CN110195106A - For detecting probe groups, kit and its application of PD-L1 gene unconventionality - Google Patents
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- CN110195106A CN110195106A CN201910391638.5A CN201910391638A CN110195106A CN 110195106 A CN110195106 A CN 110195106A CN 201910391638 A CN201910391638 A CN 201910391638A CN 110195106 A CN110195106 A CN 110195106A
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Abstract
The invention discloses a kind of for detecting the probe groups and kit of PD-L1 gene unconventionality, wherein probe groups include PD-L1 gene break probe and PD-L1 gene unconventionality amplification probe, PD-L1 gene break probe is a pair of of probe, target sequence is distributed in the two sides of breaking point, abnormal amplification probe targets the gene coding region PD-L1, and PD-L1 gene break probe and PD-L1 gene unconventionality amplification probe are all made of phi29 archaeal dna polymerase and carry out probe label.Using PD-L1 gene unconventionality quick detection kit of the invention, the expression of PD-L1 gene in tumour cell can be accurately detected；Meanwhile it can accurate interpretation PD-L1 gene unconventionality (fracture, amplification)；Using the quick hybridization system of the kit, hybridizing for tissue samples and detection probe can be quickly completed (in 2 hours)；It can effectively assist existing method to carry out the accurate layering of tumour using kit of the invention, precisely diagnose and predict anti-tumor drug therapeutic effect.
Description
Technical field
The present invention relates to technical field of gene detection, especially a kind of probe groups for detecting PD-L1 gene unconventionality, examination
Agent box and its application.
Background technique
PD-L1 (CD274) wide expression is the ligand of PD-1 in cell surface, and PD-1 is a negativity of CD28 family
Costimulatory molecules are expressed in lymphocyte, especially tumor infiltrating lymphocyte.Studies have shown that PD-L1 optionally exists
The expression of lung carcinoma cell surface height, and specifically bound by the PD-1 on the T cell surface with activation, activate the downstream PD-1/PD-L1
Access transmits negativity adjustment signal, and then leads to the apoptosis for activating T cell and immunocompetent forfeiture.PD-1/PD-L1 access
It is considered as the key molecule for the mediated immunity escape being present in tumor microenvironment, blocks the access that can release tumour cell
Inhibition to T lymphocyte, identification lethal effect of the booster immunization system to tumour.Therefore, the table of tumor cell surface PD-L1
Up to the important outcome prediction marker that may be anti-PD-1/PD-L1 immunization therapy.
Currently, there is two classes to block PD-1/PD-L1 pathway inhibitor, monoclonal antibody for PD-1 and for PD-L1's
Monoclonal antibody.The clinical test carried out late succeeds in NSCLC treatment, the research of multinomial III clinical trial phase
It has been shown that, can be improved advanced NSCLC ORR and OS.FDA in the end of the year 2014 ratify 2 drug nivolumab (BMS-936558) and
Pembrolizumab (MK-3475) (for the monoclonal antibody of PD-1) is used for the immune targeted therapy of advanced melanoma.
2015 have approved the two drugs for the metastatic NSCLC after platinum containing regimens chemotherapy progression of disease【1】.Clinical test at present or
Tumour involved in research has included lung cancer, kidney, gastric cancer, colon cancer, oophoroma, breast cancer, neoplastic hematologic disorder and brain tumor
Deng.
Although the study found that anti-PD-1/PD-L1 drug effect in the patients with lung cancer of part is quite significant, in gland cancer
Effective percentage still less than 50%, about 20% in squamous carcinoma, and medical expense is fairly expensive, therefore needs to select reliable marker to screen
Adaptation population out.In addition, the patient of PD-L1 feminine gender still has partial agonistic PD-1/PD-L1 drug effective, it may be there is also other
For instructing the biomarker of anti-PD-1/PD-L drug therapy.
The gene C D274 of PD-L1 albumen is located at 9p24.1, can be by FISH method to tumour cell chromosome 9p 24.1
Middle related gene copy number is detected, to assess expression of the PD-L1 in tumor tissues.Immunohistochemistry is to examine at present
The main method of PD-L1 protein expression level in tumor tissues is surveyed, the positive rate of pertinent literature report is 32%~60%, due to
Various antibody difference itself with determine that the cutoff value of the PD-L1 positive is different causes testing result inconsistent.
