CN107058593A - A kind of hypotype Ph likeALL detection method - Google Patents
A kind of hypotype Ph likeALL detection method Download PDFInfo
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- CN107058593A CN107058593A CN201710462205.5A CN201710462205A CN107058593A CN 107058593 A CN107058593 A CN 107058593A CN 201710462205 A CN201710462205 A CN 201710462205A CN 107058593 A CN107058593 A CN 107058593A
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of hypotype Ph likeALL detection method, comprise the following steps：1）Ethanol solution, pretreatment fluid, washing lotion and enzyme buffer liquid are prepared respectively；2）Fluorescence probe dissolves；3）Bone marrow cell is solidified, smear is made；4）By probe solution pre-temperature to room temperature, centrifugation is added drop-wise to hybridising region, sealed；5）Denaturation hybridization；6）Mounting glue is removed, Kao Pulin cylinder wash-ins is placed in and removes cover glass, take out smear, is rinsed with washing lotion, ethanol solution, lucifuge is air-dried；7）Redye；8）Scanning, determines whether sample has heterogeneity.This detection method have the advantages that simply, quick, cost is low, sensitivity is high, high specificity, detects comprehensive, being capable of simple and quick comprehensively detection Ph likeALL.
Description
Technical field
The present invention relates to biology field, and in particular to a kind of hypotype Ph-likeALL detection method.
Background technology
ALL（Abbreviation ALL）It is the most common tumour of children, the incidence of disease drops with the growth at age
Low, current reappearance chromosomal change is ALL important symbol, including Chromosome recombination, structural change etc..For many years, it is clinical
By combining above-mentioned chromosomal variation feature, risk stratification treatment is carried out to ALL so that ALL therapeutic effect has obtained very big
It must improve, wherein the 5 of children ALL year survival rate has brought up to 85%, but still there are some patientss to treat poor prognosis, ALL's answers
Send out intractable and cause the disease to be still one of main fatal disease of children and adults.
Ph-likeALL, is the one group of disease identified according to gene expression profile, and its gene expression profile is positive with BCR-ABL1
ALL is similar, but does not express BCR-ABL1, so referred to as BCR-ABL1-like ALL, also known as Ph-likeALL.Such ALL
It is mainly seen in acute precursor B cells lymphocytic leukemia（BCP- ALL）, typically lack typical gene and dye in BCP-ALL
Colour solid makes a variation（ETV6-RUNX1, E2A-PBX1, ERG and MLL rearrangement, hyperdiploid etc.）And it is often clinical special with excessive risk
Levy.Research finds that Ph-likeALL accounts for SR（Mark danger）And HR（It is high-risk）The 15% of children B-ALL, in young ALL about
27.4%.Similar to BCR-ABL1 positives ALL, Ph-likeALL patient's poor prognosis, conventional chemotherapy effect is not good, easily recurrence.Cell
ABL1, ABL2, CSF1R and the PDGFRB fusion that strain and human leukaemia cell express are in vitro to Dasatinib
（dasatinib）Sensitivity, EPOR and JAK2 replace Buddhist nun to phosphoric acid Luso（ruxolitinib）Sensitivity, ETV6-NTRK3 fusions
Buddhist nun is replaced to gram azoles（crizotinib）It is sensitive.Said medicine plays inhibitory action only for indivedual abnormal fusions, uses
There is certain limitation；In detection or cell processes, it is necessary to which multi-medicament repeated detection, cumbersome.
The content of the invention
The technical problems to be solved by the invention are to overcome the defect of above prior art there is provided a kind of hypotype Ph-
LikeALL detection method, simple and quick can comprehensively detect Ph-likeALL.
