
Heat.Section after hybridization is taken out from hybridization instrument, mounting glue is removed, is placed in 5min in the lavation buffer solution I of room temperature, washes lid off
Slide, then 5min in 76 ± 1 DEG C of lavation buffer solution II is placed in, it during which can gently rock section 1-3s；Repeat above-mentioned purge step
Rapid 1 time, finally repeated and washed once with washing fliud flushing I, time during every kind of lavation buffer solution repeated washing with using for the first time
The time is identical when the buffer solution is washed.Section is uprightly air-dried after washing.
5th, DAPI is redyed (lucifuge operation)：10 μ L, which are added, in sample hybridising region redyes liquid, covered, room temperature lucifuge
Can micro- Microscopic observation fluorescence signal after 5-10min.Section after DAPI is redyed can also be placed in -25 DEG C to -18 DEG C lucifuge guarantors
(holding time is no more than 72h) is deposited to be observed to room temperature, it is necessary to put slide when detecting.
3rd, result judgement standard
Kit A result judgement standards of the present invention are as follows：
1st, 100 cells of every sample counting, if positive cell number is less than 3 (3/100 or ＜ 3%), the sample judges
For feminine gender.
2nd, 100 cells of every sample counting, if positive cell number is more than 5 (5/100 or ＞ 5%), the sample judges
For the positive.
3rd, 100 cells of every sample counting, if positive cell number is between 3-5 (3-5%), need another diagosis people in addition
Count 100 cells.Collect two diagosis people and count cell number and positive cell number, that is, amount in 200 cells, if positive thin
Born of the same parents' sum is less than 8 (＜ 4%), and the sample is determined as feminine gender, if positive cell sum is more than or equal to 8 (>=4%), the sample
It is determined as the positive.
The positive of cell and negative judgement：
Field of microscope is moved up and down, lookup is all to be present in endonuclear signal.
ABL be danger signal (Red, O), BCR be green (Green, G), danger signal and green it is overlapping or
Person's contact is fusion signal (F), represents BCR-ABL or ABL-BCR fusions.
1st, signals below pattern can determine that as BCR/ABL fusions positive cell (referring to Fig. 1)：
(1) M types fracture (M-BCR)：Occurred that in the interphase nucleus of typical Ph chromosome translocations 2 red, 1 green and
1 red green fusion hybridization signal (2R1G1F)；
(2) m types fracture (m-BCR)：Occurred that in the interphase nucleus of typical Ph chromosome translocations 1 red, 1 green and
2 red green fusion hybridization signals (1R1G2F)；
(3) fracture of M types occurs with the Ph chromosomes of variation：2 red, 1 green and 2 are occurred that in interphase nucleus
Red green fusion hybridization signal (2R1G2F)；
(4) fracture of m types occurs with the Ph chromosomes of variation：1 red, 1 green and 3 are occurred that in interphase nucleus
Red green fusion hybridization signal (1R1G3F)；
(5) fracture of M types is with ASS gene delections：Occur that 1 red, 1 green and 1 red green are melted in interphase nucleus
Close hybridization signal (1R1G1F)；
(6) fracture of m types is with ASS gene delections：Occur that 1 red, 2 greens and 1 red green are melted in interphase nucleus
Close hybridization signal (1R2G1F).
2nd, signals below pattern can determine that as BCR/ABL fusions negative cells (referring to Fig. 2)：Occur in interphase nucleus
It is 4 hybridization signals (2R2G) of 2 red and 2 green random dispersions.
Note：Black represents red point in accompanying drawing, and white represents green point.
4th, Analysis of test results
15 mutations in leukemia patients by peripheral blood samples (numbering 1-15) are detected using the kit A of embodiment 1, selected simultaneously
With K562 cell lines as positive control, NB4 cell lines as negative control, those skilled in the art are only it is to be understood that cell line
Title can be by being commercially available.About 2000 K562 and NB4 cells (being determined by cell counter) are respectively taken respectively, and mixing is equal
Sample is respectively divided into 5 parts, numbering 16-20 and 21-25 after even.Specific testing result such as table 3：
The pattern detection result of table 3
From above-mentioned testing result, detection of the kit of the present invention to BCR-ABL fusions has sensitive well
Degree and specificity, can realize detection and the broken site parting of mutations in leukemia patients by peripheral blood sample, while ASS genes can also be detected
Deletion condition.All positive control K562 cells of the present embodiment are that BCR-ABL fusions are positive, and all feminine genders
Control NB4 cells are that BCR-ABL fusions are negative.Illustrate this kit design probe can specifically with target gene
Hybridization reaction occurs for sequence, and the fluorescence signal of generation is by force and stably, it is ensured that the specificity and accuracy of testing result.
