CN110172511A - For detecting probe groups, kit and its application of HER-2 gene magnification level - Google Patents
For detecting probe groups, kit and its application of HER-2 gene magnification level Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses the probe groups for detecting HER-2 gene magnification level, including targeting the probe of HER-2 gene order and referring to probe, it include the probe for targeting RAI1 gene order and/or the probe of CSP17 gene order referring to probe, wherein, it targets the probe of HER-2 gene order and is all made of phi29DNA polymerase referring to probe and carry out probe label.It include the dimethyl sulfoxide as promotor the invention also discloses the kit for detecting HER-2 gene magnification level, including above-mentioned probe groups and hybridization buffer, in hybridization buffer.The present invention provides the kits for accurately detecting HER-2 gene magnification level, realize accurate interpretation and tissue samples and the probe quick hybridization in 2 hours of HER-2 amplification；Existing method can be assisted quickly to carry out the accurate layering of tumour, accurate diagnosing and treating using detection kit of the invention.
Description
Technical field
The present invention relates to technical field of gene detection, in particular for detecting probe groups, the examination of HER-2 gene magnification level
Agent box and its application.
Background technique
Breast cancer is the great public health problem of current social.According to National Cancer Center and Ministry of Public Health's prevention and control of diseases
Office's pathogenesis of breast carcinoma data in 2009 announced in 2012 are shown: national tumour registration area breast cancer incidence occupies women evil
Property the 1st of tumour, female mammary gland cancer morbidity (rough and careless) whole nation adds up to 42.55/10 ten thousand, and city is 51.91/10 ten thousand, agriculture
Village is 23.12/10 ten thousand.And morbidity has the tendency that rising and rejuvenation.The treatment of breast cancer is a kind of complex treatment, including hand
The multiple means such as art, radiotherapy, chemotherapy, endocrine therapy, Biological target therapy.
A kind for the treatment of means that breast cancer targeted therapy is taken primarily directed to HER-2 gene high expression.HER-2 gene
The detection method of state is made a definite diagnosis clinically using IHC as prescreening method, for the suggestion of 2+ sample using FISH method.But it is same
Sample there is also it is equivocal, be difficult to the problem of interpreting.ASCO/CAP breast cancer HER2 guide 2018 editions further update,
For the refinement of " HER2 is uncertain " various situations, the diagnosis of " uncertain " is eliminated, in clinical detection definitely.So
And usually using No. 17 centromeric probes as internal control probe in clinical diagnosis, pass through gene/No. 17 HER-2 average signal ratio
Value determines the HER-2 gene with the presence or absence of abnormal.This judgment method is not easy to assess whether by more bodies or X chromosome centric
False negative result caused by amplification makes patient miss best medication time.In addition, currently on the market for detecting patient HER-2
The product of gene expression dose takes long time (about 6 hours), so that patient waits the time of diagnostic result to extend.
Summary of the invention
Based on the above issues, provided it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art it is a kind of can be fast
Speed and the method that accurately detects patient's HER-2 gene magnification.
To achieve the above object, the technical solution that the present invention takes includes the following aspects:
In the first aspect, the present invention provides the probe groups for detecting HER-2 gene magnification level, the probe groups
Including targeting the probe of HER-2 gene order and referring to probe, described includes the probe of targeting RAI1 gene order referring to probe
And/or the probe of CSP17 gene order, wherein it is described targeting HER-2 gene order probe and be all made of referring to probe
Phi29 archaeal dna polymerase carries out probe label.It should be noted that for the first time phi29 archaeal dna polymerase is used to visit in the present invention
Needle label, establishes phi29 DNA random primer labelling method.
Preferably, the probe referring to probe and targeting CSP17 gene order that probe is targeting RAI1 gene order；
The probe for targeting RAI1 gene order as a result, can make up the deficiency for targeting the probe of CSP17 gene order, thus accurate quantification
The level of amplification of HER-2 gene in sample.Preferably, when the probe groups carry out probe label, using random hexamers
It is expanded.
Preferably, when the probe groups carry out probe label, amplification temperature is 30 DEG C, and proliferation time is 12 hours.This Shen
Inventor please has found through test of many times, is expanded when amplification using random hexamers, and amplification temperature is 30 DEG C, expands
Increasing the time is 12 hours, is expanded compared to random eight mer primer, random ten mer primer, expanding effect is best, the yield of amplification
Highest.
In the second aspect, the present invention provides the kit for detecting HER-2 gene magnification level, the kits
Including above-mentioned probe groups.
Preferably, the kit further includes hybridization buffer, includes two as promotor in the hybridization buffer
Methyl sulfoxide.Present inventor has found through test of many times, when using dimethyl sulfoxide as promotor in hybridization buffer,
The better effect of hybridization, background is weaker and hybridization signal is brighter.Preferably, the temperature of hybridization is 45 DEG C.