The technical scheme is that：A kind of hypotype Ph-likeALL detection method, comprises the following steps：
1）Ethanol solution, pretreatment fluid, washing lotion and enzyme buffer liquid are prepared respectively, put 2 DEG C of -8 DEG C of condition of nitrogen gas storages；
2）Fluorescence probe dissolves：Jia 10 into probe tube ~ 20 μ l water, add 150 ~ 200 μ l hybridization solutions, 30 ~ 60s of ultrasonic dissolution,
Obtain probe solution；
3）Bone marrow cell is solidified, is applied on slide, by pretreatment, smear is made；It is described be cured as test tube of hepari processing or
Handled through EDTA- sodium acetates；
4）By probe solution pre-temperature to room temperature, whirlpool is mixed, and centrifuges 2 ~ 3s, and the probe solution for taking 5 ~ 15 μ l to mix is added drop-wise to hybridization
Region, covered is sealed with mounting glue；
5）Setting program：First 70 ~ 80 DEG C are denatured 5 ~ 10 minutes, and then 35 ~ 40 DEG C hybridize 16 ~ 18h；
6）Mounting glue is removed, 1 ~ 5min washes away cover glass in the room temperature washing lotion being placed in Kao Pulin cylinders, take out smear, with warmed-up
1 ~ 3min is rinsed to 72 ~ 74 DEG C of washing lotions, then 1 ~ 3min, lucifuge natural air drying are rinsed with the ethanol solution of room temperature 65 ~ 75%；
7）Dropwise addition redyes liquid to hybridising region, and covered is put -25 ~ -15 DEG C of dark places and redyed more than 20 minutes；It is described to redye
Liquid is made up of the component of following parts by weight：5 ~ 7 parts of gram staining liquid, 1 ~ 3 part of magnesium chloride, 0.2 ~ 0.6 part of citric acid, fluorescence
0.5 ~ 0.7 part of element, 0.1 ~ 0.3 part of sodium alginate；
8）Sample is scanned using object lens, determines whether sample has heterogeneity.
Specifically, the volume fraction of the ethanol solution is 70 ~ 85%；The pretreatment fluid is 0.9 ~ 1.1mol/L sulphur
Cyanic acid sodium solution；The washing lotion is made up of the component of following percetage by weight：Sodium chloride 1.7 ~ 1.8%, sodium citrate 0.8 ~ 1%,
Nonidet P40 0.2 ~ 0.4%, surplus is water；The enzyme buffer liquid is 0.009 ~ 0.011mol/L watery hydrochloric acid.
Preferably, totally 12 groups of the fluorescence probe, respectively ABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2,
EPOR, IL2RB, NTRK3, PTK2B, TSLP and TYK2.
Most Ph-likeALL patients have chromosomal rearrangement or other hereditary changes, can active cell factor acceptor
Or kinase signal.Rearranged gene includes ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB,
PTK2B, IL2RB, TSLP or TYK2.ABL1, ABL2, CSF1R, JAK2 are expressed and PDGFRB fusions cause cell factor
Independence is expanded and phosphorylation STAT5 activation.Above-mentioned rearranged gene is marked and obtains fluorescence probe, examination is made with redying liquid
Agent box.
Redye liquid to add after denaturation hybridization, its effect embodies during redying and observing.Redye liquid and use gram
Dyeing liquor is main component, and fluorescein increase fluorescent effect, addition magnesium chloride can increase the mark color developing effect of fluorescence probe；Sea
Mosanom contributes to cell to be uniformly dispersed and redye, and citric acid then accelerates the fusion process of abnormal cell, and cell is dispersed
During redying, abnormal cell is rapidly merged each other.Mutually effectively cooperation can be quickly and accurately for each component in a word
Whether observation sample has the opposite sex.Fluorescence probe enumerate be currently known targetedly medication Ph-likeALL types, warp
Probe solution is with after denaturing samples hybridization, being added dropwise and redying liquid, can all-sidedly and accurately detect Ph-likeALL.
Preferably, the step 2）Fluorescence probe dissolving also comprises the following steps：With liquid-transfering gun rinse probe bottom of the tube and
Tube wall, vibration mixes 20 ~ 40s, centrifuges 20 ~ 40s, puts 35 ~ 40 DEG C of water-bath insulations and dissolves 5 ~ 15 minutes；Repeat said process 2 ~ 3
It is secondary, by the probe solution prepared it is standby or packing frost be kept in dark place.
