CN103952493B - The fluorescence in situ hybridization polyclone separate probe of renal carcinoma and test kit application thereof - Google Patents
The fluorescence in situ hybridization polyclone separate probe of renal carcinoma and test kit application thereof Download PDFInfo
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Abstract
The present invention relates to the fluorescence in situ hybridization polyclone separate probe of renal carcinoma and test kit application thereof. The cloned sequence that polyclone separate probe of the present invention is selected is RP11-416B14, CTD-2522M13 and CTD-2516D6 combination respectively, and CTD-2312C1, CTD-2248C21 and RP11-959H17 combination. What instant invention overcomes that prior art exists conventional cannot be cultured to mitosis after date to tumor cell row cell and carry out the defect of karyotyping again. The present invention utilizes FISH technology for detection Xp11.2 transposition/TFE3 gene fusion distinctive gene alteration of dependency renal carcinoma thus diagnosing this tumor, the accuracy rate of this fluorescence in situ hybridization polyclone separate probe application is high, specificity is high, success rate is high, fluorescence signal is strong, simple to operation, can be applicable in paraffin section, expand the scope of detection specimen, greatly optimize the diagnostic method of Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma.
Description
Technical field
The invention belongs to the application of fluorescence in situ hybridization probe, particularly to fluorescence in situ hybridization polyclone separate probe and the test kit application thereof of renal carcinoma.
Background technology
So-called renal carcinoma refers to Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, it it is WHO in 2004 different subtypes renal cell carcinomas newly named, fracture transposition is there is with TFE3 gene on XP11.2 site, and it is fused to feature with related genes such as APSL, PRCC, PSF, NonO, CLTC, cause the unconventionality expression of Regulated Proteins based on TFE3 albumen. Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma is mainly apt to occur in teenager, accounts for the 1/3 of renal cell carcinoma in children, and adult patients is rare. Owing to its sickness rate is relatively low, the understanding for Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma is still insufficient at present, and prognosis is poor, and adult patients is worse compared with child's prognosis, main and pathological, tumor grade by stages, whether have metastasis etc. relevant.Xp11.2 transposition/TFE3 gene fusion dependency is invalid to conventional immunization therapy, and chemicotherapy is also insensitive, and operation is the therapeutic modality that it is main, and lymph node and metastasis easily occurs in patient, and only targeted therapy has been proved to certain effect. So Index for diagnosis and postoperative auxiliary treatment that Accurate Diagnosis is to judging patient are most important.
Xp11.2 transposition/most distinctive change of TFE3 gene fusion dependency renal carcinoma, is TFE3 gene generation balanced translocation on Xp11.2 site. Be currently known transposition 8 kinds multi-form and fusion, 5 kinds can clearly know gene fusion site respectively: t (X; 1) (p11.2; Q21) PRCC and the TFE3 gene fusion that causes, t (X; 17) (p11.2; Q25) ASPL and the TFE3 gene fusion that causes, t (X; 1) (p11.2; P34) PSF and the TFE3 gene fusion that causes, t (X; 17) (p11.2; Q23) CLTC and the TFE3 gene fusion caused and inv (X) (p11; Q12) NonO (p54 causednrb). But gene fusion in any shape, all causes the change of TFE3 promoter, thus causing the unconventionality expression of modulin based on TFE3 albumen. Therefore, by the method for SABC, it is the main method of current diagnosis Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma to the detection of TFE3 albumen. But ImmunohistochemistryMethods Methods is vulnerable to more multifactorial impact, it is possible to because drawing materials improper or using BouinShi liquid to cause that false negative occurs; Possibly also owing to there is false positive results in the reasons such as excessive antigen recovery, too high, the excessive signal amplification of antibody concentration. Additionally TFE3 albumen is not only specific expressed in Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, also there will be the positive findings of TFE3 SABC in the tumor tissues such as granular cell tumor, adrenocortical carcinoma, Perivascular epithelioid cell tumor and high-level myxofibrosarcoma. The method of karyotyping and RT-PCR detection is to make a definite diagnosis the goldstandard of XP11.2 transposition/TFE3 gene fusion dependency renal carcinoma. But in clinical position, conventional tumor cell row cell cannot be cultured to mitosis after date and carry out karyotyping again, and RT-PCR extracts RNA from paraffin section and is not suitable for extensive use by several factors restriction and technical sophistication, thus need badly at present one can quickly, the method for economy, Accurate Diagnosis Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma.