Preferably, in the hybridization buffer containing mass percentage be 15%~50% formamide, sulfuric acid Portugal it is poly-
20~30uL/700uL of sugar 0.2~0.25g/700uL and dimethyl sulfoxide.Present inventor has found through test of many times,
In hybridization buffer containing mass percentage be 15%~50% formamide, 0.2~0.25g/700uL of dextran sulfate with
And when 20~30uL/700uL of dimethyl sulfoxide, the crossbreeding effect of buffer (comprehensively considers) phase from signal, background, hybridization time
It is more preferable than the formamide, dextran sulfate and dimethyl sulfoxide of other contents.
Preferably, the kit further includes the placenta dna for blocking non-specific hybridization.
In the third aspect, the present invention provides the probe groups stated or kit to screen or prepare in anti-breast cancer medicines
Application.Preferably, when the probe of targeting GSP HER-2, GSP RAI1 and CSP 17 are used to detect tissue samples in the present invention,
When the cell proportion of probe signals exception is 3% or more, that is, it can determine whether tissue samples for the positive.
In conclusion the invention has the benefit that
The present invention provides the kits for detecting HER-2 gene magnification level, realize the accurate interpretation of HER-2 amplification
And tissue samples and the probe quick hybridization in 2 hours；Existing method can be assisted more using detection kit of the invention
Carry out fastly tumour it is accurate layering, accurate diagnosing and treating.
Detailed description of the invention
Fig. 1 is the pattern diagram of middle probe of the present invention；
Fig. 2 is the testing result figure using HER-2 gene magnification detection kit, wherein dotted arrow indicates GSP
HER-2 probe (red) hybridization location, solid arrow indicate the hybridization location of CSP 17 (green) probe；White edge line indicates GSP
HER-2 amplified signal area；
Fig. 3 is the testing result figure using RAI1 gene detecting kit, wherein solid arrow indicates GSP RAI1 probe
(green) hybridization location；The results of hybridization of sample and 17 probe of CSP is not shown；
Fig. 4 is the testing result figure using HER-2/RAI1 gene detecting kit, wherein dotted arrow indicates GSP
The hybridization location of HER-2 (red) probe, solid arrow indicate the hybridization location of CSP 17 (green) probe；Sample and CSP 17
The testing result of probe is not shown.
Specific embodiment
For kit for detecting HER-2 gene magnification level of the invention by optimization of C/C composites, realization 1~2 hour fast
Speed detection HER-2 gene magnification is horizontal, is conducive to optimize diagnosis and treatment structure, reduction of patient waiting time.
In some embodiments, the present invention provides a kind of HER-2 (17q12)/RAI1 (17p11)/CSP17 detection reagents
Box, wherein HER-2 is that the targeted drugs such as Herceptin are benefited from when there is HER-2 gene magnification in targeting medication site；
RAI1 and/or No. 17 centromeric probe as reference, by calculate HER-2 probe signals quantity with RAI1 and/or No. 17 silk
The ratio of grain probe signals quantity, carries out the accurate judgement of HER-2 gene magnification level.Using detection kit energy of the invention
Enough auxiliary existing method quickly carries out the accurate layering of tumour, accurate diagnosing and treating.
In some embodiments, the present invention provides the probe groups for detecting HER-2 gene magnification level, it includes 3
Probe targets 3 gene order sections, respectively HER-2, RAI1 and No. 17 chromosome centromeres on human genome respectively
Area, the design process of probe include: that BAC Immune Clone Selection → purchase (commercialization) → label, test → confirmation probing pin clone group (can
For subsequent production).
In some embodiments, the present invention provides the preparation methods of above-mentioned probe, comprising: probe label (usesphi29 Archaeal dna polymerase label) → quantitative (point sample amount, the length of # probe can all influence crossbreeding effect) → packaging/stand-by.