Preferably, the step 6）Kao Pulin cylinders are additionally operable to hold pretreatment fluid, washing lotion and enzyme buffer liquid, by the washing lotion
72 ~ 74 DEG C are preheated to, the pretreatment fluid is preheated to 75 ~ 85 DEG C, the enzyme buffer liquid is preheated to 36 ~ 38 DEG C.
Preferably, the pH value of the washing lotion at room temperature is 7.0 ± 0.2；The enzyme buffer liquid is 0.01mol/L dilute salt
Acid, pH value at room temperature is 2.0 ± 0.2.
Specifically, the step 3）Pretreatment comprises the following steps：a）By 60 ~ 70 DEG C of 5 ~ 10min of preheating of smear, waxdip is used
The transparent liquid that dewaxes is dewaxed 2 10 ~ 20min every time, and 23 ~ 5min every time are dehydrated with absolute ethyl alcohol；b）Successively in 85% ethanol, 70%
Gradient rehydration in ethanol, purified water, each 0.5 ~ 2min sucks residual solution；c）It is placed on and has been preheated with 75 ~ 85 DEG C of pretreatment
20 ~ 40min is incubated in liquid, takes out and 1 ~ 2min is soaked in purified water, suck residual solution；d）40 ~ 60mg pepsins are taken to do
Powder, which is dissolved in, to be pre-heated in 36 ~ 38 DEG C of enzyme buffer liquid, 20 ~ 30min of immersion digestion；e）1 ~ 2min is soaked in purified water,
Serial dehydration, each 0.5 ~ 2min, drying for standby in 85% ethanol, 70% ethanol, purified water successively.
Preferably, the step a）Waxdip dewaxing transparent liquid is isoparaffin type
Preferably, the step c）Described in pretreatment fluid be 0.9 ~ 1.1mol/L sodium thiocyanate solution.
Preferably, the enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, and pH value at room temperature is 2.0 ± 0.2.
Preferably, there is the feature of heterogeneity in the result of the scanning to have fused cell.
The beneficial effects of the invention are as follows：
1) this detection method have the advantages that simply, quick, cost is low, sensitivity is high, high specificity, detects comprehensive, Neng Goujian
It is single quickly comprehensively to detect Ph-likeALL；
2) fluorescence probe enumerate be currently known targetedly medication Ph-likeALL types, comprehensively detection can be
Clinical guidance medication provides favourable foundation；
3) fluorescence probe has very strong specificity, only combines the chromosome of specific site, high specificity；
4) fluorescence in situ hybridization technique is used, fluorescence signal is very clear under the microscope, and sensitivity is very high；
5) smear can be processed into rapidly after obtaining bone marrow cell, and result can be typically gone out in 2-5 days.
Brief description of the drawings
Fig. 1 is FISH normal cell schematic diagrames, 1, orange, 2, green.
Fig. 2 is FISH abnormal cells, 1, orange, 2, green, 3, fusion.
Embodiment
The present invention is described in further details with specific embodiment below, but the present invention is not only limited in detail below in fact
Apply example.
Embodiment provided below is simultaneously not used to the scope that the limitation present invention is covered, described step nor with
To limit its execution sequence.Those skilled in the art do conspicuously improved with reference to existing common knowledge to the present invention, also fall
Enter within the protection domain of application claims.