Bibliography:
1, research of the anti-PD-1/PD-L1 immunization therapy outcome prediction marker of the such as Zhao Sha in non-small cell lung cancer into
Open up tumour.
Summary of the invention
Based on the above issues, a kind of energy standard is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
Really detect the kit of PD-L1 gene expression dose and gene unconventionality expression level in tumour cell.
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 a kind of for detecting the probe groups of PD-L1 gene unconventionality, including PD-L1
Gene break probe and PD-L1 gene unconventionality amplification probe, the PD-L1 gene break probe are a pair of of probe, target sequence
The two sides of breaking point are distributed in, the exception amplification probe targets the gene coding region PD-L1, the PD-L1 gene break probe
Phi29DNA polymerase, which is all made of, with PD-L1 gene unconventionality amplification probe carries out probe label.It should be noted that in the present invention
Phi29DNA polymerase is used for probe label for the first time, establishes phi29DNA random primer labelling method.
Preferably, the probe groups further include the internal reference probe for monitoring PD-L1 gene unconventionality level of amplification.
Preferably, the internal reference probe targets No. 9 chromosome centromere nucleotide sequences.No. 9 dyes in the present invention as a result,
Colour solid centromeric probe (i.e. CSP 9), marker color is different from PD-L1 gene unconventionality amplification probe (i.e. GSP PD-L1), leads to
The ratio for crossing calculating GSP PD-L1 (red can be designated as) Yu CSP 9 (green can be designated as), judge PD-L1 whether occur amplification with
The level expanded.
Preferably, it when the probe groups carry out probe label, is expanded using random hexamers, amplification temperature is
30 DEG C, proliferation time is 12 hours.Present inventor has found through test of many times, uses random hexamers when amplification
It is expanded, amplification temperature is 30 DEG C, and proliferation time is 12 hours, is expanded compared to random eight mer primer, random ten mer primer
Increase, expanding effect is best, the yield highest of amplification.
In the second aspect, the present invention provides the kits for detecting PD-L1 gene unconventionality, including above-mentioned probe
Group.
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, 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 above-mentioned probe groups or above-mentioned kit in preparation tumour layering diagnosis
Application in the reagent of agent or/and prediction anti-tumor drug curative effect.Preferably, the tumour is lung cancer, kidney, gastric cancer, colon
Cancer, oophoroma, breast cancer, neoplastic hematologic disorder or brain tumor.
In conclusion the invention has the benefit that
(1) PD-L1 gene unconventionality quick detection kit of the invention is used, PD- in tumour cell can be accurately detected
The expression of L1 gene；Meanwhile it can accurate interpretation PD-L1 gene unconventionality (fracture, amplification)；Using the quick of the kit
Hybridization system can quickly be completed (in 2 hours) hybridizing for tissue samples and detection probe；
(2) existing method can be effectively assisted to carry out the accurate layering of tumour, precisely diagnosis using kit of the invention
With prediction anti-tumor drug therapeutic effect.
Detailed description of the invention
Fig. 1 is middle probe of the present invention and corresponding positional diagram on No. 9 chromosomes；
Fig. 2 is middle probe of the present invention and corresponding positional diagram on No. 9 chromosomes；
Fig. 3 is middle probe of the present invention and corresponding positional diagram on No. 9 chromosomes；
Fig. 4 is middle probe of the present invention and corresponding positional diagram on No. 9 chromosomes；
Fig. 5 is the photo of the testing result of fracture double-color probe group under the microscope, and wherein left figure is people's Peripheral blood culture
Cell, right figure are tissue samples；
Fig. 6 is the photo of the testing result of amplification double-color probe group under the microscope, and wherein left figure is people's Peripheral blood culture
Cell, right figure are tissue samples；
Fig. 7 is the photo of the testing result of four color probe groups under the microscope, wherein solid arrow shows 2RAG fusion letter
Number, dotted arrow shows 2Gold signal.
