CN104388423B - ASPL TFE3 amalgamation kidney gene probes and its kit application - Google Patents
ASPL TFE3 amalgamation kidney gene probes and its kit application Download PDFInfo
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Abstract
The present invention relates to ASPL TFE3 amalgamation kidney gene probes and its kit application.The present invention is that RP11 634L10, RP11 51H16 and RP11 475F12 are combined respectively for the cloned sequence that gene probe is selected, and CTD 2311N12, RP11 416B14, CTD 2522M13, CTD 2516D6, CTD 2312C1, CTD 2248C21 and RP11 959H17 combinations.Instant invention overcomes cumbersome existing for the RT PCR of past application and cell method of karyotype analysis and time-consuming the defects of being not suitable for applying in clinical position.The present invention is using distinctive ASPL TFE3 fusions in FISH technology detection ASPL TFE3 amalgamation kidneys so as to diagnosing the tumour, the accuracy rate height of gene probe application, specificity are high, success rate is high, fluorescence signal is strong, it is simple to operation, diagnosis is quick, can be applicable in paraffin section, has expanded the scope of detection sample, the new method of accurately and reliably easy diagnosis ASPL TFE3 amalgamation kidneys is established, has started the beginning using FISH detection ASPL TFE3 amalgamation kidneys.
Description
Technical field
The invention belongs to the application field of fluorescence in situ hybridization probe, more particularly to ASPL-TFE3 amalgamations kidney gene
Probe and its kit application.
Background technology
Xp11.2 transpositions/TFE3 Gene Fusion associated renal cell carcinomas are apt to occur in teenager, account for the 1/3 of renal cell carcinoma in children,
The 15% of less than 45 years old kidneys, prognosis is poor, and adult patients are worse compared with children's prognosis, mainly divides with pathological, tumor grade
Phase, whether there is DISTANT METASTASES IN etc. relevant.Immunization therapy of the Xp11.2 transpositions/TFE3 Gene Fusions correlation to routine is invalid, right
Chemicotherapy is also insensitive, and operation is its main therapeutic modality, and lymph node and DISTANT METASTASES IN, only targeted therapy easily occurs in patient
It is proved to have certain effect.
ASPL-TFE3 amalgamation kidneys are sent out in Xp11.2 transpositions/8 kinds of partings of TFE3 Gene Fusions associated renal cell carcinoma
Sick rate highest is a kind of, and its most significant feature is the ASPL genes on TFE3 genes and No. 17 chromosomes on Xp11.2 sites
Generation is broken transposition, and forms new ASPL-TFE3 fusions, and thus triggers cell-signaling pathways and cell metabolism regulation and control
A kind of clear-cell carcinoma caused by disorder.Meanwhile ASPL-TFE3 amalgamation kidneys are also grade malignancy highest, tumor prognosis is worst
Xp11.2 transpositions/TFE3 Gene Fusion associated renal cell carcinomas.The growth of tumour cell propagation of ASPL-TFE3 amalgamation kidneys
Speed is fast and invasion is high, and adhesion is poor between cell, often just has occurred and that lymph node or DISTANT METASTASES IN in diagnosis,
Poor prognosis at a specified future date is, it is necessary to take positive treatment means to extend patient survival and improve life quality, so ASPL-TFE3
Amalgamation kidney Accurate Diagnosis is most important to the prognosis and postoperative auxiliary treatment for judging patient.
Before making the present invention, the method that can diagnose ASPL-TFE3 amalgamation kidneys only has RT-PCR and cell caryogram point
Analysis.Karyotyping must be cultivated to m period tumour cell, and tumour cell is carried out using technologies such as the aobvious bands of G
Karyotyping, this method is cumbersome and time-consuming is not suitable for applying in clinical position.Likewise, merged in view of ASPL-TFE3
Property the rare property of kidney and the complexity of PCR experiment processes be can not routinely using RT-PCR to all renal carcinoma tissues carry out
The detection of ASPL-TFE3 fusions, and RNA can only be extracted from paraffin section when suspecting ASPL-TFE3 amalgamation kidneys,
RNA fast degradation and a lot of other factors limit RT-PCR application, and technical sophistication is not suitable for extensive use.This
A little limitations, which result in ASPL-TFE3 amalgamations kidney, accurately, timely to be diagnosed, and influence clinician and ASPL-TFE3 is melted
The judgement of conjunction property prognosis of patients with renal cell carcinoma and post surgery treatment.At present need badly one kind can accurately, quickly, economic diagnosis ASPL-
The method of TFE3 amalgamation kidneys.