In some embodiments, the present invention provides the optimization process of probe hybrid system, hybridization promoter is related generally to
Selection, reduce hybridization time, to realize quick hybridization.Specific optimization process is as follows:
(1) the choice of accelerator:
In hybrid process, the formation of base stacking force and hydrogen bond can be influenced by increasing denaturant, increases the hydrophobic of water
Property and reduce the capacity volume variance between double-strand and single stranded DNA, reduce Tm value.It at present include formamide in conventional hybridization buffer,
Hybridization temperature is at 37 DEG C.In addition, comprising dextran sulfate can make contact surface with adsorption of DNA probe molecule because of its microparticle surfaces
Product increases, and advantageous hybridization carries out.There is the nucleic acid hybridization promoter type of record more, but main in fluorescence in situ hybridization technique
Using is dextran sulfate and formamide, also certain basic demand for completing FISH, but by the longer shadow of hybridization time
It rings, limits its clinical application.This case tests its influence to hybridization by other inert polymers of screening or chemical reagent,
Filter out the hybridization promoter for being beneficial to shorten hybridization time；
(2) accelerator dosage adjusts:
When in view of carrying out hybridization in 6 hours and 16 hours, hybrid context has regional raising.Hybridization Buffer formula of liquid is adjusted,
It is wherein matched by optimization, including formamide content, dextran sulfate dosage, sodium salt content, DMSO dosage etc., reduces background,
Improve signal-to-noise ratio.The results show that can be improved luminance signals, but as hybridization time is prolonged when dextran sulfate content increases
It is long, hybrid context may be will increase；When formamide dosage reduces, with the amount for increasing DMSO, hybridization signal can make up for it, it is full
When being expected to require enough, but being lower than 15%, crossbreeding effect in short-term will affect.Thereby determine that formamide additive amount 15%~50%,
0.2~0.25g/700uL of dextran sulfate, 20~30uL/700uL of dimethyl sulfoxide.
In some embodiments, the hybridization conditions of sample and probe are optimized in the present invention, and in particular to such as the following table 1
Parameter:
The optimization of 1 hybridization conditions of table
| Optimal conditions | Parameter |
| Hybridization temperature | 37、42、45℃ |
| Denaturation temperature | 78℃、82℃、85℃、90℃ |
| Hybridization time | 1 hour, 2 hours, 16 hours |
The single factor experiment and combination comparative test of parameter are carried out by the hybridization conditions to table 1,85 DEG C of discovery denaturation is miscellaneous
45 DEG C are handed over, the quick hybridization of probe and sample to be tested of the invention may be implemented for 2 hours in hybridization.
In some embodiments, the present invention provides probe groups Quality Control and the methods for establishing threshold value, including use people periphery
Blood culture cell is for sensitivity and specificity assessment (conventional method), and then judgment threshold when establishing probe in detecting sample,
And the using effect of application sample of bone marrow assessment probe (matrix samples sources are in clinic).In some embodiments, in order to right
The kit of detection HER-2 gene magnification level of the invention carries out Clinical application evaluation, and 20 or so samples is selected to be somebody's turn to do
The application assessment of kit, and result interpretation is carried out using the threshold value that above-described embodiment is established.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.Unless otherwise instructed, the test method in the application is conventional method；Unless otherwise instructed,
Reagent concentration in the present invention is mass concentration.
The design of 1 probe of embodiment and preparation
A kind of embodiment of probe groups for detecting HER-2 gene magnification level of the invention, including 3 probes, point
It Ba Xiang not 3 sequence sections, respectively HER-2, RAI1 and No. 17 X chromosome centrics on human genome.
1) probe design process includes:
BAC Immune Clone Selection → purchase (commercialization) → label, test → confirmation probing pin clone group (can be used for subsequent production).
BAC colony screening method is referring to p26-28 such as " Medical Genetics database resource utilizes example study course " Zhao Jia.
BAC Immune Clone Selection (referring to table 2): gene loci probe is typically designed the probe comprising the gene and is detected.
Probing pin clone selection prepares probe by the clone that verifying selection fluorescence intensity is met the requirements it is noted that the specificity cloned.For
Guarantee luminance signals, probe length can be appropriately extended, by the detection of clinical sample, meet the requirement of accuracy in detection.
The selection of the corresponding BAC clone of the middle probe group of the present invention of table 2
It should be noted that the combination of BAC clone can be changeable, as long as meeting sensitivity and specificity after a test
It is required that.Sensitivity refers to that detection probe is located at corresponding position, is not in the signal (non-specific binding) of non-binding target spot, special
When the opposite sex refers to probe for pattern detection, corresponding positive and negative can be distinguished very well.Two schemes are provided in table 2, that is, are embodied
The flexibility of Immune Clone Selection.The clone of two schemes selection can achieve the desired purpose after label.
2) prepared by probe
The preparation process of above-mentioned probe is related to: probe marks (marking using phi29 archaeal dna polymerase) → quantitative (# probe
Point sample amount, length can all influence crossbreeding effect) → packaging/stand-by.
The present embodiment is basic template with BAC clone, using phi29 archaeal dna polymerase, using random hexamers, often
The lower isothermal duplication of temperature.The label of gene probe carries out room temperature label using phi29 archaeal dna polymerase.Phi29 archaeal dna polymerase is
The mesophilic archaeal dna polymerase cloned from bacteriophage phi29 has 3' → 5' excision enzyme proofreading ability, and with special
Multiple displacement and continuous composite character.It is expanded in label using short random hexamers, expands 30 DEG C of temperature, label
Time is 12 hours.