Specific embodiment
The present invention provides PD-L1 gene unconventionality detection method and kits to use wherein abnormal includes fracture and amplification
Whether PD-L1 gene is abnormal in the accurate interpretation sample of kit energy, and existing method can preferably be assisted to carry out the standard of tumour
Really layering, accurate diagnosing and treating.Detection kit of the invention realizes 1~2 hour quickly detection tissue by optimization of C/C composites
Sample is conducive to optimize diagnosis and treatment structure, reduces patient's waiting time.
In some embodiments, the present invention provides a kind of quick hybridization system, it is directed to the new opplication of DMSO；One
In a little embodiments, the present invention provides a kind of preparation method of PD-L1 gene unconventionality detection probe and a kind of new opplications of enzyme；
In some embodiments, the present invention provides four kinds of detection kits, solve diagnostic requirements before clinical application, and realization is precisely examined
It treats.
In some embodiments, the present invention provides the probe for detecting PD-L1 gene unconventionality, which is used for simultaneously
Amplification and rearrangement detection, include two schemes:
Scheme one, four color probe groups are visited for red, the green probe of breaking point and the amplification expanded extremely for PD-L1
Needle；
Scheme two, dichromatism probe groups, i.e. one group of probe are used for for detecting the rearrangement of PD-L1 gene break, another group of probe
Detect PD-L1 gene magnification.The advantage and disadvantage of two groups of probes are as shown in table 1 below:
The characteristics of 1 two groups of probes of table
In some embodiments, the present invention provides the preparation methods of probe, comprising steps of
(1) probe designs: BAC Immune Clone Selection → purchase (commercialization) → label, test → confirmation probing pin clone group are (available
In subsequent production)；
(2) prepared by probe:
Probe label (usesPhi29DNA polymerase tag(point sample amount, the length of # probe can all influence to hybridize) → quantitative
Effect) → packaging/stand-by；
(3) probe hybrid system optimizes:
It relates generally to the use of hybridization promoter, reduce hybridization time, realize quick hybridization.
In some embodiments, the present invention provides the optimization process of hybridization system, include the following:
(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, can also complete the basic demand of FISH, but is influenced by hybridization time is longer, is limited
Its clinical application.
The present invention tests its influence to results of hybridization, filters out by screening other inert polymers or chemical reagent
It is beneficial to shorten the hybridization promoter of hybridization time.
(2) accelerator dosage adjusts
When in view of carrying out hybridization in 6 hours and 16 hours in some embodiment, 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 results show that can be improved luminance signals when dextran sulfate content increases, but with
Hybridization time extend, hybrid context may be will increase；It, can be more with the amount for increasing DMSO when formamide dosage reduces
Hybridization signal is mended, when meeting expected requirement, but being lower than 15%, will affect crossbreeding effect in short-term.Determine that formamide additive amount exists
15%~50%, 0.2~0.25g/700uL of dextran sulfate, 20~30uL of dimethyl sulfoxide.
(3) sample hybridization conditions optimize
The selection of 2 sample 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 |
Single factor test and combination comparative test are carried out using the corresponding parameter of hybridization conditions each in table 2,85 DEG C of discovery denaturation is miscellaneous
45 DEG C are handed over, hybridizes 2 hours, the quick hybridization of 2 hours probes and sample may be implemented.
In some embodiments, the present invention provides the process that probe Quality Control and threshold value are established, it is related to human peripheral culture
Cell is for sensitivity and specificity assessment (conventional method), to establish threshold value；And sample of bone marrow is commented for using effect
Estimate (matrix samples sources are in clinic) two aspects.
In some embodiments, the present invention provides the kits and preparation method thereof for detecting PD-L1 gene unconventionality.
In some embodiments, the present invention to for detecting PD-L1 gene unconventionality probe and kit carried out clinic
Applicating evaluating has been selected 20 or so samples to carry out the application assessment of probe and kit, and has been tied using the threshold value of foundation
Fruit interpretation.
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 method in the present invention is conventional method.