Embodiment 3 is detected using the kit B of embodiment 1 to clinical sample
First, sample preprocessing
It is consistent with embodiment 2
2nd, FISH is detected
It is consistent with embodiment 2.
3rd, result judgement standard
Kit B result judgement standards of the present invention are as follows：
1st, 100 cells of every sample counting, if positive cell number is less than 3 (3/100 or ＜ 3%), the sample judges
For feminine gender.
2nd, 100 cells of every sample counting, if positive cell number is more than 5 (5/100 or ＞ 5%), the sample judges
For the positive.
3rd, 100 cells of every sample counting, if positive cell number is between 3-5 (3-5%), need another diagosis people in addition
Count 100 cells.Collect two diagosis people and count cell number and positive cell number, that is, amount in 200 cells, if positive thin
Born of the same parents' sum is less than 8 (＜ 4%), and the sample is determined as feminine gender, if positive cell sum is more than or equal to 8 (>=4%), the sample
It is determined as the positive.
The positive of cell and negative judgement：
Field of microscope is moved up and down, lookup is all to be present in endonuclear signal.
ABL be danger signal (Red, O), BCR be green (Green, G), danger signal and green it is overlapping or
Person's contact is fusion signal (F), represents BCR-ABL or ABL-BCR fusions.
1st, signals below pattern can determine that as BCR/ABL fusions positive cell (referring to Fig. 3)：
(1) typical case's Ph chromosome translocations：1 red, 1 green and 2 red green fusion hybridization are occurred that in interphase nucleus
Signal (1R1G2F)；
(2) make a variation Ph chromosome translocations：2 red, 2 greens and 1 red green fusion hybridization are occurred that in interphase nucleus
Signal (2R2G1F)；
ABL and BCR partial sequences near (3) No. 9 chromosome breakpoints are lacked simultaneously：1 is occurred that in interphase nucleus
Red, 1 green and 1 red green fusion hybridization signal (1R1G1F)；
ABL partial sequences missing near (4) No. 9 chromosome breakpoints：Occurred that in interphase nucleus 1 red, 2 it is green
Color and 1 red green fusion hybridization signal (1R2G1F)；
BCR partial sequences missing near (5) No. 9 chromosome breakpoints：Occurred that in interphase nucleus 2 red, 1 it is green
Color and 1 red green fusion hybridization signal (2R1G1F)；
2nd, signals below pattern can determine that as BCR/ABL gene mutations negative cells (referring to Fig. 2)：Occur in interphase nucleus
It is 4 hybridization signals (2R2G) of 2 red and 2 green random dispersions.
Note：Black represents red point in accompanying drawing, and white represents green point.
4th, Analysis of test results
15 mutations in leukemia patients by peripheral blood samples (numbering 26-40) are detected using the kit B of embodiment 1, simultaneously
From K562 cell lines as positive control, NB4 cell lines as negative control, those skilled in the art are only it is to be understood that cell line
Title can be by being commercially available.About 2000 K562 and NB4 cells (being determined by cell counter) are respectively taken respectively, are mixed
Sample is respectively divided into 5 parts, numbering 41-45 and 46-50 after uniform.Specific testing result such as table 3：
The pattern detection result of table 4
From above-mentioned testing result, detection of the kit of the present invention to BCR-ABL gene mutations has sensitive well
Degree and specificity, can realize the detection of No. 9 chromosomes of mutations in leukemia patients by peripheral blood sample and No. 21 various variation types of chromosome.
All positive control K562 cells of the present embodiment are BCR-ABL positive gene mutations, and all negative control NB4 cells
It is that BCR-ABL gene mutations are negative.Illustrate that the probe of this kit design can be specifically miscellaneous with target gene sequence generation
Reaction is handed over, the fluorescence signal of generation is by force and stably, it is ensured that the specificity and accuracy of testing result.
Embodiment 4 hybridizes influence of the duration to kit Detection results
First, hybridization time
The design of kit probe length of the present invention and dye labeling methodologies is that inventor passes through many experiments, and experiment is tied
Fruit be compared with the optimal case drawn after statistical analysis, can reach optimal flat between detection specificity and hybridization time
Weighing apparatus, both can guarantee that result specificity and sensitivity, and can shorten hybridization time again, and improved detection efficiency.
Influence for the check cross time to testing result of the present invention, the present embodiment is by taking kit A as an example, there is provided different
4 experimental groups of hybridization time, are specifically shown in Table 5, detection probe used and reagent are consistent with the kit A of embodiment 1.