Random hexamers contain the random sequence of 6 bases.The end 5` has phosphate, and the end 3` is hydroxyl.Sequence
Be classified as 5`-P-d (NNNNNN) -3`, N=G A C T.The combining form of random primer is more, and therefore, specificity is lower, but for
It is long or preferable with secondary structure region template expanding effect.When probe marks, because the cloned template of selection removes carrier sequence
(carrier sequence is non-human genome) outside, remaining sequence is the special complementary segment of target region.Labeling process need in amplification anamorphic zone
The sequence of fluorescent marker.To improve amplification efficiency, random hexamers, random eight mer primer, random ten have been used respectively
Mer primer is tested, and the expanding effect of random hexamers is best as the result is shown, yield highest.
Probe label is carried out using the label system optimized, the usage amount of plasmid mixture is respectively 1ug, fluorescein
The dosage of dUTP/dCTP is as shown in table 3 below, and wherein internal reference probe, probe HER-2 and RAI1 to be checked are respectively labeled as cyan, red
Color and green.
3 probe of table marks system *
| Ingredient names | Dosage |
| dATP(1mM) | 1 |
| dGTP(1mM) | 1 |
| dTTP(1mM) | 0.4 |
| dCTP(1mM) | 0.7 |
| Spectrum-O or G-dUTP | 0.6 |
| Spectrum-O or G-dCTP | 0.3 |
| NNNNNN(10pmol/μL) | 2 |
| Plasmid DNA (200ng/ μ L) | 1 |
| Water | Supply 10 (uL) |
* cyan probe is marked using DEAC-dUTP, and the ratio with dTTP is 0.5:(0.4~0.6).
Prepare the system of table 3 as above, then sequentially add in the above solution 10 × Reaction b μ ffer, 2 μ L,
Phi29 archaeal dna polymerase (10U/ μ L) 4.0~6.0 μ L, 1 Tween20 μ L, H2O supplies 20 μ L.With complete rear mixing, 30 DEG C of labels
12 hours, 65 DEG C of 10 minutes inactivators.It takes 20 μ L marked products that 1.5mL centrifuge tube is added, then sequentially adds 3mol/L acetic acid
50 μ l of 2 μ l of sodium and dehydrated alcohol, mixing are placed in -70 DEG C of refrigerators at least 2 hours, and 4 DEG C of 13000rpm are centrifuged 30 minutes, are gone
Clearly；70% ethyl alcohol of 500 μ l is added, 4 DEG C 13000 revs/min are centrifuged 15 minutes, remove supernatant, are protected from light drying.
It is dissolved and is precipitated using 100 μ L purified waters.Dissolved product is placed in Covaris M220 ultrasonic wave DNA to be crushed
Instrument, 20 DEG C are handled 40 seconds.After completing DNA fragmentation, product is taken out, it is pure using Ampure XP purifying magnetic bead according to 1:1.8 ratio
Change product, is finally dissolved in 20 μ L purified waters to get the probe after label of the invention.1 μ l is taken to use 2% Ago-Gel electricity
Swimming, it is desirable that there are dispersion platings in 200bp or so.
2 probe hybrid system of embodiment
The probe according to the form below 4 that embodiment 1 has marked carries out hybridization system preparation:
Table 4 hybridizes system
| Composition | Every test |
| Green probe (50ng/ μ l) | 0.3~0.6 μ l |
| Red probe (50ng/ μ l) | 0.3~0.5 μ l |
| Cyan probe (50ng/ μ l) | 0.2~0.4 μ l |
| Hybridization buffer | 7μl |
| COT Human DNA(1mg/ml)* | 1μl |
| H2O | It mends to 10 μ l |
* COT Human DNA is placenta dna, and length is mainly 50 to 300bp, and is rich in duplicate DNA sequence dna, than
As Alu and Kpn family member inhibits reiterated DNA sequences commonly used in blocking non-specific hybridization.
The optimization of 3 probe hybrid system of embodiment
The use of hybridization promoter is related generally to, hybridization time is reduced, realizes quick hybridization.
Optimizing each formula, detailed process is as follows:
(1) the choice of accelerator
In hybrid process, the formation of base stacking force and hydrogen bond can be influenced by increasing denaturant, increases the hydrophobic of water
Property and reduce the capacity volume variance between double-strand and single stranded DNA, reduce Tm value.It at present include formamide in conventional hybridization buffer,
Hybridization temperature is at 37 DEG C.In addition, comprising dextran sulfate can make contact surface with adsorption of DNA probe molecule because of its microparticle surfaces
Product increases, and advantageous hybridization carries out.There is the nucleic acid hybridization promoter type of record more, but main in fluorescence in situ hybridization technique
Using is dextran sulfate and formamide, is also able to satisfy the basic demand of FISH really, but longer by hybridization time, is usually needed
10~16 hours are wanted, its clinical application is limited.