Summary of the invention
It is an object of the invention to the defect for above-mentioned diagnostic method, it is provided that the fluorescence in situ hybridization polyclone separate probe of a kind of renal carcinoma and test kit application thereof.
The technical scheme is that
The fluorescence in situ hybridization polyclone separate probe of renal carcinoma, its cloned sequence being mainly characterized by the selection of polyclone separate probe is RP11-416B14, CTD-2522M13 and CTD-2516D6 combination respectively, and CTD-2312C1, CTD-2248C21 and RP11-959H17 combination.
Described polyclone separate probe, wherein RP11-416B14, CTD-2522M13 and CTD-2516D6 integrated positioning is in TFE3 telomere side, and three is labeled as identical green florescent signal.
Described polyclone separate probe, wherein CTD-2312C1, CTD-2248C21 and RP11-959H17 integrated positioning is in TFE3 centromere side, and three is labeled as identical red fluorescent.
The color of the described cloned sequence combination institute labelling being positioned at TFE3 telomere side, TFE3 centromere side is respectively green and red.
The present invention another solution is that
The fluorescence in situ hybridization polyclone separate probe test kit of renal carcinoma, this test kit is by probe hybridization liquid and 4 ', 6-diamidino-2-phenylindone after stain composition, and it is mainly characterized by:
(1) TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence;TFE3 centromere side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, is labeled as red fluorescence;
(2) probe hybridization liquid is to be mixed in proportion configuration formed by described polyclone separate probe and HumanCot-1DNA, hybridization buffer, purified water, it is necessary to-20 DEG C of lucifuge freezen protective;
(3) 4 ', 6-diamidino-2-phenylindone after stain is mainly used in nucleus DNA dyeing.
Described TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 centromere side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, it is labeled as red fluorescence, and above-mentioned polyclone separate probe and HumanCot-1DNA, hybridization buffer, purified water are mixed in proportion are configured to probe hybridization liquid.
The present invention also a kind of technical scheme is:
The fluorescence in situ hybridization polyclone separate probe test kit application of renal carcinoma, it is mainly characterized by the application in diagnosis Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma.
Advantages of the present invention and effect are in that to utilize FISH technology for detection Xp11.2 transposition/TFE3 gene fusion distinctive gene alteration of dependency renal carcinoma thus diagnosing this tumor, the accuracy rate of this fluorescence in situ hybridization polyclone separate probe application is high, specificity is high, success rate is high, fluorescence signal is strong, simple to operation, can be applicable in paraffin section, expand the scope of detection specimen, greatly optimize the diagnostic method of Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma.
Accompanying drawing explanation
Fig. 1 fluorescence in situ hybridization polyclone separate probe station-keeping mode schematic diagram.
Fig. 2 women's Papillary Renal Cell Carcinoma negative control case application schematic diagram.
Fig. 3 male's Papillary Renal Cell Carcinoma negative control case application schematic diagram.
Fig. 4 women's clear cell carcinoma of kidney negative control case application schematic diagram.
Fig. 5 male's kidney transparent renal cell carcinoma negative control case application schematic diagram.
Fig. 6 male Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma application schematic diagram.
Fig. 7 male Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma application schematic diagram.
Fig. 8 women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma application schematic diagram.
Fig. 9 women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma application schematic diagram.
Figure 10 women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma application schematic diagram.
Figure 11 women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma application schematic diagram.
Figure 12 women's case, negates Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma pathological diagnosis schematic diagram.