The preparation of 1 probe of embodiment
(1) probe designs
Comprising 4 probes, it is related to four PD-L1 gene, gene both ends extension sequence, No. 9 chromosome centromeres sections, if
Meter process includes: that BAC Immune Clone Selection → purchase (commercialization) → label, test → confirmation probing pin clone group (can be used for subsequent life
It produces).BAC colony screening (referring to table 3) method is referring to p26- such as " Medical Genetics database resource utilizes example study course " Zhao Jia
28。
The combination of the preferred BAC of table 3 clone
(2) prepared by probe
Preparation flow: probe label (using phi29DNA polymerase tag) → quantitative optimization (point sample amount, length of # probe
Degree can all influence crossbreeding effect) → packaging/stand-by.
1) probe marks: it is basic template with BAC clone, using phi29DNA polymerase, and using random hexamers,
It is expanded under room temperature.The label of gene probe carries out room temperature label using phi29DNA polymerase.Phi29DNA polymerase is from biting
The mesophilic archaeal dna polymerase cloned in thallus phi29.With 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, mark the time
(i.e. PCR expansion time) 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.In probe label, 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
Fluorescent marker sequence.To improve amplification efficiency, random hexamers, random eight mer primer, random ten is used to gather respectively
Body primer is tested (result is referring to table 4), (red with PD-L1 amplification probe (red GSP PD-L1) and PD-L1 fracture probe
Green dichromatism) cloned plasmids are as amplification template, by quantitatively determining amplification yield to products probe after amplification, as the result is shown at random
The expanding effect of six mer primers is best, probe yield highest, compares random eight mer primer, random ten mer primer, at random
At least 110.5%, 125% and 227% has been respectively increased in six mer primers amplification yield.
4 different primers of table amplification yield compares
R is represented labeled as red；G is represented labeled as green；Amp is the abbreviation of amplification amplification, represents amplification
Probe；Ba is the abbreviation of break apart fracture separation, represents fracture probe；Thereafter t is the abbreviation of telomere telomere,
Telomere end of the probe close to gene is represented, c represents the abbreviation in the centromere centrosome, represents centromere of the probe close to gene
End.
2) probe label, the usage amount of plasmid mixture the selection of reagent dosage: are carried out using the label system optimized
The dosage of respectively 1ug, fluorescein dUTP/dCTP are as shown in table 4 below, and wherein internal reference probe is respectively labeled as green with probe to be checked
Color and red, fracture probe both ends are respectively labeled as green and red.
Table 5 marks system
| Ingredient names | Dosage/uL |
| 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 and golden probe are marked using DEAC-dUTP and Spectrum-gold-dUTP respectively, with dTTP's
Molar ratio is respectively 0.5:(0.4~0.6) and 0.4:(0.6~0.8).
Prepare system shown in table 5 as above, then sequentially add in the above solution 10 × Reaction b μ ffer, 2 μ L,
Phi29DNA 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 marked.1 μ l is taken to use 2% agarose gel electrophoresis, it is desirable that
In 200bp or so, there are dispersion platings.
The probe according to the form below 5 marked carries out hybridization system preparation, is several probe combinations forms in the following table 6, if needed
It wants that the label that above-mentioned steps complete probe can be applied respectively.
Table 6 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) (if any) | 0.5~0.8 μ l |
| Golden probe (50ng/ μ l) (if any) | 0.5~0.8 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.
Embodiment 2 hybridizes system optimization
The use for relating generally to hybridization promoter reduces hybridization time, realizes quick hybridization.
The specific optimization process of different condition 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.
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 7).Test probe is uniformly selected as the mixing of 100bp~500bp nucleic acid
Object.
The optimization of promotor in 7 hybridization buffer of table
In order to test the influence of different hybridization promoters, probe is same probe, according to 8 preparing hybrid liquid of table:
Table 8 hybridizes liquid system
Using A2~E2 as hybridization system, human peripheral culture cell is sample, is hybridized.When hybridization time sets 3
Between point, respectively 1 hour, 2 hours, 6 hours and 16 hours.DAPI is redyed after the completion of hybridization, and hybridization signal, background are assessed under mirror
Situation.
The results are shown in Table 9, and A-Z and B-Z, that is, ethylene carbonate and dimethyl sulfoxide have better crossbreeding effect.Consider
There is clinical application to ethylene carbonate, and had selected dimethyl sulfoxide (DMSO) as the emphasis of subsequent research and development test.