The present embodiment is tested its influence to hybridization, has been filtered out by screening other inert polymers or chemical reagent
Beneficial to shortening the hybridization promoter of hybridization time (referring to table 5).Test probe is uniformly selected as 500bp or less mixtures of nucleic acids.
The formula of 5 hybridization buffer of table
The formula of 6 hybridization solution of table
* B500 refers to that probe fragment is 500bp mixtures of nucleic acids below.
Using A2~E2 in table 6 as hybridization system, human peripheral culture cell is sample, is hybridized.Hybridization time is set
Fixed 3 time points, respectively 1 hour, 2 hours, 6 hours and 16 hours.DAPI is redyed after the completion of hybridization, assessment hybridization letter under mirror
Number, background.Statistical result is as shown in Table 7, and A-Z and B-Z, that is, ethylene carbonate and dimethyl sulfoxide have preferable hybridization
Effect.In view of ethylene carbonate had had clinical application, select dimethyl sulfoxide (DMSO) as subsequent research and development test
Emphasis.
7 results of hybridization of table statistics
(2) accelerator dosage adjusts
When carrying out hybridization in 6 hours and 16 hours in view of the B1 formula in table 8, hybrid context has regional raising.It adjusts miscellaneous
Buffer formulation is handed over, is wherein matched by optimization, including formamide content, dextran sulfate dosage, sodium salt content, DMSO dosage
Deng, reduction background, improvement signal-to-noise ratio.
The hybridization buffer of 8 different formulations of table
8 example of table as above distinguishes preparing hybrid liquid.Using human peripheral culture cell as sample, hybridized.Hybridization time
Set 3 time points, respectively 1 hour, 2 hours, 6 hours and 16 hours.DAPI is redyed after the completion of hybridization, and hybridization is assessed under mirror
Signal, background, statistical result are as shown in table 9.
9 results of hybridization of table statistics
As a result as shown in Table 9, when dextran sulfate content increases, it can be improved luminance signals, but with hybridization time
Extend, hybrid context may be will increase；When formamide dosage reduces, with the amount for increasing DMSO, hybridization signal can make up for it,
When meeting expected requirement, but being lower than 15%, crossbreeding effect in short-term will affect.Thereby determine that formamide additive amount in hybridization buffer
In 15%~50%, 0.2~0.25g/700uL of dextran sulfate, 20~30uL/700uL of dimethyl sulfoxide.
(3) sample hybridization conditions optimize: carrying out the single factor experiment of parameter by the hybridization conditions to table 1 and combination compares
Test, 85 DEG C of discovery denaturation hybridize 45 DEG C, and the quick hybridization of probe and sample to be tested of the invention may be implemented for 2 hours in hybridization.
4 probe Quality Control of embodiment and threshold value are established
The present embodiment assesses the sensitivity and specificity of the probe of embodiment 1 using human peripheral culture cell, and establishes
The judgment thresholds of probe groups of the invention when detecting sample.(matrix samples sources are in facing using sample of bone marrow for the present embodiment
Bed) assessment embodiment 1 probe groups using effect.Specific processing method includes the following steps:
4.1 film-making
It takes 2~3ml of marrow (heparin sodium is anticoagulant) 2000rpm to be centrifuged 5 minutes, removes most of supernatant；
Take 1ml cell that the 0.075M KCl of 10ml is added, piping and druming mixes, and stands 2 minutes；
37 ± 1 DEG C of thermostat water baths hypotonic 20 minutes；
Add Ka Nuoshi fixer 1ml, piping and druming mixes, and room temperature pre-fixes 10 minutes；
Piping and druming mixes, and 2000rpm is centrifuged 5 minutes；
Supernatant is removed, precipitating is fixed liquid 10ml, and piping and druming mixes, and -20 DEG C stand 30 minutes；
2000rpm is centrifuged 5 minutes, removes supernatant；
Appropriate fixer is added, cell is resuspended, 3 μ l suspensions is taken to be added drop-wise on clean glass slide；
4.2 pretreatments/denaturation hybridization
The slide dripped is placed in room temperature 2 × SSC solution and is impregnated 2 minutes；
It successively impregnates 2 minutes and is dehydrated in the ethyl alcohol of room temperature 70%, 90%, 100%；Slide is then taken out, room temperature is dried in the air
It is dry；
The sample chips handled well are stand-by.
Take out the hybridization solution comprising probe, balance to room temperature；
Hybridization solution drops to coverslip, then inverts sample chips, gently closes after alignment.Light pressure is uniformly distributed probe, keeps away
Exempt to generate bubble；
Wet in situ hybridization instrument humidity item, slide is placed in situ hybridization instrument, closes in situ hybridization instrument lid, setting
" Denat&Hyb " program is denaturalized 78 ± 1 DEG C 2 minutes, and 37 ± 1 DEG C of hybridization (if amixia instrument, can be used substitution instrument in 1~16 hour
Device, if Thermostatic platform is denaturalized, Electric heat oven/or water-bath are hybridized, and should be noted that temperature is accurate and keeps hybridization wet
Degree).
