CN105603109A - Probe labeling method for detecting BCR/ABL gene fusion under single channel - Google Patents

Abstract

The invention discloses an application of a quantum dot fluorescent dye in a probe for detecting the BCR/ABL gene fusion. The invention also discloses a labeling method of the probe for detecting the BCR/ABL gene fusion and a probe and a kit for detecting the BCR/ABL gene fusion. The quantum dot fluorescent dye is used for performing the fluorescent labeling on two genes, namely, BCR and ABL, the two genes emit different fluorescence under the exciting light of the same wavelength, and when FISH detection is used, observation can be performed in one fluorescent channel, so that the detection efficiency is improved.

Description

Under a kind of single channel, detect the probe mark method of BCR/ABL gene fusion

Technical field

The invention belongs to biology field, particularly a kind of labeling method of fluorescence in situ hybridization probeAnd detection kit, by using the means of FISH, detect in the sample genome in sampleBCR/ABL gene fusion.

Background technology

Chronic myelocytic leukemia (CML) is the malignant hematologic disease that originates from pluripotential hemopoietic stem cell, 9 and 22The BCR/ABL fusion that number chromosome translocation forms is to make a definite diagnosis and the important evidence of curative effect judgement.BCR/ABL is positioned respectively No. 22 chromosomes and No. 9 chromosomes, and BCR/ABL fusion is a kind of anti-Apoptotic gene, its expression product gets muddled cell regulate and control by anti-apoptotic effect. 95%Patient CML have this fusion, and the expressed albumen of this fusion is targeted therapy medicineThe effect target of thing imatinib mesylate. So, the BCR/ABL of detection leukemia patient peripheral blood or marrowIt is particularly important that fusion seems.

Compared with other detection method, the distinctive accuracy of FISH (FISH) and intuitive make itBecome the goldstandard that detects BCR/ABL fusion. But traditional FISH kit uses the organic of routineFluorescent dye carries out probe mark, is difficult to overcome two weakness:

1) organic fluorescent dye (as Cy3, FAM etc.) fluorescence intensity a little less than, must be with larger genomeDNA fragmentation is template label probe, stronger fluorescence signal just can be detected. Examine with BCR/ABL geneSurvey as example, traditional FISH probe must be respectively with the gene of 600kB BCR, two genes of ABL nearGroup DNA fragmentation is that template is carried out mark, just can obtain comparatively desirable fluorescence signal. But due to 600kB'sDNA fragmentation is too large, and the subclone of having to split into about 100kB is preserved. Thereby each probe markNote all must be distinguished mark to more than 10 subclones, has greatly increased BCR/ABL fusion and has detectedThe production difficulty of kit, thus production cost improved.

2) organic fluorescent dye of different colours, has different excitation wavelengths, if Cy3 is at 548nm wavelengthUnder exciting light, send red fluorescence, FAM sends green fluorescence under 492nm wavelength exciting light, and transfect cellThe DAPI of core sends blue-fluorescence under 340nm wavelength exciting light. So in FISH testing result, representativeThe red fluorescence of BCR gene and represent that the green fluorescence of abl gene cannot occur under same fluorescence channel,Can not directly judge by visually observing whether red green fluorescence signal has coincidence, and whether BCR/ABL gene hasMerge, can only under red/green fluorescence passage, take pictures respectively, after processing by image analysis software, judged again,Detection efficiency is lower.

Summary of the invention

On the one hand, the object of the invention is to overcome the weak point of prior art existence and quantum dot is providedThe application of fluorescent dye in the probe that detects BCR/ABL gene fusion.

Preferably, described quantum dot fluorescence dyestuff is CdSe/ZnS and CdTe/ZnS; Described detection BCR/ABLThe probe of gene fusion comprises the probe that detects BCR gene and the probe that detects abl gene, more preferably,Described CdSe/ZnS and CdTe/ZnS are marked at respectively and detect the probe of BCR gene and detect abl geneProbe on.