9 crossbreeding effect of table statistics
(2) accelerator dosage adjusts
When being formulated progress hybridization in 6 hours and 16 hours in view of B1 (referring to table 10), hybrid context has regional raising.Adjustment
Hybridization Buffer formula of liquid is wherein matched by optimization, including formamide content, dextran sulfate dosage, sodium salt content, DMSO are used
Amount etc. reduces background, improves signal-to-noise ratio.
10 Hybridization Buffer formula of liquid of table
10 example of table as above distinguishes preparing hybrid liquid.Using human peripheral culture cell as sample, hybridized.Hybridization time
Set 4 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, as a result as shown in table 11.
11 results of hybridization of table statistics
As a result table 11 as above is shown, when dextran sulfate content increases, can be improved luminance signals, but when with hybridization
Between extend, hybrid context may be will increase；When formamide dosage reduces, with the amount for increasing DMSO, hybridization letter can make up for it
Number, when meeting expected requirement, but being lower than 15%, it will affect crossbreeding effect in short-term.Thereby determine that: formamide additive amount 15%~
50%, 0.2~0.25g/700uL of dextran sulfate, 20~30uL/700uL of dimethyl sulfoxide.
(3) sample hybridization conditions optimize, wherein the parameter selected is as shown in table 12 below:
The selection of 12 Crossbreeding parameters of table
| Optimal conditions | Parameter |
| Hybridization temperature | 37、42、45℃ |
| Denaturation temperature | 78℃、82℃、85℃、90℃ |
| Hybridization time | 1 hour, 2 hours, 16 hours |
Single factor test and combination experiment are carried out by using the parameter in table 12, finds 85 DEG C of denaturation temperature, hybridization temperature 45
DEG C, the experiment effect of hybridization 2 hours is able to satisfy the demand of accurate detection PD-L1 gene unconventionality.
(4) probe dosage optimization
It include 4 discrepant probes of marker color in probe groups, in order to optimize to obtain optimal reaction system, first single
The amount ranges (referring to table 13) of preresearch estimates single probe in system determine that (i.e. signal reaches 2 for the optimum amount of single parameter
+, background 0 or 1+), it is then carried out respectively by two schemes again reaction system optimization (referring to table 14 and 15).
The amount ranges of 13 probe of table
14 4 color probe (scheme one) of table
Referring to table 14, Red and Green probe all achieves better result, but 2 hours and overnight hybridization between 0.5~0.8
Slightly difference, that is, the probe amount hybridization signal of high concentration is more stable；It can satisfy testing requirements when Aqua probe dosage 0.6, increase
Add dosage to signal without improvement, background is similar；It can satisfy requirement when Gold probe dosage 0.5, partial region has powerful connections when 0.7
Increase situation, signal has section poor when 0.4.Overnight or quick hybridization is unobvious to background influence；Overnight hybridization can improve probe
Signal when low concentration dosage helps to improve luminance signals that is, when signal performance is general by extending hybridization time.COT
Human DNA is placenta dna, and length is mainly 50 to 300bp, and is rich in duplicate DNA sequence dna, such as Alu and Kpn family
Member inhibits reiterated DNA sequences commonly used in blocking non-specific hybridization.The certain improvement background effect of COT-1DNA measurer
Fruit, but continue to increase and obvious optimization is had no to background.1.0uL additive amount is selected in the present embodiment, can preferably inhibit background.It is comprehensive
It is upper described, it selects D formula as optimization formula, prepares PD-L1 hybridization solution (four colors).
15 double-color probe group (scheme two) of table
Referring to table 15, in the double-color probe group of detection of broken, the dosage of R/G probe has preferable noise between 0.6~0.8
Than, and 2 hours or overnight hybridization signal show stabilization；When G probe dosage is reduced to 0.5, the unstability of hybridization can be showed,
Especially performance is obvious in quickly hybridization in short-term.To sum up, GSPPD-L1-ba-t-R and GSP PD-L1-ba-c-G dosage selects
0.6~0.8.It selects B formula as optimization formula, prepares PD-L1 (ba) hybridization solution (double-colored).