4.3 washings are redyed
Hybridization instrument power supply is closed, slide is taken out, removes coverslip；
● sample chips are put into 72 ± 1 DEG C of 0.3%NP-40/SSC 2 minutes；
● it takes out, puts it into room temperature 0.1%NP-40/2 × SSC 30 seconds；
● it takes out, then puts it into each 2 minutes in 70%, 90%, 100% ethyl alcohol of room temperature and be dehydrated；
● it takes out, dark place spontaneously dries slide；
Room temperature, is added dropwise 10 μ lDAPI counterstains, and dark place storage is to be seen.
Testing result shows that probe specificity prepared by embodiment 1 is 100%, and sensitivity 100% can satisfy pre-
Phase, in the detection of sample of bone marrow, the detection signal of probe is bright.
When three kinds of probes mark different colours, signal type is as shown in the following table 10:
10 signal probe type of table
| Metaphase chromosome | Sample of bone marrow | |
| HER-2 | Two danger signals (2R) | Two danger signals (2R) |
| RAI1 | Two greens (2G) | Two greens (2G) |
| 17 | Two cyan signals (2A) | Two cyan signals (2A) |
* R shows danger signal；G shows green；A shows cyan signal.
100 bone marrow cells are counted, typical abnormal and normal cell ratio is calculated separately, calculates abnormal cell average value,
Standard deviation is calculated, using average value ± 3SD as threshold range (referring to table 11).
The corresponding threshold value of 11 probe in detecting of table
| Average value | Standard deviation SD | 3±SD | |
| HER-2 | 0.500 | 0.688 | 3% |
| RAI1 | 0.550 | 0.759 | 3% |
| 17 | 0.800 | 0.768 | 3% |
By the detection to negative sample, confirm the threshold value of three targets 3%.Believe when occurring >=3 in pattern detection
Number abnormal cell number > 3% when, assess probe ratio, carry out the judgement of sample positive and negative；When the abnormal cell number of >=3 signals
When < 3%, the appearance of abnormal signal may be related with random signal fracture or nucleus stereochemical structure or non-specific impurity absorption,
It is not calculated as the positive.
5 gene detecting kit of embodiment
Gene detecting kit of the invention includes hybridization buffer and probe, and the kit for packaging, explanation
Book.Wherein, formamide (additive amount is 15%~50%), 0.2~0.25g/ of dextran sulfate are contained in hybridization buffer
700uL, 20~30uL/700uL of dimethyl sulfoxide and sodium citrate buffer solution (2 × SSC).
According to purpose difference is used, the kit of three types can be formed, comprising:
(1) HER-2 gene magnification detection kit, for HER-2 gene magnification detect, comprising targeting GSP HER-2 and
The probe of CSP 17, the preparation method for being all made of embodiment 1 carry out the label of probe；Using HER-2 gene magnification detection reagent
Box detects the result figure of sample referring to fig. 2, and left figure is people's Peripheral blood culture cell, including metaphase chromosome and Interphase cells,
Middle dotted arrow is HER-2 gene signal, and solid arrow show No. 17 centromere signals；Right figure is breast cancer tissue FFPE sample
This, wherein white edge part is irised out as HER-2 gene signal, and arrow show No. 17 centromere signals, as can be seen from Figure 2 probe
Hybridization signal brightness it is high, be easily recognized；
(2) RAI1 gene detecting kit is detected for RAI1 gene unconventionality (amplification/missing), includes targeting GSP
The probe of RAI1 and CSP 17, probe preparation method is referring to embodiment 1；Using the knot of RAI1 gene detecting kit detection sample
Fruit figure illustrates human peripheral culture cell detection results referring to Fig. 3, and solid arrow show RAI1 gene signal, can be with from Fig. 3
Find out that the hybridization signal of probe is very bright, it is readily identified；
(3) HER-2/RAI1 gene detecting kit is used for the doubtful positive case of HER2 result in conjunction with RAI1 amplification situation
Detection, including targeting GSP HER-2, GSP RAI1 and CSP 17 probe, probe preparation method is referring to embodiment 1；Using
HER-2/RAI1 gene detecting kit detects the result figure of sample referring to fig. 4, and left figure is people's Peripheral blood culture cell, including in
Phase chromosome and Interphase cells, right figure are breast cancer tissue FFPE sample, and wherein dotted arrow is RAI1 gene signal, solid line arrow
Head show No. 17 centromere signals, and probe hybridization location signal compares the cell that surrounding does not hybridize, letter as can be seen from Figure 4
Number brightness contrast is obvious, easily distinguishable.