The content of the invention
The purpose of the present invention is that the defects of overcoming above-mentioned diagnostic method, there is provided a kind of ASPL-TFE3 amalgamations kidney
Gene probe and its kit application.
The technical scheme is that：
The gene probe of ASPL-TFE3 amalgamation kidneys, it is mainly characterized by clone's piece of gene probe selection
Section is that RP11-634L10, RP11-51H16 and RP11-475F12 are combined respectively, and CTD-2311N12, RP11-416B14,
CTD-2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined.
Described gene probe, wherein RP11-634L10, RP11-51H16 and RP11-475F12 integrated positioning are in No. 17
On chromosome ASPL genes, three is labeled as identical red fluorescent.
Described gene probe, wherein CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2516D6,
CTD-2312C1, CTD-2248C21 and RP11-959H17 integrated positioning are on X chromosome TFE3 genes, and mark is
Green florescent signal.
What the described combination of the cloned sequence on No. 17 chromosome ASPL genes, X chromosome TFE3 genes was marked
Color is respectively red and green, color can exchange.
The present invention another technical scheme be：
ASPL-TFE3 amalgamation kidney gene probe kits, the kit is by probe hybridization solution and 4 ', 6 one two amidines
Base -2-phenylindone counterstain composition, it is mainly characterized by：
(1) RP11-634L10, RP11-51H16 and RP11-475F12 group being positioned on No. 17 chromosome ASPL genes
Close, labeled as red fluorescent；It is positioned at CTD-2311N12, RP11-416B14, CTD- on X chromosome TFE3 genes
2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined, and are believed labeled as green fluorescence
Number；
(2) probe hybridization solution is mixed in proportion with Human Cot-1DNA, hybridization buffer, purified water by the probe
Configuration is formed, it is necessary to -20 DEG C of lucifuge freezen protectives；
(3) 4 ', 6 one diamidinos -2-phenylindone counterstain is mainly used in nuclear targeting.
Described RP11-634L10, RP11-51H16 and RP11-475F12 for being positioned on No. 17 chromosome ASPL genes
Combination, labeled as red fluorescent；It is positioned at CTD-2311N12, RP11-416B14, CTD- on X chromosome TFE3 genes
2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined, and are believed labeled as green fluorescence
Number, and above-mentioned probe and Human Cot-1DNA, hybridization buffer, purified water are mixedly configured into probe hybridization in proportion
Liquid.
The present invention also has a kind of technical scheme to be：
The application of ASPL-TFE3 amalgamation kidney gene probe kits, it is mainly characterized by diagnosis ASPL-
Application in TFE3 amalgamation kidneys.
Advantages of the present invention and effect are distinctive in taking the lead in using FISH technology detection ASPL-TFE3 amalgamation kidneys
For ASPL-TFE3 fusions so as to diagnose the tumour, the accuracy rate height of gene probe application, specificity are high, success rate is high, glimmering
Optical signal is strong, simple to operation, and diagnosis is quick, can be applicable in paraffin section, has expanded the scope of detection sample, and it is accurate to establish
The new method of reliable easy diagnosis ASPL-TFE3 amalgamation kidneys, start and utilized FISH detection ASPL-TFE3 amalgamation kidneys
The beginning.
Brief description of the drawings
Fig. 1 --- gene probe station-keeping mode schematic diagram.
Fig. 2 --- male's clear cell carcinoma of kidney negative control case application schematic diagram.
Fig. 3 --- women clear cell carcinoma of kidney negative control case application schematic diagram.
Fig. 4 --- the Xp11.2 transpositions of the non-ASPL-TFE3 fusions of male/TFE3 Gene Fusion correlations kidney application signal
Figure.
Fig. 5 --- the Xp11.2 transpositions of the non-ASPL-TFE3 fusions of women/TFE3 Gene Fusion correlations kidney application signal
Figure.
Fig. 6 --- male's ASPL-TFE3 amalgamation kidney application schematic diagrams.
Fig. 7 --- women ASPL-TFE3 amalgamation kidney application schematic diagrams.
Fig. 8 --- male's ASPL-TFE3 amalgamation kidney application schematic diagrams.
Fig. 9 --- women ASPL-TFE3 amalgamation kidney application schematic diagrams.
Figure 10 PCR experiments result confirms ASPL-TFE3 fusions be present in tumor tissues.