Embodiment one
A kind of hypotype Ph-likeALL detection method, comprises the following steps：
1）Ethanol solution, pretreatment fluid, washing lotion and enzyme buffer liquid are prepared respectively, put 2 DEG C of -8 DEG C of condition of nitrogen gas storages；The ethanol
The volume fraction of solution is 70 ~ 85%；The pretreatment fluid is 0.9 ~ 1.1mol/L sodium thiocyanate solution；The washing lotion by with
The component composition of lower percetage by weight：Sodium chloride 1.7 ~ 1.8%, sodium citrate 0.8 ~ 1%, Nonidet P40 0.2 ~
0.4%, surplus is water；The enzyme buffer liquid is 0.009 ~ 0.011mol/L watery hydrochloric acid；
2）Fluorescence probe dissolves：Jia 10 into probe tube ~ 20 μ l water, add 150 ~ 200 μ l hybridization solutions, 30 ~ 60s of ultrasonic dissolution,
Obtain probe solution；Totally 12 groups of the fluorescence probe, respectively ABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2,
EPOR, IL2RB, NTRK3, PTK2B, TSLP and TYK2；
3）Bone marrow cell is solidified, is applied on slide, by pretreatment, smear is made；It is described be cured as test tube of hepari processing or
Handled through EDTA- sodium acetates；
4）By probe solution pre-temperature to room temperature, whirlpool is mixed, and centrifuges 2 ~ 3s, and the probe solution for taking 5 ~ 15 μ l to mix is added drop-wise to hybridization
Region, covered is sealed with mounting glue；
5）Setting program：First 70 ~ 80 DEG C are denatured 5 ~ 10 minutes, and then 35 ~ 40 DEG C hybridize 16 ~ 18h；
6）Mounting glue is removed, 1 ~ 5min washes away cover glass in the room temperature washing lotion being placed in Kao Pulin cylinders, take out smear, with warmed-up
1 ~ 3min is rinsed to 72 ~ 74 DEG C of washing lotions, then 1 ~ 3min, lucifuge natural air drying are rinsed with the ethanol solution of room temperature 65 ~ 75%；
7）Dropwise addition redyes liquid to hybridising region, and covered is put -25 ~ -15 DEG C of dark places and redyed more than 20 minutes；It is described to redye
Liquid is made up of the component of following parts by weight：5 ~ 7 parts of gram staining liquid, 1 ~ 3 part of magnesium chloride, 0.2 ~ 0.6 part of citric acid, fluorescence
0.5 ~ 0.7 part of element, 0.1 ~ 0.3 part of sodium alginate；
8）Sample is scanned using object lens, determines whether sample has heterogeneity.
The step 2）Fluorescence probe dissolving also comprises the following steps：Probe bottom of the tube and tube wall are rinsed with liquid-transfering gun, is shaken
20 ~ 40s of mixing is swung, 20 ~ 40s is centrifuged, 35 ~ 40 DEG C of water-bath insulations is put and dissolves 5 ~ 15 minutes；Repeat said process 2 ~ 3 times, will match somebody with somebody
The probe solution made is standby or packing frost is kept in dark place.
The step 6）Kao Pulin cylinders are additionally operable to hold pretreatment fluid, washing lotion and enzyme buffer liquid, and the washing lotion is preheated to
72 ~ 74 DEG C, the pretreatment fluid is preheated to 75 ~ 85 DEG C, the enzyme buffer liquid is preheated to 36 ~ 38 DEG C.
The pH value of the washing lotion at room temperature is 7.0 ± 0.2；The enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, at room temperature
PH value be 2.0 ± 0.2.
The step 3）Pretreatment comprises the following steps：a）By 60 ~ 70 DEG C of 5 ~ 10min of preheating of smear, dewaxed with waxdip saturating
Bright liquid is dewaxed 2 10 ~ 20min every time, and 23 ~ 5min every time are dehydrated with absolute ethyl alcohol；The waxdip dewaxing transparent liquid is isomery
Alkane type；b）Gradient rehydration, each 0.5 ~ 2min in 85% ethanol, 70% ethanol, purified water, suck residual solution successively；c）Put
20 ~ 40min is incubated in 75 ~ 85 DEG C of pretreatment fluid is had been preheated with, takes out and 1 ~ 2min is soaked in purified water, suck remnants
Solution；d）Take 40 ~ 60mg pepsin dry powder to be dissolved in be pre-heated in 36 ~ 38 DEG C of enzyme buffer liquid, immersion digestion 20 ~
30min；e）1 ~ 2min is soaked in purified water, successively the serial dehydration in 85% ethanol, 70% ethanol, purified water, each 0.5 ~
2min, drying for standby.
The step c）Described in pretreatment fluid be 0.9 ~ 1.1mol/L sodium thiocyanate solution.
The enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, and pH value at room temperature is 2.0 ± 0.2.