Detailed description of the invention
The technical thought of the present invention is:
Fluorescence in situ hybridization is that utilization combines with chromosome and (or) gene loci with being marked with the probe specificity of fluorescein, type by fluorescence microscope fluorescence signal, thus detecting chromosome and the method for corresponding gene change, have safe, quick, economical, highly sensitive, signal is strong, hybrid specificities is high, can show the advantages such as multiple color simultaneously in detection, and compensate for the defect that Interphase cells, complex karyotype cell and microdeletion cannot be diagnosed by traditional method. Meanwhile, fluorescence in situ hybridization technique can apply to carry out on paraffin-embedded sample retrospective study, greatly reduces the requirement to research sample.Fast development based on fluorescence in situ hybridization technique in recent years, the present invention just principle according to fluorescence in situ hybridization, it is proposed that one is used for diagnosing the fluorescence in situ hybridization polyclone separate probe of Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma and test kit application thereof.
The invention will be further elaborated by the examples below.
The polyclone separate probe that the present invention adopts is different from prior art and existing probe, be based on to the specific BAC cloned sequence binding site in TFE3 gene two ends comprehensive analyze after the prioritization scheme that filters out.
In the present invention, TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14 (fragment length is about 182Kb), CTD-2522M13 (fragment length is about 182Kb) and CTD-2516D6 (fragment length is about 218Kb); TFE3 centromere side BAC cloned sequence is the combination of CTD-2312C1 (fragment length is about 210Kb), CTD-2248C21 (fragment length is about 88Kb) and RP11-959H17 (fragment length is about 147Kb). These fragments are bacterial artificial chromosome clone (BAC clone), and its location on human chromosomal is all disclosed.
Maintain a certain distance between TFE3 gene and selected, to be positioned at its both sides (telomere side and centromere side) BAC cloned sequence and will not be overlapping, and the shortest in only about 36Kb between the BAC cloned sequence of two ends, two kinds of signals can be in close proximity to when transposition does not occur gene to make probe, will not because of design reasons so that two sitgnal distancel are excessive and Signal separator occur. TFE3 gene both sides are respectively arranged with the BAC cloned sequence combination of 3, require between the cloned sequence that homonymy is adjacent, there is a small amount of fragment overlapping, prevent homonymy cloned sequence from occurring that signal ruptures because spacing is relatively big, cause the appearance of independent signal, cause the erroneous judgement of testing result.
BAC cloned sequence at X chromosome TFE3 gene both sides 3 same colors of labelling respectively is to strengthen fluorescence intensity and scope, improves the accuracy of detection. Each BAC cloned sequence selected in probe combinations both passes through independent test, the cloned sequence that finishing screen select-out signal intensity is strong, specificity is good. Although it is to be noted that the cloned sequence length of CTD-2248C21 only has about 88Kb, but signal intensity is stronger in testing, CTD-2248C21 both sides respectively connected CTD-2312C1 and RP11-959H17 simultaneously, disappearance CTD-2248C21 is likely to result in the fracture of centromere side signal, therefore it is just selected as one of labeled fragment.
Select two groups totally 6 cloned sequences have some advantage following: (1) most cloned sequence size is substantially all at optimum about mark lengths 150-200Kb, not only signal intensity is similar each other for such cloned sequence, and does not have and cause that the strength difference of signal is bigger because cloned sequence is different in size; (2) there is a small amount of overlapping sequences between homonymy adjacent segment so that same color signal is mutually continuous, do not have the fracture of homonymy signal and the appearance of independent signal, reduce the probability of erroneous judgement; Farthest about 1100Kb apart between (3) two groups of cloned sequences, less than 1500Kb so that TFE3 gene does not occur two kinds of signals of transposition time institute labelling to occur merging, and so can form sharp contrast with feminine gender group when Signal separator occurs in positive group. If spacing is farther out, then no matter gene whether transposition, is all likely to occur separation signal, it is impossible to realize the effect of probe; Shortest spacing between (4) two groups of cloned sequences is less, and about 36Kb occurs that fusion signal on non-translocation chromosome, not easily with separate signal and obscure mutually.
BAC cloned sequence mark fluorescent, it is simple to use fluorescence microscope, convenient directly perceived, meanwhile, if desired adopt other detection techniques can also be marked as corresponding detection label.