Embodiment
The present invention technical thought be：
FISH be using be marked with fluorescein probe specificity with chromosome and (or) gene loci phase
With reference to by the type of fluorescence microscope fluorescence signal, so as to detect the method for chromosome and corresponding gene change, having
Safety, quick, economy, high sensitivity, detection signal is strong, hybrid specificities are high, can show the advantages that multiple color simultaneously, and
It compensate for the defects of conventional method can not diagnose to Interphase cells, complex karyotype cell and microdeletion.Meanwhile fluorescence
Hybridization in situ technique can apply to carry out retrospective study on the sample of FFPE, greatly reduces and research sample is wanted
Ask.Based on the fast development of fluorescence in situ hybridization technique in recent years, the principle of the invention according to FISH, it is proposed that one
Kind is used for gene probe and its kit application for diagnosing ASPL-TFE3 amalgamation kidneys.
Below by embodiment, the invention will be further elaborated.
The gene probe that the present invention uses is different from prior art, and belongs to first Application FISH technology detection ASPL-
TFE3 amalgamation kidneys, be based on the research to ASPL-TFE3 amalgamation kidney gene alteration features and to TFE3 genes with
And the scheme that the comprehensive analysis of the BAC cloned sequence binding sites of ASPL gene specifics filters out.
In the present invention, BAC cloned sequences on ASPL genes are RP11-634L10 (fragment length is about 172Kb),
RP11-51H16 fragment lengths are about 166Kb) and RP11-475F12 (fragment length is about 185Kb) combination；On TFE3 genes
BAC cloned sequences for CTD-2311N12 (fragment length is about 210Kb), RP11-416B14 (fragment length is about 182Kb),
CTD-2522M13 (fragment length is about 182Kb), CTD-2516D6 (fragment length is about 218Kb), CTD-2312C1 (fragments
Length is about 210Kb), CTD-2248C21 (fragment length is about 88Kb) and RP11-959H17 (fragment length is about 147Kb)
Combination.These fragments are bacterial artificial chromosome clone (BAC clones), and its positioning on human chromosomal is public
Open.
BAC cloned sequences on selection ASPL genes and TFE3 genes must are fulfilled for the place gene at least completely covered,
It so just can guarantee that two fusions newly formed in ASPL-TFE3 amalgamation kidneys can show two fusion signals.
Meanwhile the combination of the BAC cloned sequences of 3 on ASPL genes, it is desirable to a small amount of fragment weight between adjacent cloned sequence be present
It is folded, it is broken to prevent homonymy cloned sequence signal, causes the appearance of independent signal, influences the judgement of testing result；In TFE3
The combination of the BAC cloned sequences of 7 on gene, in the case where TFE3 genetic profiles are completely covered, meets the adjacent clone of homonymy as far as possible
It is overlapping or both at a distance of being no more than 10Kb to there is a small amount of fragment between fragment, it is ensured that the integrality of monochrome signal.Each
It is the accuracy that detection is improved to strengthen fluorescence intensity and scope that multiple BAC cloned sequences are marked on gene.Probe combinations
In selected each BAC cloned sequence by individually test, the clone that finishing screen select-out signal intensity is strong, specificity is good
Fragment.
Select two groups totally 10 cloned sequences have some following advantage：(1) most cloned sequence sizes are substantially in optimum
Mark lengths 150-200Kb or so, not only signal intensity is similar each other for such cloned sequence, and be not in because gram
The strength difference that grand fragment is different in size to cause signal is larger；(2) exist between homonymy adjacent segment a small amount of overlapping sequences or
Apart it is no more than 10Kb so that same color signal is mutually continuous, is not in fracture and the independent signal of homonymy signal
Occur, reduce the probability of erroneous judgement；(3) two groups of cloned sequences completely covering selected by gene, in ASPL-TFE3 amalgamation kidneys
Two fusion signals can be formed.
BAC cloned sequence mark fluorescent signals, fluorescence microscope is easy to use, it is convenient directly perceived, meanwhile, if desired adopt
Corresponding detection label can also be marked as with other detection techniques.