In technique scheme, use amount in the labeling process of probe that detects BCR/ABL gene fusionSon point fluorescent dye CdSe/ZnS and CdTe/ZnS substitute conventional fluorescent dyestuff, the BCR after mark is completedProbe sends red fluorescence under exciting light, fluorophor excitation wavelength 340nm, and emission wavelength is 570nm;And ABL probe sends green fluorescence under Same Wavelength monochromatic excitation light, fluorophor excitation wavelength is340nm, emission wavelength is 510nm.

On the other hand, the present invention also provides a kind of labeling method of probe of the BCR of detection gene, uses nucleic acidProbe mark method is carried out probe mark to BCR gene, and the template sequence using when mark is human chromosome22q11.21～22q11.26 district, the fluorescent dye of use is quantum dot fluorescence dyestuff.

Preferably, described quantum dot fluorescence dyestuff is CdSe/ZnS.

Described labeled nucleic acid probe method can be labeled nucleic acid probe method conventional in prior art, preferably,Described labeled nucleic acid probe method is nick-translation, random priming or PCR method.

On the one hand, the present invention also provides a kind of labeling method of the probe that detects abl gene, uses nucleic acid againProbe mark method is carried out probe mark to abl gene, and the template sequence using when mark is human chromosome9q34.62～9q34.78 district, the fluorescent dye of use is quantum dot fluorescence dyestuff.

Preferably, described quantum dot fluorescence dyestuff CdTe/ZnS.

Described labeled nucleic acid probe method can be labeled nucleic acid probe method conventional in prior art, preferably,Described labeled nucleic acid probe method is nick-translation, random priming or PCR method.

Another aspect, the present invention also provides a kind of mark side of probe of the BCR/ABL of detection gene fusionMethod, the probe of described detection BCR/ABL gene fusion comprises the probe and the detection ABL that detect BCR geneThe probe of gene, by labeled nucleic acid probe method to respectively BCR gene and abl gene being carried out to probe markNote, the template behaviour Chromosome 22q11 .21～22q11.26 district using while detecting the probe mark of BCR gene,The template using while detecting the probe mark of abl gene is human chromosome 9q34.62～9q34.78 district, detectsThe fluorescent dye that the probe of BCR gene uses from the probe that detects abl gene is that two kinds of different quantum dots are glimmeringPhotoinitiator dye, two kinds of quantum dot fluorescence dyestuffs send the fluorescence of different colours under the monochromatic excitation light of Same Wavelength.

Preferably, the quantum dot fluorescence dyestuff that the probe of described detection BCR gene uses is CdSe/ZnS, instituteThe quantum dot fluorescence dyestuff of stating the probe use that detects abl gene is CdTe/ZnS.

Described labeled nucleic acid probe method can be labeled nucleic acid probe method conventional in prior art, preferably,Described labeled nucleic acid probe method is nick-translation, random priming or PCR method.

The present invention also provides according to the labeling method system of the probe of described detection BCR/ABL gene fusionThe probe of the detection BCR/ABL gene fusion obtaining.

When fluorescence in situ hybridization technique is carried out probe mark, in order to obtain comparatively desirable fluorescence signal intensity,The template that conventionally need to use very large genomic DNA fragment to serve as a mark, as traditional FISH probeConventionally selecting the genomic DNA fragment of BCR, near the 600kB of two genes of ABL is template. SoLarge DNA fragmentation cannot be saved in a clone, can only split into multiple clones and preserve with follow-upProbe mark, has increased production difficulty and the production cost of BCR/ABL fusion detection kit greatly.Because the fluorescence intensity of quantum dot fluorescence dyestuff (CdSe/ZnS, CdTe/ZnS) is dyed higher than organic fluorescence far awayMaterial (Cy3, FAM etc.), so the genomic DNA fragment of selection of small carries out probe mark as templateAlso can obtain comparatively desirable effect. The present invention, by great many of experiments, is BCR mould by the suitableeest zone locationPlate: human chromosome 22q11.21～22q11.23 district; ABL template: human chromosome 9q34.62～9q34.68 district.Two template regions are all no more than 60kB size, are only conventional BCR/ABLFISH detection kit template region1/10 of territory, the production difficulty greatly reducing, and improved the stability of product.