There is the feature of heterogeneity in the result of the scanning to have fused cell.
Embodiment two
A kind of hypotype Ph-likeALL detection method, comprises the following steps：
1）Ethanol solution, pretreatment fluid, washing lotion and enzyme buffer liquid are prepared respectively, put 2 DEG C of -8 DEG C of condition of nitrogen gas storages；The ethanol
The volume fraction of solution is 70 ~ 85%；The pretreatment fluid is 0.9 ~ 1.1mol/L sodium thiocyanate solution；The washing lotion by with
The component composition of lower percetage by weight：Sodium chloride 1.7 ~ 1.8%, sodium citrate 0.8 ~ 1%, Nonidet P40 0.2 ~
0.4%, surplus is water；The enzyme buffer liquid is 0.009 ~ 0.011mol/L watery hydrochloric acid；
2）Fluorescence probe dissolves：Jia 10 into probe tube ~ 20 μ l water, add 150 ~ 200 μ l hybridization solutions, 30 ~ 60s of ultrasonic dissolution,
Obtain probe solution；Totally 12 groups of the fluorescence probe, respectively ABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2,
EPOR, IL2RB, NTRK3, PTK2B, TSLP and TYK2；
3）Bone marrow cell is solidified, is applied on slide, by pretreatment, smear is made；It is described be cured as test tube of hepari processing or
Handled through EDTA- sodium acetates；
4）By probe solution pre-temperature to room temperature, whirlpool is mixed, and centrifuges 2 ~ 3s, and the probe solution for taking 5 ~ 15 μ l to mix is added drop-wise to hybridization
Region, covered is sealed with mounting glue；
5）Setting program：First 70 ~ 80 DEG C are denatured 5 ~ 10 minutes, and then 35 ~ 40 DEG C hybridize 16 ~ 18h；
6）Mounting glue is removed, 1 ~ 5min washes away cover glass in the room temperature washing lotion being placed in Kao Pulin cylinders, take out smear, with warmed-up
1 ~ 3min is rinsed to 72 ~ 74 DEG C of washing lotions, then 1 ~ 3min, lucifuge natural air drying are rinsed with the ethanol solution of room temperature 65 ~ 75%；
7）Dropwise addition redyes liquid to hybridising region, and covered is put -25 ~ -15 DEG C of dark places and redyed more than 20 minutes；It is described to redye
Liquid is made up of the component of following parts by weight：5 ~ 7 parts of gram staining liquid, 1 ~ 3 part of magnesium chloride, 0.2 ~ 0.6 part of citric acid, fluorescence
0.5 ~ 0.7 part of element, 0.1 ~ 0.3 part of sodium alginate；
8）Sample is scanned using object lens, determines whether sample has heterogeneity.
The step 2）Fluorescence probe dissolving also comprises the following steps：Probe bottom of the tube and tube wall are rinsed with liquid-transfering gun, is shaken
20 ~ 40s of mixing is swung, 20 ~ 40s is centrifuged, 35 ~ 40 DEG C of water-bath insulations is put and dissolves 5 ~ 15 minutes；Repeat said process 2 ~ 3 times, will match somebody with somebody
The probe solution made is standby or packing frost is kept in dark place.
The step 6）Kao Pulin cylinders are additionally operable to hold pretreatment fluid, washing lotion and enzyme buffer liquid, and the washing lotion is preheated to
72 ~ 74 DEG C, the pretreatment fluid is preheated to 75 ~ 85 DEG C, the enzyme buffer liquid is preheated to 36 ~ 38 DEG C.
The pH value of the washing lotion at room temperature is 7.0 ± 0.2；The enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, at room temperature
PH value be 2.0 ± 0.2.