The effect of the present invention：
The present invention utilizes FISH principle according to the characteristics of the group translocations of ASPL-TFE3 amalgamation kidneys, will
TFE3 genes and ASPL genes use the gene probe of not homochromy fluorescence labeling respectively, by detecting in paraffin-embedded tissue section
Fusion or separation signal, judge whether tumor tissues ASPL-TFE3 genes occur the fusion of specific group translocation, improve
Accuracy rate of diagnosis, to judge that patient's prognosis and postoperative adjuvant therapy provide accurate information and guidance.After the completion of probe design,
It is applied to the detection of ASPL-TFE3 amalgamation kidneys, while using the actual effect of PCR result verification probes, according to us
Experimental result, the accuracy rate of the probe application is high, specificity is high, success rate is high, and experimental signal is strong, simple and quick, especially
It is can be applied in paraffin section, has expanded the scope of detection sample, is provided for Accurate Diagnosis ASPL-TFE3 amalgamation kidneys
New method and instrument.
Embodiment 1：
Step 1：The preparation of gene probe：
Pass through http：//genome.ucsc.edu/ searches bacterium people corresponding to X chromosome TFE3 genes and ASPL genes
Work chromosome (BAC clones), corresponding BAC cloned sequences are selected respectively, control BAC cloned sequences selected by homonymy completely to cover phase
For gene, and homonymy fragment has the overlapping of certain sequence between each other or is smaller than 10Kb.Chosen by above-mentioned requirements
BAC cloned sequences on ASPL genes are RP11-634L10 (chr17：79796813-79969288, fragment length are about
172Kb)、RP11-51H16(chr17：79931578-80097452, fragment length are about 166Kb) and RP11-475F12
(chr17：80032268-80216871, fragment length are about 185Kb) combination；BAC cloned sequences on TFE3 genes are
CTD-2311N12(chrX：48713289-48923401, fragment length are about 210Kb), CTD-2516D6 (chrX：
48265836-48484403, fragment length are about 218Kb), CTD-2522M13 (chrX：48420425-48602847, fragment
Length is about 182Kb), RP11-416B14 (chrX：48580872-48763198, fragment length are about 182Kb), CTD-
2312C1(chrX：48959513-49170367, fragment length are about 210Kb), CTD-2248C21 (chrX：49154756-
49242991, fragment length is about 88Kb), RP11-959H17 (chrX：49293025-49440119, fragment length are about
147Kb).And specific analysis is carried out to selected BAC clones, the specificity of clearly selected BAC fragments on chromosome.This
Gene probe station-keeping mode in invention is as shown in Figure 1.Corresponding BAC clones are purchased from Invitrogen companies, BAC is cloned
Plasmid is extracted after culture, using nick-translation, 3 BAC cloned sequences of ASPL genes side are used
Tetramethylrhodamine-5-dUTP is labeled as red fluorescence, and the BAC cloned sequences on TFE3 genes are used
Fluorescein-12-dUTP is labeled as green fluorescence, and both ends color can exchange.In order to detect the correctness of structure clone,
The cloned sequence of each mark fluorescent signal is individually dripped into piece (by the peripheral blood cells of healthy human body by training in cell respectively
Support, colchicine processing is hypotonic, obtains the suspension containing a large amount of mitotic cells after fixed and is made) on to carry out hybridization positioning real
Test, the positioning of cloned sequence on chromosome is observed on mitosis metaphase cell.Above-mentioned 10 groups of cloned sequences pass through respectively
Checking, can be accurately positioned on ASPL genes and TFE3 genes, finally by cloned sequence mark purification after with Human
Cot-1DNA, hybridization buffer, purified water are configured to probe hybridization solution, -20 DEG C of lucifuge freezen protectives in proportion.
In next step, by the probe of preparation use FISH method, clinical diagnosis be Xp11.2 transpositions/
Tested in TFE3 Gene Fusion correlations kidney (including four PCR turn out to be ASPL-TFE3 amalgamations kidney) and clear cell carcinoma
Demonstrate,prove its diagnosis effect.