Probe marker color is general preferred red, green, green；General centromeric probe is labeled as green or cyan；Gene probe
Labeled as red.The label combination selected in the present invention is merely illustrative, is not limited to this label.
6 Clinical application evaluation of embodiment
Selected sample defines the sample for being judged as that ISH is suspicious according to 2018CAP HER-2[1]It is suspicious is defined as: each cell
Interior HER2 copy number Zhi≤4.0 Ping Jun and < 6.0, and the infiltrating carcinoma of ratio < 2.0 HER2/CEP17.
Interpretation is carried out by the research of internal control of RAI1 according to what document referred to[2], standard are as follows:
(1) HER2 is not expanded: ratio < 2.0 HER2/RAI1, and every cell RAI1 average is greater than or equal to 1.5, and 40
Having no more than 16 cells shows in cell is only 1 RAI1 signal；
(2) HER2 is expanded: ratio >=2.0 HER2/RAI1；
(3) HER2 is uncertain: (RAI1 individual cells average has more than less than 1.5, and in 40 cells when RAI1 is lacked
17 cells shows are only 1 RAI1 signal).
20 suspicious samples of HER-2 ISH are selected in the present embodiment, using probe (ideograph such as Fig. 1 institute of embodiment 1
Show) it is detected, RAI1 is added as new internal control gene auxiliary judgment.The abnormal cell number of three targets is all larger than threshold value 3%.
Testing result is as shown in table 12 below.The processing method of 20 suspicious samples of HER-2 ISH, comprises the following specific steps that:
1, slide pre-processes
1.1 slides are put into 65 ± 5 DEG C of insulating boxs or bake piece on heated at constant temperature platform and stay overnight；
1.2 take out slide, put it into room temperature dimethylbenzene (or dimethylbenzene substituting agent), 10 minutes；
1.3 take out slide, put it into another cylinder dimethylbenzene of room temperature (or dimethylbenzene substituting agent), 10 minutes；
1.4 take out slide, then put it into room temperature dehydrated alcohol 10 minutes；
1.5 take out slide, then put it into 100% ethyl alcohol of room temperature, 90% ethyl alcohol, 70% ethyl alcohol each 3 minutes；
1.6 take out slide, then put it into room temperature purified water 3 minutes, get rid of excessive moisture；
1.7 put it into 100 ± 5 DEG C of purified water and boil piece 25 minutes (ensuring that sample areas is not contacted with container)；
1.8 take out slide, and room temperature is dried；
1.9 are added dropwise pepsin working solution (the pepsin work of 200 μ l by the face-up horizontal of slide, in sample areas
Make liquid before use, preheating 10~15 minutes at 37 ± 1 DEG C；The whole tissue parts of covering of being subject to), digestion (can lead to for 5~20 minutes
It crosses trial test and determines enzyme effect)；
1.10 get rid of surplus liquid, and slide is put into 2 × SSC of room temperature 3 minutes；
1.11 take out slide, then are sequentially placed into room temperature 70%, and 90%, 100% graded ethanol is dehydrated each 2 minutes；
1.12 take out slide, and room temperature is dried.
2, sample and the same time variation/hybridization of probe (being protected from light operation)
Probe is taken out in 2.1 from -20 ± 5 DEG C of refrigerators, concussion mixes, brief centrifugation；
The probe of 2.2 plus 10 μ l covers rapidly 22 × 22mm coverslip to hybridising region, and light pressure is uniformly distributed probe,
It avoids generating bubble；
2.3 use rubber glue along coverslip edge mounting, and the position of coverslip and glass slide contact is completely covered；
2.4 are put into slide in hybridization instrument, moisten in situ hybridization instrument humidity item, are inserted into wet bar, cover hybridization instrument upper cover, if
" Denat&Hyb " program is set, is denaturalized 85 DEG C 5 minutes, hybridizes 37 DEG C 10~18 hours, (if amixia instrument, substitution instrument can be used
Device, if Thermostatic platform is denaturalized, Electric heat oven or water-bath are hybridized, and be should be noted that temperature is accurate and are kept hybridizing humidity).
3, it post-hybridization washing and redyes and (is protected from light operation)
3.1 first 30 minutes of washings, 2 × SSC, 0.1%NP-40/2 × SSC are put into 37 ± 1 DEG C of thermostat water bath,
Measurement is to ensure that temperature is suitable；
3.2 close hybridization instrument power supply, and slide is taken out, and gently tear rubber glue off, remove coverslip (if coverslip is difficult to
It removes, can put it into 2 × SSC and rock slightly, so that it falls off)；
3.3 slides are put into 37 ± 1 DEG C of 2 × SSC 10 minutes；
3.4 take out slide, then put it into 37 ± 1 DEG C of 0.1%NP-40/2 × SSC 5 minutes；
3.5 take out slides, 3 minutes in 70% ethyl alcohol of room temperature；
3.6 take out slide, dry in the dark；
3.7 room temperatures are added dropwise on 10 μ l in situ hybridization indigo plants dye dyeing liquor to dry 22 × 22mm coverslip, invert sample
Piece contacts coverslip with the target area of glass slide respectively, gently presses after reversion, avoids generating bubble, store in the dark, wait see
It examines.