The effect of the present invention:
The present invention feature according to the gene alteration of Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, utilize fluorescence in situ hybridization principle, not homochromy fluorescently-labeled polyclone probe is used respectively in TFE3 gene both sides, by the fusion in detection paraffin-embedded tissue section or separation signal, judge whether tumor tissues gene specific group translocation occurs, improve accuracy rate of diagnosis, for judging that patient's prognosis and postoperative adjuvant therapy provide information and guidance accurately. Experimental result according to us, the accuracy rate of this probe application is high, specificity is high, success rate is high, experimental signal is strong, simple and quick, especially can apply in paraffin section, expand the scope of detection specimen, provide new instrument for Accurate Diagnosis Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma.
Embodiment 1:
Step 1: the preparation of polyclone DNA probe:
The bacterial artificial chromosome (BAC clone) that X chromosome TFE3 gene both sides (i.e. telomere side and centromere side) are corresponding is searched by http://genome.ucsc.edu/, both sides select the BAC cloned sequence that 3 sizes are close respectively, control the maximum distance between both sides BAC cloned sequences within 1500Kb and non-overlapping copies, and 3 fragments of homonymy have the overlap of a fixed sequence each other. Choosing TFE3 telomere side BAC cloned sequence by above-mentioned requirements is CTD-2516D6 (chrX:48265836-48484403, fragment length is about 218Kb), CTD-2522M13 (chrX:48420425-48602847, fragment length is about 182Kb), RP11-416B14 (chrX:48580872-48763198, fragment length is about 182Kb); TFE3 centromere side BAC cloned sequence is CTD-2312C1 (chrX:48959513-49170367, fragment length is about 210Kb), CTD-2248C21 (chrX:49154756-49242991, fragment length is about 88Kb), RP11-959H17 (chrX:49293025-49440119, fragment length is about 147Kb). And at http://projects.tcag.ca/cgi-bin/efish/index.cgi, selected BAC clone is carried out specificity analyses, clearly selected BAC fragment specificity on X chromosome. Fluorescence in situ hybridization polyclone separate probe station-keeping mode in the present invention is as shown in Figure 1. Corresponding BAC clone is purchased from Invitrogen company, plasmid is extracted after being cultivated by BAC clone, utilize nick-translation, the 3 of telomere side BAC cloned sequence Sptectrumgreen-dUTP are labeled as green fluorescence, 3, centromere side BAC cloned sequence Sptectrumorange-dUTP is labeled as red fluorescence, and two ends color can be exchanged. And probe good for labelling and HumanCot-1DNA, hybridization buffer, purified water are configured to probe hybridization liquid in proportion ,-20 DEG C of lucifuge freezen protective.
Next step, the method that the probe of preparation is adopted fluorescence in situ hybridization, in the Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma and non-Xp11.2 group translocation renal carcinoma tissue of clinical diagnosis, verify its diagnosis effect.
Step 2: fluorescence in situ hybridization process:
3um tumor tissue section is placed in the roasting sheet of 65 �� 1 DEG C of calorstats overnight. Put into xylene soak 30min after taking-up, place into soaked in absolute ethyl alcohol 10min.It is subsequently placed in 100%, 85%, 70% graded ethanol and purified water each 3min of rehydration, places in purified water 100 �� 5 DEG C and boil sheet 20min. After taking-up is dried, drip 100ul pepsin reactant liquor in sample areas, digest 5min. Putting into rapidly 2 �� SSC and wash 5min, be finally putting into room temperature 70%, 85%, the 100% graded ethanol each 3min of dehydration successively, taking-up is dried. Adding the hybridization solution of 10 �� l to dry 22 �� 22mm coverslip, overturn by sample chips, make sample down, cover rapidly, after reversion, light cap slide makes hybridization solution be uniformly distributed; With rubber cement along coverslip edge mounting, edge that coverslip with microscope slide contact is completely covered; Slide is placed in the thermal station of 85 �� 1 DEG C, degeneration 5min; Rapidly slide is put in the hybridizing box of preheating, lucifuge, 37 �� 1 DEG C of overnight incubation. 2xSSC solution, 0.1%NP-40/2xSSC solution are put into preheating in the water-bath of 37 �� 1 DEG C; Remove rubber cement and coverslip, slide is put into washing 10min in 2xSSC solution; Proceed to washing 5min in 0.1%NP-40/2xSSC solution again; Then room temperature 70% ethanol dehydration 3min. After taking-up slide in the dark dries, dripping 10 �� lDAPI after stains to dry 22 �� 22mm coverslip, reverse sample chips, makes the target area of coverslip microscope slide contact, gently presses after reversion, it is to avoid produces bubble, in the dark deposits, to be seen.