Step 2：FISH process：
(1) histotomy pre-processes：The tumor tissue section previously prepared is baked into piece in 65 ± 1 DEG C of insulating boxs to stay overnight, taken
Xylene soak 30min is put into after going out, places into soaked in absolute ethyl alcohol 10min.It is subsequently placed into 100%, 85%, 70% gradient second
Each 3min of rehydration in alcohol and purified water, it is put into purified water 100 ± 5 DEG C and boils piece 20min.After section taking-up is dried, in sample area
100ul stomach cardia enzyme liquids are added dropwise in domain, digest 5min.Rapid be put into 2 × SSC solution washs 5min, is finally putting into room temperature
70%th, 85%, 100% graded ethanol is dehydrated each 3min successively, and taking-up is dried.(2) histotomy and probe co-variation：Add 10 μ l
Probe hybridization solution to drying on 22 × 22mm cover glasses, the histotomy sample that step (1) treats is faced lower cover and covered
On slide, light cap slide is uniformly distributed hybridization solution after reversion, with rubber cement along cover glass edge mounting.Slide is placed on
5min is denatured in 85 ± 1 DEG C of thermal station, is put into the hybridizing box of preheating rapidly, 37 ± 1 DEG C of lucifuges are incubated overnight.(3) after hybridizing
Wash and redye：In 37 ± 1 DEG C of water bath, 2 × SSC of preheating solution, 0.1%NP-40/2 × SSC solution.By step (2)
Treated histotomy removes rubber cement and cover glass, is put into 2 × SSC solution and washs 10min, places into 0.1%NP-
5min is washed in 40/2 × SSC solution, then 70% ethanol dehydration 3min, dark place are dried at room temperature.10 μ l DAPI are added dropwise to redye
It is on agent to another dry 22 × 22mm cover glass, the histotomy sample previously dried is face-down, connect target area
Lid fragmentation is touched, light cap slide removes bubble removing after reversion, and dark place is deposited to be seen.
Result judgement：
Excited in darkroom with the color optical filterings of 0lympus B × 5l fluorescence microscopes DAPI/FIFC/TexasRed tri-, 100
Times thing Microscopic observation FISH fluorescence signals, the fish analysis software analysis image provided with lmstar companies, assess whole section
Probe hybridisation events.
Using fluorescence microscope it is observed that two kinds of signal types：(1) red green separation signal：TFE3 genes are not occurring
With in the tumor tissues of ASPL group translocations, because X chromosome and No. 17 chromosomes are apart from each other, then distinguishing respectively on chromosome
Mark red and green signals independently occur, and the quantity of red and green signals depends on No. 17 chromosome numbers and X chromosome in nucleus, normally
Women is due to containing 2 No. 17 chromosomes and 2 X chromosomes, just occurring+2 greens of 2 danger signals, and male is only
There is the green conjunction signal of 2 danger signals+1；(2) double fusion signals：When TFE3 telomere side bases in ASPL-TFE3 amalgamation kidneys
After balanced translocations occur with No. 17 chromosome ASPL gene telomeres sides, formed new X chromosome by former TFE3 centromeres side with
ASPL gene telomeres side forms, and No. 17 new chromosomes then by ASPL genes centromere side and transposition and Lai TFE3 telomeres side
Form, red and green signals cause point after Gene Fusion according to the TFE3 genes and ASPL genes that mark principle is complete covering full section
The gene location for being is not marked mutually to adjoin, therefore (red and green color is folded for the red green continuous signal of formation or yellow signal
The effect added).Only simultaneously when two, which merge signal, occurs in same nucleus, double fusion signals can be just designated as.Women
ASPL-TFE3 amalgamation patients with renal cell carcinoma may occur in which 2 fusion+1 greens of danger signal of signal+1, show in the nucleus
TFE3 genes on ASPL genes and an X chromosome on interior No. 17 autosomes there occurs mutual transposition fusion,
Also retain a normal X chromosome and normal No. 17 chromosome simultaneously.Male patient occurs that 2 fusion signals+1 are red
Chrominance signal, show No. 17 autosomes in the nucleus in two and unique 1 X chromosome of male occur ASPL with
The transposition fusion of TFE3 genes, only retains normal No. 17 chromosome.
Each section is at least observed 100 in the region that nuclear targeting is clear, hybridization signal is stronger and not weighed mutually
Folded nucleus and there are clear and definite FISH signals just to can determine that FISH detections are effective.There are double fusions in neoplastic cell nuclei more than 2%
Signal, it is determined as that ASPL-TFE3 fusions FISH detections are positive, ASPL-TFE3 amalgamation kidneys can be diagnosed as；Only occur
Red green separation signal or double fusion signals are not up to threshold value, then are determined as that ASPL-TFE3 fusions FISH detections are negative, no
ASPL-TFE3 amalgamation kidneys can be diagnosed as.