Further, the present invention also provides a kind of kit of the BCR/ABL of detection gene fusion, comprises instituteThe probe of the detection BCR/ABL gene fusion of stating.

Preferably, described kit also comprises hybridization solution and cleaning solution;

Wherein, described hybridization solution comprises 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 5-20%Dextran sulfate, 20-50% deionized formamide, 0.01-0.1% mercaptopropionic acid, 0.1-1% bovine serum albumin(BSA)And 0.1-1% salmon sperm dna, the pH value of hybridization solution is 6-10;

Described cleaning solution comprises 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 0.01-0.2% tenSodium dialkyl sulfate, cleaning solution pH value is 6-10.

The concentration of described probe is 1-10nmol/L.

Further, the present invention also provides the described kit detection BCR/ABL gene fusion of utilizationMethod, is characterized in that: comprise the following steps:

1) in sample to be tested, add described hybridization solution and probe;

2) by the sample to be tested that is added with hybridization solution and probe 90-95 DEG C of sex change after 5～10 minutes, in 40～45 DEG CConstant temperature hybridization 10-16 hour;

3), after washing, mounting, under single channel, observe colour developing result by fluorescence microscope.

The quantum dot fluorescence dyestuff using when the probe of described detection BCR gene is CdSe/ZnS, described detectionThe quantum dot fluorescence dyestuff that the probe of abl gene uses is during for CdTe/ZnS, nucleus under fluorescence microscopeBe blue-fluorescence, the BCR gene fluorescence that takes on a red color, abl gene is green fluorescence, as BCR/ABL geneMerge, be yellow fluorescence.

Preferably, utilize described kit to detect the method for BCR/ABL gene fusion, specifically comprise followingStep:

(1) sample to be checked is fixed on slide;

(2) if sample to be checked is cell, directly enter step (3), if sample to be checked is paraffin section,After dewaxing by standard dewaxing step, enter step (3);

(3) on sample, add 10 μ L hybridization solutions, and probe described in 2 μ L, concentration is 1-10nmol/L,Covered, and by glue sealing slide surrounding, to prevent hybridization solution evaporation;

(4) slide is placed in hybridization instrument, 90-95 DEG C of sex change is after 5 minutes, and 42 DEG C of constant temperature are hybridized 10-16Hour;

(5) remove cover glass, slide is put into the cleaning solution that is preheating to 60-68 DEG C, constant temp 2Inferior, each 15 minutes;

(6) dry slide, drip containing the mountant of 1-10mmol/LDAPI and carry out mounting, fluorescence microscopeLower 100 times of eyepieces, 340nm excitation channel is observed; Under fluorescence microscope, nucleus is blue-fluorescence,The BCR gene fluorescence that takes on a red color, abl gene is green fluorescence, as BCR/ABL gene merges,Be yellow fluorescence.

With respect to prior art, beneficial effect of the present invention is:

The present invention uses quantum dot fluorescent dye to carry out fluorescence labeling to BCR and two genes of ABL, makes itUnder Same Wavelength exciting light, send respectively different fluorescence, can be at a fluorescence channel while using FISH to detectMiddle observation, thus detection efficiency improved. Particularly utilize CdSe/ZnS and CdTe/ZnS respectively to BCR andTwo genes of ABL carry out fluorescence labeling, make it under 340nm wavelength exciting light, send respectively red fluorescenceAnd green fluorescence, in the time that two genes occur to merge, red fluorescence and green fluorescence signal overlap and presentYellow fluorescence signal, has improved detection efficiency greatly.

The present invention, by great many of experiments, is BCR template by the suitableeest zone location: human chromosome22q11.21～22q11.23 district; ABL template: human chromosome 9q34.62～9q34.68 district. Two template regionsTerritory is all no more than 60kB size, is only 1/10 of conventional BCR/ABLFISH detection kit template region,The production difficulty greatly reducing, and improved the stability of product.

Brief description of the drawings

Fig. 1 is the testing result of conventional organic fluorescence group mark BCR/ABL probe;

Fig. 2 is the testing result of quantum dot fluorescence dye marker BCR/ABL probe of the present invention.