Result judges:
Use standard with reference to current double-color probe, excite with OlympusB �� 5I trichroism filter lens of fluorescence microscope DAPI/FIFC/TexasRed in darkroom, 100 times of thing Microscopic observation Interphase cells, it determines fluorescence signal; The immunofluorescence software analysis image provided with Imstar company, assesses the probe hybridization situation of whole section. The whole nucleus of each example section has at least the nucleus of 100 non-overlapping copies, and observes clear and definite fluorescence signal, just can determine that the detection of immunofluorescence in situ hybridization effectively.
Use fluorescence microscope it is observed that two kinds of signal types: (1) merges signal: in the tumor tissues that TFE3 group translocation does not occur, owing to double-colored signal is at a distance of relatively near and the yellow signal (effect of red and green color superposition) that occurs or red green continuous signal. The quantity merging signal depends on X chromosome number in nucleus, and normal female occurs that 2 merge signal, and male only occurs that 1 merges signal; (2) signal is separated: when there is TFE3 genic balance transposition, TFE3 gene telomere lateral translocation is to other chromosome, and X chromosome only retains former TFE3 centromere side gene after transposition, cause that separating occurs in the red and green signals being marked at TFE3 gene two ends respectively. Only hot green Signal separator occurs and when more than one signal diameter of distance simultaneously, just can be designated as separation signal. Women Xp11.2 transposition/TFE3 gene fusion dependency patients with renal cell carcinoma may occur in which that 1 merges signal and 1 pair of separation signal, and male patient only there will be 1 pair and separates signal.
The whole nucleus of each example section has at least the nucleus of 100 non-overlapping copies, and red green separation signal occurs in the neoplastic cell nuclei having more than 10% at nucleus, it is judged that fluorescence in situ hybridization is positive; Red green separation signal does not occur, then judges that fluorescence in situ hybridization is negative; Individually occur that red or green does not then count.
Result:
30 example tumor of kidney specimen are utilized this polyclone separate probe to detect by we, and wherein 10 example clinical diagnosises are Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, 10 example papillary renal cell tumors, 10 example clear cell carcinoma of kidneys.If separating signal and reaching fluorescence in situ hybridization diagnosis positive criteria occur in patient's pathological section, Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma can be made a definite diagnosis. In 10 example Xp11.2 transpositions/TFE3 gene fusion dependency renal carcinoma, 9 examples meet fluorescence in situ hybridization diagnostic criteria, it is judged that for the positive, 1 example is negative; Papillary renal cell tumor and renal clear cell carcinoma section all do not detect separation signal, it is judged that for feminine gender. Fig. 2 is women Papillary Renal Cell Carcinoma negative control case, sees 2 red green fusion signals, represent 2 X chromosomes that transposition does not occur in tumor tissue cell's core; Fig. 3 is male's Papillary Renal Cell Carcinoma negative control case, sees 1 red green fusion signal, represent 1 X chromosome that transposition does not occur in tumor tissue cell's core; Fig. 4 is women clear cell carcinoma of kidney negative control case, sees 2 red green fusion signals, represent 2 X chromosomes that transposition does not occur in tumor tissue cell's core; Fig. 5 is the transparent renal cell carcinoma negative control case of male's kidney, sees 1 red green fusion signal, represent 1 X chromosome that transposition does not occur in tumor tissue cell's core; Fig. 6 is male Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, sees 1 pair of red green separation signal in tumor tissue cell's core, represents the X chromosome that transposition occurs; Fig. 7 is male Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, sees 1 pair of red green separation signal in tumor tissue cell's core, represents the X chromosome that transposition occurs; Fig. 8 is women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, sees 1 pair of red green separation signal and 1 fusion signal, represent 1 X chromosome that transposition occurs and 1 X chromosome that transposition does not occur respectively in tumor tissue cell's core; Fig. 9 is women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, sees 1 pair of red green separation signal and 1 fusion signal, represent 1 X chromosome that transposition occurs and 1 X chromosome that transposition does not occur respectively in tumor tissue cell's core; Figure 10 is women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, sees 1 pair of red green separation signal and 1 fusion signal, represent 1 X chromosome that transposition occurs and 1 X chromosome that transposition does not occur respectively in tumor tissue cell's core; Figure 11 is women Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, sees 1 pair of red green separation signal and a fusion signal, represent 1 X chromosome that transposition occurs and 1 X chromosome that transposition does not occur respectively in tumor tissue cell's core; Figure 12 is women case, clinical pathology and immunodiagnosis are Xp11.2 transposition/TFE3 gene fusion dependency Diagnosis of Renal Cell Carcinoma schematic diagram, but this fluorescence in situ hybridization polyclone separate probe detection feminine gender, 2 pairs of red green fusion signals are seen in tumor tissue cell's core, represent 2 X chromosomes that transposition does not occur, thus negative pathology and immunodiagnosis.