As a result：
We are detected to 30 tumor of kidney samples using the gene probe, wherein 15 by FISH detect or
RT-PCR is diagnosed as Xp11.2 transpositions/TFE3 Gene Fusion correlations kidney, and (four patients turn out to be ASPL- through RT-PCR experiments
TFE3 amalgamations kidney), 15 clear cell carcinoma of kidney.If there are double fusion signals and reaches fluorescent in situ in patient's pathological section
Hybridization diagnosis positive criteria, that is, be diagnosed as ASPL-TFE3 amalgamation kidneys.15 Xp11.2 transpositions/TFE3 Gene Fusions are related
Property kidney in, 4 there are double fusion signals by the PCR ASPL-TFE3 amalgamations kidneys confirmed and reach positive threshold value,
ASPL-TFE3 fusions FISH detections are positive, meet the diagnosis of ASPL-TFE3 amalgamation kidneys；11 other Xp11.2
Transposition/TFE3 Gene Fusion correlation kidneys and renal clear cell carcinoma section are not detected to separate signal, are judged as
It is negative.Fig. 2 is male's clear cell carcinoma of kidney negative control case, sees that 2 danger signals+1 are green in tumor tissue cell's core
Chrominance signal, 2 chromosomes of No. 17 that transposition does not occur and the X chromosome of 1 non-transposition are represented respectively；Fig. 3 is that women kidney is transparent
Cell cancer negative control case ,+2 greens of 2 danger signals are seen in tumor tissue cell's core, 2 is represented and does not occur
No. 17 chromosomes and 2 normal X chromosomes of group translocation；The Xp11.2 transpositions that Fig. 4 merges for the non-ASPL-TFE3 of male/
TFE3 Gene Fusion correlation kidney cases ,+1 green of 2 danger signals is shown in tumor tissue cell's core, represents 2
No. 17 chromosomes and 1 normal X chromosome of the non-producer transposition of bar；Fig. 5 is the non-ASPL-TFE3 fusions of women
Xp11.2 transpositions/TFE3 Gene Fusion correlation kidneys ,+2 green letters of 2 danger signals are seen in tumor tissue cell's core
Number, represent No. 17 chromosomes and X chromosome of 2 non-producer transpositions；Fig. 6 is male's ASPL-TFE3 amalgamation kidneys,
See in tumor tissue cell's core 2 it is red it is green fusion+1 danger signal of signal, two fusion signals represent respectively occur ASPL with
The X chromosome of TFE3 genic balance transpositions and No. 17 chromosomes, 1 danger signal represent intracellular also one and transposition do not occur
No. 17 autosomes；Fig. 7 is women ASPL-TFE3 amalgamation kidneys, and 2 red green fusion letters are seen in tumor tissue cell's core
Number+1 green of+1 danger signal, two fusion signals represent ASPL and the X of TFE3 genic balance transpositions occurs respectively
Chromosome and No. 17 chromosomes, 1 danger signal represents normal No. 17 autosome into the cell, 1 green letter
Number represent another X chromosome that transposition does not occur in two X chromosomes of women；Fig. 8 is male's ASPL-TFE3 amalgamation kidneys
Cancer, is shown in 2 red green fusion+1 danger signals of signal in tumor tissue cell's core, and two fusion signals represent generation respectively
ASPL and the X chromosome and No. 17 chromosomes of TFE3 genic balance transpositions, 1 danger signal represent intracellular also one and not sent out
No. 17 autosomes of raw transposition；Fig. 9 is women ASPL-TFE3 amalgamation kidneys, see in tumor tissue cell's core 2 it is red green
The green of danger signal+1 of signal+1 is merged, it is easy that two fusion signals represent generation ASPL and TFE3 genic balances respectively
The X chromosome and No. 17 chromosomes of position, 1 danger signal represent intracellular also No. 17 that transposition does not occur and often dyed
Body, 1 green represent another X chromosome that transposition does not occur in two X chromosomes of women；Figure 10 demonstrate,proves for PCR experiment
ASPL-TFE3 fusions in real tumor tissues be present, No. 5 bands are to utilize specially to design for ASPL-TFE3 fusions
PCR primer enter the product band of performing PCR, amplify 310bp or so target stripe, meet the type of ASPL-TFE3 fusions 1
Standard, be diagnosed as ASPL-TFE3 amalgamation kidneys, to verify FISH detection accuracy.
Evaluation：
Using FISH technology, autonomous Design gene probe detection ASPL-TFE3 amalgamation kidneys are domestic first Applications.