Detailed description of the invention

The general principle of FISH (FluorescenceInSituHybridization is called for short FISH)Being use mark, the single stranded DNA of fluorescence (probe) and the DNA (sample) complementary with it are hybridized, glimmering by observingOptical signal is at nucleus, and the position on chromosome and quantity reflect the situation of corresponding gene. Because it is directly perceived,Fast, sensitiveness is high and convenient, flexible, in cancer, heredity, more and more obtains in hematological diagnosis extensivelyApplication is the goldstandard of kinds of tumors and blood disease Clinical detection.

The feature of FISH technology is to use multicolor fluorescence to carry out mark and detection to multiple genes, but shortcomingThe fluorescence that is every kind of color all must be used under its specific fluorescence channel, enters with the excitation source of specific wavelengthRow excites, so the fluorescence labeling of a kind of color of every increase just must increase a kind of glimmering under fluorescence microscopeOptical channel is observed, and has increased testing cost. The result of simultaneously seeing under different fluorescence channels must be passed throughProfessional image software can be seen final result after processing, and has reduced the intuitive and the inspection that detectSurvey efficiency.

The present invention uses quantum dot fluorescent dye CdSe/ZnS and CdTe/ZnS respectively to BCR and ABL twoIndividual gene carries out fluorescence labeling, makes it under 340nm wavelength exciting light, sends respectively red fluorescence and greenFluorescence, in the time that two genes occur to merge, red fluorescence and green fluorescence signal overlap and present yellow glimmeringOptical signal, has improved detection efficiency greatly.

When fluorescence in situ hybridization technique is carried out probe mark, in order to obtain comparatively desirable fluorescence signal intensity,The template that conventionally need to use very large genomic DNA fragment to serve as a mark, as traditional FISH probeConventionally selecting the genomic DNA fragment of BCR, near the 600kB of two genes of ABL is template. SoLarge DNA fragmentation cannot be saved in a clone, can only split into multiple clones and preserve with follow-upProbe mark, has increased production difficulty and the production cost of BCR/ABL fusion detection kit greatly.Because the fluorescence intensity of quantum dot fluorescence dyestuff (CdSe/ZnS, CdTe/ZnS) is dyed higher than organic fluorescence far awayMaterial (Cy3, FAM etc.), so the genomic DNA fragment of selection of small carries out probe mark as templateAlso can obtain comparatively desirable effect. The present invention, by great many of experiments, is BCR mould by the suitableeest zone locationPlate: human chromosome 22q11.21～22q11.23 district; ABL template: human chromosome 9q34.62～9q34.68 district.Two template regions are all no more than 60kB size, are only conventional BCR/ABLFISH detection kit template region1/10 of territory, the production difficulty greatly reducing, and improved the stability of product.

Middle probe preparation method of the present invention includes but not limited to nick-translation, random priming, PCR method etc.These probe mark methods are that those skilled in the art grasp.

The sample that probe of the present invention and kit detect, includes but not limited to that cell drips sheet, bone marrow smear, bloodLiquid smear and paraffin section etc., the pre-treating method of these samples is that those skilled in the art grasp.

For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment pairThe present invention is described further.

Embodiment 1

Random priming preparation detects the probe of BCR/ABL gene fusion

BCR and abl gene are carried out to probe mark with random primering, wherein BCR template is behavedChromosome 22q11 .21～22q11.23 district, ABL template is human chromosome 9q34.62～9q34.68 district, reactionSystem is as follows:

Add according to the above ratio all the components except pol polymerase, 100 DEG C are heated 5 minutes, place on ice5 minutes, add pol polymerase, 37 DEG C are reacted 30 minutes, add final concentration 10mmol/LEDTA to stopReaction, makes the probe that detects BCR/ABL gene fusion.