Evaluate:
Two groups of 6 cloned sequences that this probe adopts not yet have same probe to use, and cloned sequence size is relatively uniform. Each cloned sequence marking signal intensity is more similar, the probe signals of homonymy labelling occurs without interruption, the more general probe of signal intensity is strong, specificity is high, fully take into account reliability and the effectiveness of probe when being applied to diagnostic reagent and prepare into diagnostic kit, meet the requirement being applied to clinical diagnosis. Utilize fluorescence in situ hybridization technique, use TFE3 gene double-colored separation fluorescence in situ hybridization polyclone probe diagnostics Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma rate accurate, quick, economic and successful high, optimize the diagnostic method of Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma greatly.From accompanying drawing 2-5, can be seen that it is all negative for utilizing all of papillary renal cell tumor of this probe in detecting and clear cell carcinoma of kidney, and show in accompanying drawing 6-11 and the tumor tissue section of this probe in detecting Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma all can show fluorescence signal in most nucleus, and fluorescence signal is clear, luminance signals is high, and all successfully carries out FISH detection in each example tissue slice. The experimental result display present invention is in the high advantage of high specificity and sensitivity, and this probe penetrance is strong simultaneously, and signal is clear, and luminance signals height meets the requirement preparing into diagnostic kit.
Embodiment 2:Xp11.2 transposition/TFE3 gene fusion dependency Diagnosis of Renal Cell Carcinoma test kit
The fluorescence in situ hybridization polyclone separate probe test kit of renal carcinoma, this test kit is by probe hybridization liquid and 4 ', 6-diamidino-2-phenylindone after stain composition, it is characterised in that:
(1) TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 centromere side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, is labeled as red fluorescence.
(2) two kinds of polyclone separate probe are configured to probe hybridization liquid with being mixed in proportion with HumanCot-1DNA, hybridization buffer, purified water ,-20 DEG C of lucifuge freezen protective.
(3) 4 ', 6-diamidino-2-phenylindone after stain is mainly used in nucleus DNA dyeing.
The fluorescence in situ hybridization polyclone separate probe test kit of this renal carcinoma is mainly used in diagnosis Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, it is characterized in that: TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 centromere side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, it is labeled as red fluorescence, and above-mentioned polyclone separate probe and HumanCot-1DNA, hybridization buffer, purified water are mixed in proportion are configured to probe hybridization liquid.