Cloned sequence size used is relatively uniform on the gene probe that we design, each cloned sequence marking signal intensity relatively phase
Seemingly, the probe signals of homonymy mark occur without interruption, and the more general probe of signal intensity is strong, and specificity is high, fully takes into account application
The reliability and validity of probe when diagnostic reagent and reagent preparation box, meet the requirement of clinical diagnosis.Utilize fluorescence
Hybridization in situ technique, have accurate, quick, economic and successful rate high using gene probe diagnosis ASPL-TFE3 amalgamation kidneys
Characteristic, greatly optimize the diagnostic methods of ASPL-TFE3 amalgamation kidneys.It can be seen that utilizing the spy from accompanying drawing 2-5
Pin detects all clear cell carcinoma of kidney and Xp11.2 transpositions/TFE3 Gene Fusion correlation kidneys of non-ASPL-TFE3 fusions
Cancer is negative, and shown in accompanying drawing 6-9 in the tumor tissue section of probe in detecting ASPL-TFE3 amalgamation kidneys into
Work(carries out FISH detections, double fusion signals occurs, while F I SH testing results are consistent with PCR testing results.Experimental result shows
Show the present invention in the high advantage of high specificity and sensitiveness, while probe hybridization success rate is high, signal is clear, and brightness is high, symbol
Close the requirement for being prepared into diagnostic kit.
Embodiment 2：ASPL-TFE3 amalgamation Diagnosis of Renal Cell Carcinoma kits
The gene probe kit of ASPL-TFE3 amalgamation kidneys, the kit is by probe hybridization solution and the amidine of 4 ', 6- bis-
Base -2-phenylindone counterstain composition, it is characterised in that：
(1) RP11-634L10, RP11-51H16 and RP11-475F12 group being positioned on No. 17 chromosome ASPL genes
Close, labeled as red fluorescent；It is positioned at CTD-2311N12, RP11-416B14, CTD- on X chromosome TFE3 genes
2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined, and are believed labeled as green fluorescence
Number；
(2) probe hybridization solution is mixed in proportion with Human Cot-1DNA, hybridization buffer, purified water by the probe
Configuration is formed, it is necessary to -20 DEG C of lucifuge freezen protectives；
(3) 4 ', 6- diamidino -2-phenylindone counterstain are mainly used in nuclear targeting.
Described RP11-634L10, RP11-51H16 and RP11-475F12 for being positioned on No. 17 chromosome ASPL genes
Combination, labeled as red fluorescent；It is positioned at CTD-2311N12, RP11-416B14, CTD- on X chromosome TFE3 genes
2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined, and are believed labeled as green fluorescence
Number, and above-mentioned probe and Human Cot-1DNA, hybridization buffer, purified water are mixedly configured into probe hybridization in proportion
Liquid.
Detection kit application method of the present invention is as follows：
1. the sample comprising tumor tissues is fixed through 10% neutral formalin, 3um tissues are prepared into after FFPE
Section；
Stayed overnight 2. section is placed in into 65 ± 1 DEG C of insulating boxs and bakes piece, xylene soak 30mi n are put into after taking-up, place into nothing
Water-ethanol immersion 10mi n.Each 3min of rehydration in 100%, 85%, 70% graded ethanol and purified water is subsequently placed into, is placed into pure
100 ± 5 DEG C are boiled piece 20min in change water；
3. after taking-up are dried, 100ul pepsin reaction solutions being added dropwise in sample areas, digest 5min, it is put into 2 rapidly ×
SSC washs 5min, is finally putting into the graded ethanol of room temperature 70%, 85%, 100% and is dehydrated each 3min successively, taking-up is dried；
4. the probe hybridization solution of the 10 μ l present invention is added to overturn sample chips to drying on 22 × 22mm cover glasses, make sample
Down, cover rapidly, light cap slide is uniformly distributed hybridization solution after reversion, complete with rubber cement along cover glass edge mounting
The edge that all standing cover glass contacts with slide；
5. slide is placed in 85 ± 1 DEG C of thermal station, 5min is denatured, slide is put into the hybridizing box of preheating rapidly, kept away
Light, 37 ± 1 DEG C of overnight incubations；
6. 2 × SSC solution, 0.1%NP-40/2 × SSC solution to be put into 37 ± 1 DEG C of water-bath and be preheated, remove
Rubber cement and cover glass in section, slide is put into 2 × SSC solution and washs 10min, then be transferred to 0.1%NP-40/2 ×
5min is washed in SSC solution；Then the ethanol dehydration 3min of room temperature 70%；
After 7. taking-up slide dries in the dark, it is added dropwise on 10 μ lDAPI counterstains to dry 22 × 22mm cover glasses, instead
Turn sample chips, contact the target area of cover glass slide, gently pressed after reversion, avoid producing bubble, deposit in the dark, use
Fluorescence microscope fluorescence signal.Each section is at least seen in the region that nuclear targeting is clear, hybridization signal is stronger
Observe the nucleus of 100 non-overlapping copies and there are clear and definite FISH signals just to can determine that FISH detections are effective.It is effective when being determined as
In the section of FISH detections, red and green signals individually occur or double threshold values for merging signal and being not up to 2%, then are determined as ASPL-
TFE3 fusions FISH detections are negative, it is impossible to can be diagnosed as ASPL-TFE3 amalgamation kidneys；When being determined as effective FISH inspection
In the section of survey, there are double fusion signals in the neoplastic cell nuclei for having more than 2%, then is determined as ASPL-TFE3 fusions FISH
Detection is positive, can be diagnosed as ASPL-TFE3 amalgamation kidneys.