Embodiment 2

Detect the kit of BCR/ABL gene fusion

A kit that detects BCR/ABL gene fusion, comprising:

(1) hybridization solution: 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 5-20% sulfuric acid PortugalGlycan, 20-50% deionized formamide, 0.01-0.1% mercaptopropionic acid, 0.1-1% bovine serum albumin(BSA), 0.1-1%Salmon sperm dna, pH value is 6-10;

(2) probe of the detection BCR/ABL gene fusion of preparation in embodiment 1, concentration is 1-10nmol/L;

(3) cleaning solution: 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 0.01-0.2% tenSodium dialkyl sulfate, pH value is 6-10.

Embodiment 3

Use the probe in detecting BCR/ABL gene fusion of detection BCR/ABL gene fusion of the present invention

The probe in detecting BCR/ABL gene of detection BCR/ABL gene fusion prepared by use embodiment 1 meltsClose, comprise the following steps:

(1) sample to be checked is fixed on slide;

(2) by conventional dewaxing step, paraffin section is dewaxed;

(3) on sample, add 10 μ L hybridization solutions, and the probe of 2 μ L detection BCR/ABL gene fusion,Concentration is 1-10nmol/L; Covered, and by glue sealing slide surrounding, to prevent hybridization solution evaporation;

(4) slide is placed in hybridization instrument, 90-95 DEG C of sex change is after 5 minutes, and 42 DEG C of constant temperature are hybridized 10-16Hour;

(5) remove cover glass, slide is put into the cleaning solution that is preheating to 60-68 DEG C, constant temp 2Inferior, each 15 minutes;

(6) dry slide, drip containing the mountant of 1-10mmol/LDAPI and carry out mounting, fluorescence microscopeLower 100 times of eyepieces, observe the blue-fluorescence signal of DAPI, abl gene under 340nm wavelength excitation channelGreen fluorescence signal and the red fluorescence signal of BCR gene, notice whether red fluorescence and green fluorescence haveOverlapping is yellow fluorescence signal. Detection method of the present invention can directly be observed three looks under 340nm exciting lightFluorescence, as shown in Figure 2, bright spot is wherein fluorescence signal to the testing result of mark BCR/ABL probe.

Wherein, hybridization solution: 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 5-20% sulfuric acidGlucan, 20-50% deionized formamide, 0.01-0.1% mercaptopropionic acid, 0.1-1% bovine serum albumin(BSA), 0.1-1%Salmon sperm dna, pH value is 6-10;

Cleaning solution: 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 0.01-0.2% dodecaneBase sodium sulphate, pH value is 6-10.

Comparative example 1

Use traditional F ISH probe in detecting BCR/ABL gene fusion

Use traditional FISH probe in detecting BCR/ABL gene fusion, comprise the following steps:

(1) sample to be checked is fixed on slide;

(2) by conventional dewaxing step, paraffin section is dewaxed;

(3) on sample, add 10 μ L hybridization solutions, and 2 μ L traditional F ISH probes, covered,And by glue sealing slide surrounding, to prevent hybridization solution evaporation;

(4) slide is placed in hybridization instrument, 90-95 DEG C of sex change is after 5 minutes, and 42 DEG C of constant temperature are hybridized 10-16Hour;

(5) remove cover glass, slide is put into the cleaning solution that is preheating to 60-68 DEG C, constant temp 2Inferior, each 15 minutes;

(6) dry slide, drip containing the mountant of 1-10mmol/LDAPI and carry out mounting, fluorescence microscopeLower 100 times of eyepieces, observe the blue-fluorescence signal of DAPI under 340nm wavelength excitation channel, take pictures; PreserveThe visual field is constant, switches to the green fluorescence signal of observing abl gene under 490nm wavelength excitation channel, takes pictures;The preservation visual field is constant, switches to the red fluorescence signal of observing BCR gene under 548nm wavelength excitation channel,Take pictures; Whether three photos are overlapped with image processing software, observing red fluorescence and green fluorescence hasOverlapping is yellow fluorescence signal. The testing result of conventional organic fluorescence group mark BCR/ABL probe is as Fig. 1Shown in, wherein in the photo of 340nm, show blue-fluorescence signal, in the photo of 490nm, show greenFluorescence signal, has shown red fluorescence signal in the photo of 550nm.