The step 3）Pretreatment comprises the following steps：a）By 60 ~ 70 DEG C of 5 ~ 10min of preheating of smear, dewaxed with waxdip saturating
Bright liquid is dewaxed 2 10 ~ 20min every time, and 23 ~ 5min every time are dehydrated with absolute ethyl alcohol；The waxdip dewaxing transparent liquid is isomery
Alkane type；b）Gradient rehydration, each 0.5 ~ 2min in 85% ethanol, 70% ethanol, purified water, suck residual solution successively；c）Put
20 ~ 40min is incubated in 75 ~ 85 DEG C of pretreatment fluid is had been preheated with, takes out and 1 ~ 2min is soaked in purified water, suck remnants
Solution；d）Take 40 ~ 60mg pepsin dry powder to be dissolved in be pre-heated in 36 ~ 38 DEG C of enzyme buffer liquid, immersion digestion 20 ~
30min；e）1 ~ 2min is soaked in purified water, successively the serial dehydration in 85% ethanol, 70% ethanol, purified water, each 0.5 ~
2min, drying for standby.
The step c）Described in pretreatment fluid be 0.9 ~ 1.1mol/L sodium thiocyanate solution.
The enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, and pH value at room temperature is 2.0 ± 0.2.
There is the feature of heterogeneity in the result of the scanning to have fused cell
Embodiment three
A kind of hypotype Ph-likeALL detection method, comprises the following steps：
1）Ethanol solution, pretreatment fluid, washing lotion and enzyme buffer liquid are prepared respectively, put 2 DEG C of -8 DEG C of condition of nitrogen gas storages；The ethanol
The volume fraction of solution is 70 ~ 85%；The pretreatment fluid is 0.9 ~ 1.1mol/L sodium thiocyanate solution；The washing lotion by with
The component composition of lower percetage by weight：Sodium chloride 1.7 ~ 1.8%, sodium citrate 0.8 ~ 1%, Nonidet P40 0.2 ~
0.4%, surplus is water；The enzyme buffer liquid is 0.009 ~ 0.011mol/L watery hydrochloric acid；
2）Fluorescence probe dissolves：Jia 10 into probe tube ~ 20 μ l water, add 150 ~ 200 μ l hybridization solutions, 30 ~ 60s of ultrasonic dissolution,
Obtain probe solution；Totally 12 groups of the fluorescence probe, respectively ABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2,
EPOR, IL2RB, NTRK3, PTK2B, TSLP and TYK2；
3）Bone marrow cell is solidified, is applied on slide, by pretreatment, smear is made；It is described be cured as test tube of hepari processing or
Handled through EDTA- sodium acetates；
4）By probe solution pre-temperature to room temperature, whirlpool is mixed, and centrifuges 2 ~ 3s, and the probe solution for taking 5 ~ 15 μ l to mix is added drop-wise to hybridization
Region, covered is sealed with mounting glue；
5）Setting program：First 70 ~ 80 DEG C are denatured 5 ~ 10 minutes, and then 35 ~ 40 DEG C hybridize 16 ~ 18h；
6）Mounting glue is removed, 1 ~ 5min washes away cover glass in the room temperature washing lotion being placed in Kao Pulin cylinders, take out smear, with warmed-up
1 ~ 3min is rinsed to 72 ~ 74 DEG C of washing lotions, then 1 ~ 3min, lucifuge natural air drying are rinsed with the ethanol solution of room temperature 65 ~ 75%；
7）Dropwise addition redyes liquid to hybridising region, and covered is put -25 ~ -15 DEG C of dark places and redyed more than 20 minutes；It is described to redye
Liquid is made up of the component of following parts by weight：5 ~ 7 parts of gram staining liquid, 1 ~ 3 part of magnesium chloride, 0.2 ~ 0.6 part of citric acid, fluorescence
0.5 ~ 0.7 part of element, 0.1 ~ 0.3 part of sodium alginate；
8）Sample is scanned using object lens, determines whether sample has heterogeneity.
The step 2）Fluorescence probe dissolving also comprises the following steps：Probe bottom of the tube and tube wall are rinsed with liquid-transfering gun, is shaken
20 ~ 40s of mixing is swung, 20 ~ 40s is centrifuged, 35 ~ 40 DEG C of water-bath insulations is put and dissolves 5 ~ 15 minutes；Repeat said process 2 ~ 3 times, will match somebody with somebody
The probe solution made is standby or packing frost is kept in dark place.