Table 5 hybridizes duration
| Packet | Experimental group 1 | Experimental group 2 | Experimental group 3 | Experimental group 4 |
| Hybridization time | 2h | 4h | 8h | 16h |
2nd, pattern detection
The kit prepared using above-mentioned design, detection process and method to Leukemia Patients periphery as described in embodiment 2
Blood sample 51-65 is detected that, wherein each experimental group hybridization time is consistent with the hybridization time of table 5, specific experiment result is as follows：
Influence of the hybridization time of table 6 to testing result
The testing result for contrasting 4 experimental groups understands that experimental group 6, can be with sample in 4h using 100-500bp probe
This target gene fully hybridizes, and realizes the detection of sample and ensures the specificity and accuracy of result.2h hybridization time due to
Probe can not fully hybridize with sample, and cause some positive cell missing inspections, cause false negative result, and testing result is unstable
It is fixed；And 8h and 16h hybridization duration testing result and 4h are completely the same, and fluorescence signal intensity is basically identical.Illustrate the present invention
Probe length and dye marker mode can significantly shorten the hybridization time of probe and target gene, improve Detection results, protect simultaneously
Demonstrate,prove the specificity and accuracy of detection.Influence of the hybridization time to kit B testing results is consistent with kit A, specific data
Omit.
Influence of the probe length of embodiment 5 to kit Detection results
First, probe length
The design of kit probe length of the present invention and dye labeling methodologies is that inventor passes through many experiments, and experiment is tied
Fruit be compared with the optimal case drawn after statistical analysis, can reach optimal flat between detection specificity and hybridization duration
Weighing apparatus, both can guarantee that result specificity and sensitivity, and can shorten hybridization time again, and improved detection efficiency.
Influence for checking probe length to testing result of the present invention, the present embodiment is by taking kit A as an example, there is provided different
4 experimental groups of probe length, are specifically shown in Table 7,4 experimental group amplification region positions identical.Detection probe preparation method used
It is consistent with the kit A of embodiment 1 with reagent.
The probe length of table 7
| Packet | Experimental group 5 | Experimental group 6 | Experimental group 7 | Experimental group 8 |
| Probe length | 20-100bp | 100-500bp | 500-1000bp | ＞ 1000bp |
2nd, pattern detection
The kit prepared using above-mentioned design, detection process and method to Leukemia Patients periphery as described in embodiment 2
Blood sample 61-75 is detected that specific experiment result is as follows：
Influence of the probe length of table 8 to testing result
The testing result for contrasting 4 experimental groups understands that experimental group 6, can be with sample in 4h using 100-500bp probe
This target gene fully hybridizes, and realizes the detection of sample and ensures the specificity and accuracy of result.Too short (the experiment of probe length
5) group, can reduce detection specificity, cause higher background noise, while the fluorescence signal produced is very weak, cause the mistake of result
Sentence；Probe length long (experimental group 7 and 8) can reduce intake ability of the cell to probe, at the same extend hybridization required for when
Between, cause with target gene can not fully to hybridize in 4h probes, influence the Stability and veracity of testing result.Probe length pair
The influence of kit B testing results is consistent with kit A, and specific data are omitted.
Embodiment 6 builds influence of the probe library primer pair type and quantity to kit Detection results
First, the selection of primer pair
Kit of the present invention includes three groups of probe libraries, and every group of probe library is obtained by 15 pairs of primer amplifications respectively, amplification region
Respectively fragment non-duplicate and highly conserved in object detection area, the present invention is preferred for the structure difference of every group of probe library
3 non-duplicate and highly conserved amplification regions in object detection area, 5 pairs of specific bases are devised for each region
Cause.
Influence of the primer pair type and quantity to testing result of the present invention, this implementation used in probe library are built for detection
Example is by taking the structure of first group of probe library as an example, and the present embodiment is by taking kit A as an example, there is provided 3 experimental groups, are specifically shown in Table 9, institute
It is consistent with embodiment 1 with detection probe preparation method and reagent.