Claims (4)
1.ASPL-TFE3 amalgamation kidney gene probes, it is characterised in that gene probe from mark cloned sequence be respectively
RP11-634L10, RP11-51H16 and RP11-475F12 are combined, and CTD-2311N12, RP11-416B14, CTD-
2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined；
RP11-634L10, RP11-51H16 and RP11-475F12 integrated positioning is on No. 17 chromosome ASPL genes, and three
Person is labeled as identical red fluorescent；
Described CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2516D6, CTD-2312C1, CTD-2248C21
With RP11-959H17 integrated positionings on X chromosome TFE3 genes, mark is green florescent signal.
2. ASPL-TFE3 amalgamations kidney gene probe according to claim 1, it is characterised in that be positioned at TFE3 bases
Cause, the cloned sequence combination institute marker color of ASPL genes are respectively labeled as green and red.
3.ASPL-TFE3 amalgamation kidney gene probe kits, the kit by probe hybridization solution and 4 ', 6- diamidino-
2-phenylindone counterstain forms, it is characterised in that：
(1) RP11-634L10, RP11-51H16 and RP11-475F12 combination being positioned on No. 17 chromosome ASPL genes, mark
It is designated as red fluorescent；Be positioned at CTD-2311N12, RP11-416B14 on X chromosome TFE3 genes, CTD-2522M13,
CTD-2516D6, CTD-2312C1, CTD-2248C21 and RP11-959H17 are combined, labeled as green florescent signal；
(2) probe hybridization solution is by the probe and Human Cot-1DNA, hybridization buffer, purified water mixed configuration in proportion
Into, it is necessary to -20 DEG C of lucifuge freezen protectives；
(3) 4 ', 6- diamidino -2-phenylindone counterstain are mainly used in nuclear targeting.
4. ASPL-TFE3 amalgamations kidney gene probe kit according to claim 3, it is characterised in that：It is positioned at
RP11-634L10, RP11-51H16 and RP11-475F12 combination on No. 17 chromosome ASPL genes, labeled as red fluorescence
Signal；Be positioned at CTD-2311N12, RP11-416B14 on X chromosome TFE3 genes, CTD-2522M13, CTD-2516D6,
CTD-2312C1, CTD-2248C21 and RP11-959H17 combine, labeled as green florescent signal, and by above-mentioned probe with
Human Cot-1DNA, hybridization buffer, purified water are mixedly configured into probe hybridization solution in proportion.
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| CN107267608B (en) * | 2017-06-16 | 2018-03-09 | 中国人民解放军南京军区南京总医院 | A kind of MATR3 TFE3 fusions and its detection primer and application |
| CN109825582A (en) * | 2019-03-01 | 2019-05-31 | 南京鼓楼医院 | NONO-TFE3 amalgamation kidney gene probe group and detection kit |
| CN109988836B (en) * | 2019-03-18 | 2022-06-07 | 厦门艾德生物技术研究中心有限公司 | FISH probe set for detecting NTRK fusion and application thereof |
| CN110093420B (en) * | 2019-05-09 | 2022-05-24 | 首都医科大学附属北京儿童医院 | Leukemia ZNF384 gene disruption probe detection kit |
| CN110144401A (en) * | 2019-05-10 | 2019-08-20 | 广州安必平医药科技股份有限公司 | A kind of quickly detection NTRK Gene Fusion kit and its method |
| CN113005200B (en) * | 2021-04-14 | 2023-07-04 | 深圳乐土生物科技有限公司 | Primer composition for detecting sarcoma fusion gene mutation, kit and application |
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