The step 6）Kao Pulin cylinders are additionally operable to hold pretreatment fluid, washing lotion and enzyme buffer liquid, and the washing lotion is preheated to
72 ~ 74 DEG C, the pretreatment fluid is preheated to 75 ~ 85 DEG C, the enzyme buffer liquid is preheated to 36 ~ 38 DEG C.
The pH value of the washing lotion at room temperature is 7.0 ± 0.2；The enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, at room temperature
PH value be 2.0 ± 0.2.
The step 3）Pretreatment comprises the following steps：a）By 60 ~ 70 DEG C of 5 ~ 10min of preheating of smear, dewaxed with waxdip saturating
Bright liquid is dewaxed 2 10 ~ 20min every time, and 23 ~ 5min every time are dehydrated with absolute ethyl alcohol；The waxdip dewaxing transparent liquid is isomery
Alkane type；b）Gradient rehydration, each 0.5 ~ 2min in 85% ethanol, 70% ethanol, purified water, suck residual solution successively；c）Put
20 ~ 40min is incubated in 75 ~ 85 DEG C of pretreatment fluid is had been preheated with, takes out and 1 ~ 2min is soaked in purified water, suck remnants
Solution；d）Take 40 ~ 60mg pepsin dry powder to be dissolved in be pre-heated in 36 ~ 38 DEG C of enzyme buffer liquid, immersion digestion 20 ~
30min；e）1 ~ 2min is soaked in purified water, successively the serial dehydration in 85% ethanol, 70% ethanol, purified water, each 0.5 ~
2min, drying for standby.
The step c）Described in pretreatment fluid be 0.9 ~ 1.1mol/L sodium thiocyanate solution.
The enzyme buffer liquid is 0.01mol/L watery hydrochloric acid, and pH value at room temperature is 2.0 ± 0.2.
There is the feature of heterogeneity in the result of the scanning to have fused cell.
Claims (10)
1. a kind of hypotype Ph-likeALL detection method, it is characterised in that comprise the following steps：
1）Ethanol solution, pretreatment fluid, washing lotion and enzyme buffer liquid are prepared respectively, put 2 DEG C of -8 DEG C of condition of nitrogen gas storages；
2）Fluorescence probe dissolves：Jia 10 into probe tube ~ 20 μ l water, add 150 ~ 200 μ l hybridization solutions, 30 ~ 60s of ultrasonic dissolution,
Obtain probe solution；
3）Bone marrow cell is solidified, is applied on slide, by pretreatment, smear is made；It is described be cured as test tube of hepari processing or
Handled through EDTA- sodium acetates；
4）By probe solution pre-temperature to room temperature, whirlpool is mixed, and centrifuges 2 ~ 3s, and the probe solution for taking 5 ~ 15 μ l to mix is added drop-wise to hybridization
Region, covered is sealed with mounting glue；
5）Setting program：First 70 ~ 80 DEG C are denatured 5 ~ 10 minutes, and then 35 ~ 40 DEG C hybridize 16 ~ 18h；
6）Mounting glue is removed, 1 ~ 5min washes away cover glass in the room temperature washing lotion being placed in Kao Pulin cylinders, take out smear, with warmed-up
1 ~ 3min is rinsed to 72 ~ 74 DEG C of washing lotions, then 1 ~ 3min, lucifuge natural air drying are rinsed with the ethanol solution of room temperature 65 ~ 75%；
7）Dropwise addition redyes liquid to hybridising region, and covered is put -25 ~ -15 DEG C of dark places and redyed more than 20 minutes；It is described to redye
Liquid is made up of the component of following parts by weight：5 ~ 7 parts of gram staining liquid, 1 ~ 3 part of magnesium chloride, 0.2 ~ 0.6 part of citric acid, fluorescence
0.5 ~ 0.7 part of element, 0.1 ~ 0.3 part of sodium alginate；
8）Sample is scanned using object lens, determines whether sample has heterogeneity.