The double-color probe group of the detection amplification of table 16
It referring to table 16, detects in the double-color probe group of amplification, the dosage of red R probe has preferable letter between 0.6~0.8
It makes an uproar ratio, and 2 hours or overnight hybridization signal show stabilization, when overnight hybridization, background, which has, slightly to be risen, and does not influence to observe；It is green
The dosage of color G probe has a preferable signal-to-noise ratio between 0.3~0.4, and 2 hours or overnight hybridization signal show stabilization；G probe
When dosage is reduced to 0.2, the unstability of hybridization can be shown, especially performance is obvious in quickly hybridization in short-term.In conclusion
GSP PD-L1-amp-R and CSP 9-G dosage selects 0.6~0.8 and 0.3~0.4 respectively.Therefore, select C formula as optimal
Formula prepares PD-L1 (amp) hybridization solution (double-colored).
Embodiment 3 is used to detect the kit of PD-L1 gene unconventionality
According to purpose difference is used, four different kits are formed, comprising:
(1) PD-L1 Fragmentation Detection Kit (FISH) resets detection, including GSP PD- for PD-L1 gene break
L1break is broken double-color probe, is recorded as PD-L1-ba-t and PD-L1-ba-c.
(2) PD-L1 amplification detection kit (FISH) is detected for PD-L1 gene magnification, including GSP PD-L1 (record
For 9 double-color probe of PD-L1-amp) and CSP.
(3) PD-L1 gene unconventionality detection kit (DC) (FISH) is used for PD-L1 amplification and rearrangement of gene detection reagent
Box, including two groups of probes, one are GSP PD-L1 (being recorded as PD-L1-amp), CSP 9；Secondly disconnected for GSP PD-L1break
Split double-color probe (being recorded as PD-L1-ba-t and PD-L1-ba-c).DC indicates double-color probe.
(4) PD-L1 gene unconventionality detection kit (TC) (FISH) is used for PD-L1 amplification and rearrangement of gene detection reagent
Box, including GSP PD-L1 (being recorded as PD-L1-amp) (blueness), CSP 9 (gold) and GSP PD-L1break are broken double-color probe
(red green) (is recorded as PD-L1-ba-t and PD-L1-ba-c).TC indicates four color probes.
Above-mentioned marker color only makees example, can be adjusted or exchange as needed.
It should be noted that amp is the abbreviation of amplification amplification, amplification probe is represented；Ba is break apart
The abbreviation of fracture separation represents fracture probe；Thereafter t is the abbreviation of telomere telomere, represents end of the probe close to gene
Grain end, c represent the abbreviation in the centromere centrosome, represent centromere end of the probe close to gene.
In addition to above-mentioned probe, the detection kit of the present embodiment further includes hybridization buffer, contains first in hybridization buffer
Amide 15%~50% (W/W), 0.2~0.25g/700uL of dextran sulfate, 20~30uL/700uL of dimethyl sulfoxide and 2
× SSC (sodium citrate buffer solution).
4 probe Quality Control of embodiment and threshold value are established
It is sensitive for probe using human peripheral culture cell in order to find probe (embodiment 2) suitable detection threshold value
Degree and specificity assessment (conventional method)；Using effect of the sample of bone marrow for probe assesses (matrix samples sources are in clinic).
Specific processing step is as follows:
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 l DAPI counterstains, and dark place storage is to be seen.
Testing result shows visible on human peripheral culture cell metaphase chromosome referring to Fig. 5~7 and table 17, Fig. 5 left figure
Double-color probe hybridization is in expected target region, display overlapping fusion signal (solid arrow shows)；The myeloid tissue pattern detection of right figure
It can be seen that double yellow/fusion signal, indicates that PD-L1 gene is unbroken, performance overlapping fusion signal.
Fig. 6 left figure shows that visible double-color probe hybridizes in expected target region on human peripheral culture cell metaphase chromosome,
It is shown as centromere green (dotted arrow shows) and gene danger signal (solid arrow shows)；The myeloid tissue sample of right figure
Visible 2 red 2 green signal is detected, shows that PD-L1 gene does not expand.
Fig. 7 shows four color probe testing results in sample of bone marrow, and wherein solid arrow shows that 2RAG merges signal, empty
Line arrow indicates 2Gold signal, indicates that PD-L1 gene is without exception.