The selection of the primer pair of table 9
2nd, pattern detection
The kit prepared using above-mentioned design, detection process and method to Leukemia Patients periphery as described in embodiment 2
Blood sample 76-90 is detected that specific experiment result is as follows：
Table 10 builds influence of the probe library primer pair type and quantity to testing result
The testing result for comparing above-mentioned 3 experimental groups is understood, when each amplification region of target gene selects 1 pair, 2 pairs and 5
When carrying out probe library structure using 3 pairs, 6 pairs and 15 pairs primers to amplimer, i.e. probe library, detection can be completed.When using 6
To or more primer pair carry out build storehouse when, its specificity and stability it is all fine.Wherein, when building storehouse primer using all 15
Pair when, signal is stronger more stable, and Detection results are optimal.Other set up primer for first group, second group and the 3rd group probe library
Choice experiment result to type and quantity is consistent with the above results, and specific data are omitted.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of BCR/ABL Gene Fusions and ASS gene delection detection kits, it is characterised in that include for ASS and
First group of probe of abl gene sequence, for second group of probe of BCR gene M-BCR broken site upstream gene sequences, two groups
Probe is marked with dyestuff, and the dye colour of same group of probe is identical, and the dye colour of difference group probe is different；Two groups of probes
For respectively using human gene group DNA as template, obtained amplified production is expanded by primer；
It is for the amplimer of first group of probe：Selected from SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and
SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and
SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID
NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ
ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26,
At least one pair of primer in SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30；
It is for the amplimer of second group of probe：Selected from SEQ ID NO.31 and SEQ ID NO.32, SEQ ID
NO.33 and SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38, SEQ
ID NO.39 and SEQ ID NO.40, SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44,
SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID
NO.50, SEQ ID NO.51 and SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54, SEQ ID NO.55 and SEQ
At least one pair of in ID NO.56, SEQ ID NO.57 and SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60.
2. BCR/ABL Gene Fusions according to claim 1 and ASS gene delection detection kits, it is characterised in that expand
The size for increasing production thing is 100-500bp.
3. BCR/ABL Gene Fusions according to claim 1 and ASS gene delection detection kits, it is characterised in that pin
To at least three pairs of the amplimer of first group of probe in claim 1 in the corresponding amplimer, for institute
The amplimer of second group of probe is stated at least three pairs of the corresponding amplimer centering in claim 1.
4. BCR/ABL Gene Fusions according to claim 1 and ASS gene delection detection kits, it is characterised in that pin
To at least six pairs of the amplimer of first group of probe in claim 1 in the corresponding amplimer, for institute
The amplimer of second group of probe is stated at least six pairs of the corresponding amplimer centering in claim 1.
5. BCR/ABL Gene Fusions and ASS gene delection detection kits according to claim any one of 1-4, it is special
Levy and be, the fluorescent dye is selected from：FAM、TET、JOE、HEX、Cy3、TAMRA、ROX、Texas Red、LC RED640、
Cy5, LC RED705, Alexa Fluor 488 and Alexa Fluor 750, and it is mutual not for the fluorescent dye of different probe group
It is identical.
6. a kind of BCR/ABL gene mutation detection kits, it is characterised in that include dye marker, for abl gene sequence
First group of probe of row, for the 3rd group of probe of BCR complete genome sequences；Two groups of probes are marked with dyestuff, same group of probe
Dye colour is identical, and the dye colour of difference group probe is different；Two groups of probes be respectively using human gene group DNA as template,
Obtained amplified production is expanded by primer；
It is for the amplimer of first group of probe：Selected from SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and
SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and
SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID
NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ
ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26,
At least one pair of primer in SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30；
It is for the amplimer of the 3rd group of probe：Selected from SEQ ID NO.61 and SEQ ID NO.62, SEQ ID
NO.63 and SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, SEQ ID NO.67 and SEQ ID NO.68, SEQ
ID NO.69 and SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72, SEQ ID NO.73 and SEQ ID NO.74,
SEQ ID NO.75 and SEQ ID NO.76, SEQ ID NO.77 and SEQ ID NO.78, SEQ ID NO.79 and SEQ ID
NO.80, SEQ ID NO.81 and SEQ ID NO.82, SEQ ID NO.83 and SEQ ID NO.84, SEQ ID NO.85 and SEQ
At least one pair of in ID NO.86, SEQ ID NO.87 and SEQ ID NO.88, SEQ ID NO.89 and SEQ ID NO.90.
7. BCR/ABL gene mutation detection kits according to claim 6, it is characterised in that the size of amplified production
For 100-500bp.
8. BCR/ABL gene mutation detection kits according to claim 6, it is characterised in that for described first group
At least three pairs in claim 6 in the corresponding amplimer of the amplimer of probe, for the 3rd group of probe
Amplimer at least three pairs of the corresponding amplimer centering in claim 6.
9. BCR/ABL gene mutation detection kits according to claim 6, it is characterised in that for described first group
At least six pairs in claim 6 in the corresponding amplimer of the amplimer of probe, for the 3rd group of probe
Amplimer at least six pairs of the corresponding amplimer centering in claim 6.
10. the BCR/ABL gene mutation detection kits according to claim any one of 6-9, it is characterised in that described glimmering
Photoinitiator dye is selected from：FAM、TET、JOE、HEX、Cy3、TAMRA、ROX、Texas Red、LC RED640、Cy5、LC RED705、
Alexa Fluor 488 and Alexa Fluor 750, and it is different for the fluorescent dye of different probe group.
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