In summary, probe and the equal energy of traditional F ISH probe of detection BCR/ABL gene fusion of the present inventionEffectively detect the fusion of BCR/ABL gene by fluorescence in situ hybridization technique, but the inspection of conventional FISH probeAfter survey, need to observe fluorescence situations at three different fluorescence channels, and with image processing software by three passagesPhoto merge, and after probe in detecting of the present invention, only need to directly observe, take pictures,Greatly simplify testing process. In addition, can find out from testing result, fluorescence probe intensity of the present invention is also brightShow and be better than conventional FISH probe.

The last above embodiment that it should be noted that of institute is only in order to technical scheme of the present invention to be described but not to thisThe restriction of invention protection domain, although the present invention is explained in detail with reference to preferred embodiment, this areaThose of ordinary skill should be appreciated that and can modify or be equal to replacement technical scheme of the present invention, andDo not depart from essence and the scope of technical solution of the present invention.

Claims (10)

1. the application of quantum dot fluorescence dyestuff in the probe that detects BCR/ABL gene fusion.

2. application according to claim 1, is characterized in that: described quantum dot fluorescence dyestuff isCdSe/ZnS and CdTe/ZnS.

3. a labeling method that detects the probe of BCR gene, is characterized in that: use labeled nucleic acid probeMethod is carried out probe mark to BCR gene, and the template using when mark is human chromosome22q11.21～22q11.26 district, the fluorescent dye of use is quantum dot fluorescence dyestuff.

4. a labeling method that detects the probe of abl gene, is characterized in that: use labeled nucleic acid probeMethod is carried out probe mark to abl gene, and the template using when mark is human chromosome 9q34.62～9q34.78District, the fluorescent dye of use is quantum dot fluorescence dyestuff.

5. a labeling method that detects the probe of BCR/ABL gene fusion, is characterized in that: use nucleic acidProbe mark method is to respectively BCR gene and abl gene being carried out to probe mark, detects BCR geneThe template behaviour Chromosome 22q11 .21～22q11.26 district using when probe mark, detects the spy of abl geneThe pin mark template using that clocks is human chromosome 9q34.62～9q34.78 district, the probe that detects BCR gene withThe fluorescent dye that detects the probe use of abl gene is two kinds of different quantum dot fluorescence dyestuffs, two kinds of quantumPoint fluorescent dye sends the fluorescence of different colours under the monochromatic excitation light of Same Wavelength.

6. the labeling method of the probe of detection BCR/ABL gene fusion according to claim 5, itsBe characterised in that: the quantum dot fluorescence dyestuff that the probe of described detection BCR gene uses is CdSe/ZnS, described inThe quantum dot fluorescence dyestuff that detects the probe use of abl gene is CdTe/ZnS.

7. according to the labeling method of the probe of the detection BCR/ABL gene fusion described in claim 5 or 6The probe of the detection BCR/ABL gene fusion making.

8. a kit that detects BCR/ABL gene fusion, is characterized in that: comprise claim 7The probe of described detection BCR/ABL gene fusion.

9. the kit of detection BCR/ABL gene fusion according to claim 8, is characterized in that:Described kit also comprises hybridization solution and cleaning solution;

Wherein, described hybridization solution comprises 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 5-20%Dextran sulfate, 20-50% deionized formamide, 0.01-0.1% mercaptopropionic acid, 0.1-1% bovine serum albumin(BSA)And 0.1-1% salmon sperm dna, the pH value of hybridization solution is 6-10;

Described cleaning solution comprises 10-100mmol/LNaCl, 10-50mmol/L sodium citrate, 0.01-0.2% tenSodium dialkyl sulfate, cleaning solution pH value is 6-10.

The concentration of described probe is 1-10nmol/L.

10. utilize kit described in claim 8 or 9 to detect the method for BCR/ABL gene fusion, itsBe characterised in that: comprise the following steps:

1) in sample to be tested, add described hybridization solution and probe;

2) by the sample to be tested that is added with hybridization solution and probe 90-95 DEG C of sex change after 5～10 minutes, in 40～45 DEG CConstant temperature hybridization 10-16 hour;

3), after washing, mounting, under single channel, observe colour developing result by fluorescence microscope.