In conclusion detection signal of the probe of embodiment 2 in sample of bone marrow is bright, the detection of scheme one and scheme two
As a result meet expection.
Table 17 marks the testing result of three groups of probes of different colours
In table 17, F indicates red green fusion signal, i.e. shows as fusion signal when R is red and G green is close or is overlapped；
RAG shows the green fusion signal of dark purple, i.e. fusion signal is shown as when R is red, G is green and the green three kinds of color signals of A are close or is overlapped.
It include four probes, respectively PD-L1-amp, CSP 9, PD-L1-ba-t and PD-L1-ba-c in the present embodiment.
Wherein, PD-L1-amp and internal reference probe CSP 9 is used for PD-L1 augmentation detection；PD-L1-ba-t and PD-L1-ba-c is used for PD-
The detection that L1 fracture is reset.Fig. 1 shows position on relationship and corresponding No. 9 chromosomes between above-mentioned probe.
100 people's Peripheral blood culture cells are counted, typical abnormal and normal cell ratio is calculated separately, calculates abnormal cell
Average value calculates standard deviation, using average value ± 3SD as the threshold range of probe groups, as a result as shown in table 18 below, abnormal cell
Ratio is more than threshold value, that is, can determine whether testing result for the positive.
18 threshold value of table
The composition of 5 detection kit of embodiment
Kit for detecting PD-L1 gene unconventionality can be assembled into four class products, include hybridization buffer and spy
Two components of needle, and kit, specification for packaging.The component of four class kits is respectively described below: first kind reagent
Box: corresponding four color probe groups are formed as shown in table 19 and 20.
Table 19
| (uL) | Formula |
| Hybridization buffer | 7 |
| GSP PD-L1-ba-t-R | 0.6 |
| GSP PD-L1-ba-c-G | 0.6 |
| GSP PD-L1-amp-A | 0.8 |
| CSP 9-Gold | 0.5 |
| COT-1DNA | 0.5 |
| Purified water | 0 |
| Total volume | 10uL |
20 kit forms of table (5 person-portions/box)
Second class kit: corresponding double-color probe group, including fracture double-color probe group and amplification double-color probe group, composition
As shown in table 21,22 and 23.
Table 21
| It is broken double-color probe | It forms (uL) |
| Hybridization buffer | 7 |
| GSP PD-L1-ba-t-R | 0.6 |
| GSP PD-L1-ba-c-G | 0.6 |
| COT-1DNA | 0.5 |
| Purified water | 1.3 |
| Total volume | 10uL |
Table 22
23 kit forms of table (5 person-portions/box)
| Composition | Quantity | Loading amount |
| PD-L1 is broken hybridization solution (table 20) | 1 pipe | 50uL/ pipe |
| PD-L1 amplified hybridization liquid (table 21) | 1 pipe | 50uL/ pipe |
| Specification | 1 part | -- |
Third class kit: corresponding fracture double-color probe group is formed as shown in table 24 and 25.
Table 24
| It is broken double-color probe | It forms (uL) |
| Hybridization buffer | 7 |
| GSP PD-L1-ba-t-R | 0.6 |
| GSP PD-L1-ba-c-G | 0.6 |
| COT-1DNA | 0.5 |
| Purified water | 1.3 |
| Total volume | 10uL |
25 kit forms of table (5 person-portions/box)
| Composition | Quantity | Loading amount |
| PD-L1 is broken hybridization solution (table 23) | 1 pipe | 50uL/ pipe |
| Specification | 1 part | -- |
4th class kit: corresponding amplification double-color probe group is formed as shown in table 26 and 27.
Table 26
| Expand double-color probe group | It forms (uL) |
| Hybridization buffer | 7 |
| GSP PD-L1-amp-R | 0.6 |
| CSP 9-G | 0.3 |
| COT-1DNA | 0.5 |
| Purified water | 1.6 |
| Total volume | 10uL |
27 kit forms of table (5 person-portions/box)
6 Clinical application evaluation of embodiment
Application examples one: the application in lung cancer sample
Collect the lung cancer tumor tissue samples of the fixed paraffin embedding of 20 formalin, slice thickness 4um.Use embodiment
Second class " double-color probe group " kit in 5, including fracture double-color probe group and amplification double-color probe group.Respectively in two samples
PD-L1 gene magnification and/or rearrangement are detected in piece.The judgement mark positive as amplification using red green signal ratio >=2.0
Standard, to there is break signal as the judgment criteria for resetting the positive.When sample results judge, to be higher than threshold value (2%) as sun
Property judgement standard.It is judged as negative when abnormal cell ratio is lower than threshold value；Judge when abnormal cell ratio is higher than threshold value
For the positive；In Near Threshold, then increase and count cell to 100, if being still Near Threshold, is illustrated in the result.