The testing result of HER-2 level of amplification in 12 sample of table
Note: * routine interpretation refers to according to guidelines, carries out HER-2 gene appearance according to HER-2/17 signal ratio and sentences
It is disconnected；The new judging result of #, which refers to, carries out the interpretation that HER-2 does not know sample according to HER-2/RAI1 ratio.
Referring to table 12 as a result, doubtful sample positive for HER-2 ISH, which often shows as CSP17 copy number, increases (note
Meaning: it is less often to increase number, such as three-body/tetra- bodies, rather than the amplification of very high or cluster), and this may be with (1) centromere region
Regional area obtain (gain)；(2) No. 17 whole chromosomes increase one/several；Most of (3) No. 17 chromosome
Section obtains；(4) reasons such as HER2 and centromere region coamplification are related.Especially since HER2 gene is in 17q12, from
Centromere region is suffered close, feels that a possibility that coamplification occurs can be bigger.Therefore, selection galianconism is carried out with respect to conservative gene
It detects (internal control/endosmosis), can effectively monitor whether HER-2 gene expands.Think that RAI1 gene occurs in research
The probability of high level amplification is greater than only 1%, and coamplification occurs for only little 0.1% probability and HER2.So relatively
For CSP17, the probability of RAI1 amplification is very low, and can identify ratio caused by coamplification occurs due to CSP17 very well is not
It determines[2].In conclusion 20 ISH HER-2 states are not known in sample, RAI1 is used to excavate 7 amplification sun as internal standard
Property (35%).
Sample, sieve are not known using HER-2 in tri- color probe in detecting breast cancer tissue of HER-2/RAI1/17 in the present embodiment
Select positive (amplification) sample of new HER-2, can gain in targeted therapy breast cancer.
Bibliography:
1、Wolff A C,Hammond M E H,Allison K H,et al.HER2 Testing in Breast
Cancer:American Society of Clinical Oncology/College of American Pathologists
Clinical Practice Guideline Focused Update Summary[J].Journal of oncology
practice,2018,14(7):437；
2、Hui L,Geiersbach K B,Downs-Kelly E,et al.RAI1 alternate probe
identifies additional breast cancer cases as amplified following equivocal
HER2 fluorescence in situ hybridization testing:experience from a national
reference laboratory[J].Archives of pathology&laboratory medicine,2016,141
(2):274-278。
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can with modification or equivalent replacement of the technical solution of the present invention are made, without departing from technical solution of the present invention essence and
Range.
Claims (9)
1. the probe groups for detecting HER-2 gene magnification level, which is characterized in that the probe groups include targeting HER-2 base
Because of the probe of sequence and referring to probe, described referring to probe includes the probe and/or CSP17 gene sequence for targeting RAI1 gene order
The probe of column, wherein the probe of the targeting HER-2 gene order and be all made of phi29DNA polymerase referring to probe and visited
Needle label.
2. probe groups according to claim 1, which is characterized in that the reference probe is the spy for targeting RAI1 gene order
The probe of needle and targeting CSP17 gene order.
3. probe groups according to claim 1, which is characterized in that when the probe groups carry out probe label, using random
Six mer primers are expanded.
4. probe groups according to claim 1, which is characterized in that when the probe groups carry out probe label, expand temperature
It is 30 DEG C, proliferation time is 12 hours.
5. the kit for detecting HER-2 gene magnification level, which is characterized in that the kit includes Claims 1 to 4
Described in any item probe groups.
6. kit according to claim 5, which is characterized in that the kit further includes hybridization buffer, described miscellaneous
It include the dimethyl sulfoxide as promotor in friendship buffer.
7. kit according to claim 6, which is characterized in that be containing mass percentage in the hybridization buffer
15%~50% 20~30uL/700uL of formamide, 0.2~0.25g/700uL of dextran sulfate and dimethyl sulfoxide.
8. kit according to claim 5, which is characterized in that the kit further includes non-specific miscellaneous for blocking
The placenta dna of friendship.
9. the described in any item probe groups of Claims 1 to 4 or the described in any item kits of claim 5~8 in screening or
Prepare the application in anti-breast cancer medicines.
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|---|---|---|---|---|
| CN112795649A (en) * | 2021-01-05 | 2021-05-14 | 武汉友芝友医疗科技股份有限公司 | Probe set for detecting HER2 gene amplification level and application thereof |
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