2. hypotype Ph-likeALL according to claim 1 detection method, it is characterised in that the body of the ethanol solution
Fraction is 70 ~ 85%；The pretreatment fluid is 0.9 ~ 1.1mol/L sodium thiocyanate solution；The washing lotion is by following weight hundred
The component composition of fraction：Sodium chloride 1.7 ~ 1.8%, sodium citrate 0.8 ~ 1%, Nonidet P40 0.2 ~ 0.4%, surplus is
Water；The enzyme buffer liquid is 0.009 ~ 0.011mol/L watery hydrochloric acid.
3. hypotype Ph-likeALL according to claim 1 detection method, it is characterised in that the fluorescence probe totally 12
Group, respectively ABL1, ABL2, CSF1R, PDGFRB, CRLF2, JAK2, EPOR, IL2RB, NTRK3, PTK2B, TSLP and
TYK2。
4. hypotype Ph-likeALL according to claim 1 detection method, it is characterised in that the step 2）Fluorescence is visited
Pin dissolving also comprises the following steps：Probe bottom of the tube and tube wall are rinsed with liquid-transfering gun, vibration mixes 20 ~ 40s, centrifuges 20 ~ 40s,
35 ~ 40 DEG C of water-bath insulations are put to dissolve 5 ~ 15 minutes；Said process 2 ~ 3 times is repeated, by the probe solution prepared is standby or packing
Frost is kept in dark place.
5. hypotype Ph-likeALL according to claim 1 detection method, it is characterised in that the step 6）Kao Pulin
Cylinder is additionally operable to hold pretreatment fluid, washing lotion and enzyme buffer liquid, and the washing lotion is preheated into 72 ~ 74 DEG C, and the pretreatment fluid is pre-
The enzyme buffer liquid is preheated to 36 ~ 38 DEG C by heat to 75 ~ 85 DEG C.
6. hypotype Ph-likeALL according to claim 1 detection method, it is characterised in that the step 3）Pretreatment
Comprise the following steps：a）By 60 ~ 70 DEG C of smear, 5 ~ 10min of preheating, dewaxed 2 10 ~ 20min every time with waxdip dewaxing transparent liquid,
23 ~ 5min every time are dehydrated with absolute ethyl alcohol；b）The gradient rehydration in 85% ethanol, 70% ethanol, purified water successively, each 0.5 ~
2min, sucks residual solution；c）20 ~ 40min of insulation in the pretreatment fluid for have been preheated with 75 ~ 85 DEG C is placed on, is taken out in purifying
1 ~ 2min is soaked in water, residual solution is sucked；d）Take 40 ~ 60mg pepsin dry powder to be dissolved in and be pre-heated to 36 ~ 38 DEG C of enzyme
In buffer solution, 20 ~ 30min of immersion digestion；e）1 ~ 2min is soaked in purified water, successively in 85% ethanol, 70% ethanol, purified water
Middle serial dehydration, each 0.5 ~ 2min, drying for standby.
7. hypotype Ph-likeALL according to claim 8 detection method, it is characterised in that the step a）Waxdip takes off
Wax transparent liquid is isoparaffin type.
8. hypotype Ph-likeALL according to claim 8 detection method, it is characterised in that the step c）Described in
Pretreatment fluid is 0.9 ~ 1.1mol/L sodium thiocyanate solution.
9. hypotype Ph-likeALL according to claim 8 detection method, it is characterised in that the enzyme buffer liquid is
0.01mol/L watery hydrochloric acid, pH value at room temperature is 2.0 ± 0.2.
10. hypotype Ph-likeALL according to claim 1 detection method, it is characterised in that the result of the scanning
In have the feature of heterogeneity to there is fused cell.
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| CN107227359A (en) * | 2017-06-19 | 2017-10-03 | 福建万科药业有限公司 | A kind of hypotype Ph likeALL detection kit and its detection method |
| CN110218790A (en) * | 2019-05-10 | 2019-09-10 | 广州安必平医药科技股份有限公司 | For detecting probe chip, kit and its application of the rearrangement of Ph-like ALL related gene |
| CN114181995A (en) * | 2021-12-10 | 2022-03-15 | 山东博思源生物技术有限公司 | Sample pre-hybridization pretreatment reagent and use method thereof |
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