50 tumour cells are counted, positive cell number is recorded.
Above-mentioned tumor tissues sample processing method, includes the following steps:
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.
Second class kit includes two groups of double-color probes, and ideograph is as shown in Figures 2 and 3.As shown in Fig. 2, including two spies
Needle, respectively PD-L1-amp (red) and CSP 9 (green)；PD-L1-amp and internal reference probe CSP 9 is for PD-L1 amplification inspection
It surveys；As shown in figure 3, including two probes, respectively PD-L1-ba-t and PD-L1-ba-c, respectively red or green, PD-
The detection that L1-ba-t and PD-L1-ba-c is reset for PD-L1 fracture.Testing result is as shown in table 28.
28 PD-L1 gene unconventionality testing result of table
Wherein, F refers to fusion signal, i.e., red (R) and green (G) signal are adjacent or are overlapped.
Application examples two: the application in Diffuse Large B-Cell Lymphoma
Collect Diffuse Large B-Cell Lymphoma (DLBCL) tumor tissues sample (tool of the fixed paraffin embedding of 20 formalin
The tissue samples processing method of body is referring to application examples one), slice thickness 4um.It uses the first kind in embodiment 5 " four color probe groups "
It is detected, kit is based on FISH method and is detected.When sample results judge, sentenced using being higher than threshold value (2%) as the positive
Disconnected standard.It is judged as negative when abnormal cell ratio is lower than threshold value；It is judged as sun when abnormal cell ratio is higher than threshold value
Property；In Near Threshold, it is proposed that increase and count cell to 100, if being still Near Threshold, be illustrated in the result.It wants
It asks and counts 50 tumour cells, record positive cell number.
First kind kit includes four color probes, and ideograph is as shown in figure 4, Fig. 4 shows the relationship and corresponding 9 between probe
Position on number chromosome.It include four probes, respectively PD-L1-amp, CSP 9, PD-L1-ba-t and PD- in the application example
L1-ba-c；Wherein, PD-L1-amp and internal reference probe CSP 9 is used for PD-L1 augmentation detection；PD-L1-ba-t and PD-L1-ba-c
The detection reset for PD-L1 fracture.Testing result is as shown in table 29.
29 PD-L1 gene unconventionality testing result of table
Wherein, F refers to fusion signal, i.e., red (R) and green (G) signal are adjacent or are overlapped.
In conclusion, for different usage scenarios, kit of the invention can accurately be sentenced by the interpretation of testing result
The exception of disconnected sample PD-L1 gene.
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 PD-L1 gene unconventionality, which is characterized in that including PD-L1 gene break probe and PD-L1 base
Because of abnormal amplification probe, the PD-L1 gene break probe is a pair of of probe, and target sequence is distributed in the two sides of breaking point, institute
The abnormal amplification probe targeting gene coding region PD-L1 is stated, the PD-L1 gene break probe and the amplification of PD-L1 gene unconventionality are visited
Needle is all made of phi29 archaeal dna polymerase and carries out probe label.
2. probe groups according to claim 1, which is characterized in that the probe groups further include for monitoring PD-L1 gene
The internal reference probe of abnormal level of amplification.
3. probe groups according to claim 2, which is characterized in that the internal reference probe targets No. 9 chromosome centromere cores
Nucleotide sequence.
4. 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, and amplification temperature is 30 DEG C, and proliferation time is 12 hours.
5. the kit for detecting PD-L1 gene unconventionality, which is characterized in that the kit includes that Claims 1 to 4 is any
Probe groups described in.
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 are swollen in preparation
Tumor is layered the application in diagnosticum or/and the reagent of prediction anti-tumor drug curative effect.
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