Detection kit using method of the present invention is as follows:
1. the sample comprising tumor tissues is fixed through 10% neutral formalin, after paraffin embedding, prepare into 3um tissue slice;
2. section is placed in the roasting sheet of 65 �� 1 DEG C of calorstats overnight, puts into xylene soak 30min after taking-up, place into soaked in absolute ethyl alcohol 10min. It is subsequently placed in 100%, 85%, 70% graded ethanol and purified water each 3min of rehydration, places in purified water 100 �� 5 DEG C and boil sheet 20min;
3. taking out after drying, drip 100ul pepsin reactant liquor in sample areas, digest 5min, put into rapidly 2 �� SSC and wash 5min, be finally putting into room temperature 70%, 85%, the 100% graded ethanol each 3min of dehydration successively, taking-up is dried;
4. add the probe hybridization liquid of the 10 �� l present invention to dry 22 �� 22mm coverslip, sample chips is overturn, makes sample down, cover rapidly, after reversion, light cap slide makes hybridization solution be uniformly distributed, and with rubber cement along coverslip edge mounting, edge that coverslip with microscope slide contact is completely covered;
5. slide is placed in the thermal station of 85 �� 1 DEG C, degeneration 5min, rapidly slide is put in the hybridizing box of preheating, lucifuge, 37 �� 1 DEG C of overnight incubation;
6. 2xSSC solution, 0.1%NP-40/2xSSC solution are put in the water-bath of 37 �� 1 DEG C and preheat, remove the rubber cement in section and coverslip, slide is put into washing 10min in 2xSSC solution, then proceeds to washing 5min in 0.1%NP-40/2xSSC solution;Then room temperature 70% ethanol dehydration 3min;
7. after taking-up slide in the dark dries, dripping 10 �� lDAPI after stains to dry 22 �� 22mm coverslip, reverse sample chips, the target area making coverslip microscope slide contacts, and gently presses after reversion, it is to avoid produce bubble, in the dark deposit, use fluorescence microscope fluorescence signal. In non-Xp11.2 transposition/TFE3 gene fusion dependency patients with renal cell carcinoma, red and green signals merges display yellow signal, i.e. negative findings; In the tissue slice of Xp11.2 transposition/TFE3 gene fusion dependency patients with renal cell carcinoma, because X chromosome TFE3 gene occurs fracture transposition to make red and green signals separate, it is judged that for positive findings, thus clarifying a diagnosis.
Claims (2)
- The fluorescence in situ hybridization polyclone separate probe of 1.Xp12 transposition/TFE3 gene fusion dependency renal carcinoma, it is characterized in that the cloned sequence that polyclone separate probe is selected is RP11-416B14 respectively, CTD-2522M13 and CTD-2516D6 combines, and CTD-2312C1, CTD-2248C21 and RP11-959H17 combines, wherein RP11-416B14, CTD-2522M13 and CTD-2516D6 integrated positioning is in TFE3 telomere side, three is labeled as identical green florescent signal, wherein CTD-2312C1, CTD-2248C21 and RP11-959H17 integrated positioning is in TFE3 centromere side, three is labeled as identical red fluorescent.
- The fluorescence in situ hybridization polyclone separate probe test kit of 2.Xp11.2 transposition/TFE3 gene fusion dependency renal carcinoma, this test kit is by probe hybridization liquid and 4 ', 6-diamidino-2-phenylindone after stain composition, it is characterised in that:(1) TFE3 telomere side BAC cloned sequence is the combination of RP11-416B14, CTD-2522M13 and CTD-2516D6, is labeled as green fluorescence; TFE3 centromere side BAC cloned sequence is the combination of CTD-2312C1, CTD-2248C21 and RP11-959H17, is labeled as red fluorescence;(2) probe hybridization liquid is to be mixed in proportion configuration formed by described polyclone separate probe and HumanCot-1DNA, hybridization buffer, purified water, it is necessary to-20 DEG C of lucifuge freezen protective;(3) 4 ', 6-diamidino-2-phenylindone after stain is mainly used in nucleus DNA dyeing.
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| CN104388423B (en) * | 2014-10-28 | 2018-01-05 | 南京大学医学院附属鼓楼医院 | ASPL TFE3 amalgamation kidney gene probes and its kit application |
| 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 |
| CN107299152B (en) * | 2017-08-29 | 2020-03-03 | 中国人民解放军南京军区南京总医院 | Separation probe combination for diagnosing MITF translocation kidney cancer and application thereof |
| CN109825582A (en) * | 2019-03-01 | 2019-05-31 | 南京鼓楼医院 | NONO-TFE3 amalgamation kidney gene probe group and detection kit |
| 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 |
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