CN107475356A - Osteosarcoma circulating tumor cell identification kit - Google Patents
Osteosarcoma circulating tumor cell identification kit Download PDFInfo
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Abstract
The invention discloses a kind of osteosarcoma circulating tumor cell identification kit, and it includes the capture probe and signal amplifying system for every kind of marker gene mRNA, and the marker gene includes following two class：At least two epithelial cell marker gene in EpCAM, CDH1, KRT8, KRT16, KRT17, KRT18, KRT19, KRT20, at least two bone and flesh tumor metastasis correlation interstitial cell marker gene in MMP2, MMP9, VIM, CDH2, FN1, HIF 1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL.Osteosarcoma circulating tumor cell identification kit of the present invention, osteosarcoma circulating tumor cell can comprehensively be detected, and assess its transfer ability, avoid CTCs causes false negative result between various cancers type due to the difference of some marker gene expressions simultaneously, further improves the accuracy of detection.
Description
Technical field
The invention belongs to biology field, is related to medical science and biotechnology, relates particularly to a kind of osteosarcoma and follows
Ring tumour cell identification kit.
Background technology
Osteosarcoma (osteosarcoma) be also osteogenic sarcoma, is teenager or youngster of the more typical generation below 20 years old
A kind of virgin malignant bone tumor, accounts for the 60% of adolescent primary malignant bone tumor.Osteosarcoma is developed by interstitial cell system
, its grade malignancy is high, and the speed of growth is fast, and along with high-frequency DISTANT METASTASES IN, mushrooming out for tumour is due to tumour
Tumour osteoid tissue and bone tissue are directly or indirectly formed through cartilage phase.Osteosarcoma is still that Children and teenager is pernicious swollen at present
The very high disease of the knurl death rate, its grade malignancy is very high, poor prognosis, can be in occurring lung's transfer in the several months, 3~5 after amputation
Annual survival rate is only 5~20%, but early detection and treatment in time can largely improve the sick survival rate.
Circulating tumor cell (circulating tumor cells, CTCs) is because spontaneous or operation of diagnosis and treatment swells from entity
Knurl focus (primary tumor, transfer stove) comes off, into the general designation of all kinds of tumour cells in peripheral blood, it is now recognized that CTCs is probably
The early stage of metastases.Tumour cell enter Peripheral Circulation during may occur epithelial-mesenchymal transformation (ETM,
Epithelial-mesenchymal Transition), during EMT, in addition to cellular morphology and mobility change,
The expression of cellular gene expression particularly epithelium, interstitial molecular marker and its transcription factor also changes, and occurs EMT's
Stick change, migration and invasive ability enhancing between tumour cell.According to the difference of CTCs antigen markers, CTCs mainly may be used at present
It is divided into epithelium positive markers phenotype (abbreviation epithelial cell type), interstitial positive markers phenotype (abbreviation interstitial cell type) and upper
Skin mixes phenotype (abbreviation epithelial-mesenchymal cellular type) etc. with interstitial, and different type CTCs has different migrations and invasive ability.
Therefore, Patients with Osteosarcoma circulating tumor cell is identified and early diagnosis of the parting to osteosarcoma, treatment is instructed and pre-
It is afterwards etc. significant.
The identification to cancer patient CTCs and parting depend on endothelial cell marker and mesenchymal cell markers at present
Detection, conventional epithelial cell mark mainly includes epithelial cell adhesion molecule (epithelial cell adhesion
Molecule, EpCAM), keratin (keratin, KRT) and E- calcium mucin (E-cadherin) etc., mesenchymal cell markers
Mainly include N- calcium mucin (N-cadherin), vimentin (vimentin), fibronectin (fibronectin) and
EMT associated transcription factors AKT2, ZEB1, ZEB2 and SNAIL etc..According to epithelial cell mark and the table of mesenchymal cell markers
Parting is carried out to patient CTCs up to situation, CTCs transfer ability can be directly assessed from genotyping result.But with other cancer types
Difference, osteosarcoma are the malignant tumours developed by interstitial cell system, inherently express some mesenchymal cell markers.Cause
This, it is impossible to use carries out parting and transfer energy to Patients with Osteosarcoma CTCs with other cancer types identical mesenchymal cell markers
Force estimation.At present, both at home and abroad without the stable product for being adapted to Patients with Osteosarcoma CTCs partings and transfer ability assessment.Cause
This, is badly in need of filtering out the bone and flesh tumor metastasis correlation interstitial cell that can carry out Patients with Osteosarcoma CTCs transfer abilities specific assessment
Mark, and the detection method that a kind of accurate identification for realizing Patients with Osteosarcoma CTCs, parting and transfer ability are assessed is provided.
The patent application for the Application No. 201510919145.6 that we propose early stage, because screening obtains in technical scheme
Mark it is relatively fewer, have impact on the sensitivity and specificity of detection to a certain extent.Need, to technological improvement, to improve it
The sensitivity and specificity of detection.
The content of the invention
It is an object of the invention to provide the high osteosarcoma circulating tumor of a kind of high specificity, high sensitivity, the degree of accuracy is thin
Born of the same parents' identification kit.
Realize that the technical scheme of above-mentioned purpose is as follows.
A kind of osteosarcoma circulating tumor cell identification kit, including for every kind of marker gene mRNA capture probe and
Signal amplifying system, the marker gene mRNA include following two class：Selected from EpCAM, CDH1, KRT8, KRT16, KRT17,
At least two epithelial cell marker gene in KRT18, KRT19, KRT20, selected from MMP2, MMP9, VIM, CDH2, FN1,
At least two bone and flesh tumor metastasis correlation interstitial in HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL is thin
Born of the same parents' marker gene；The signal amplifying system include it is end modified have the amplification probe of fluorophor, or include amplification visit
Pin and the end modified label probe for having fluorophor, the fluorophor of variety classes target gene are different；Wherein,
The capture probe is used to connect marker gene mRNA and amplification probe, and every capture probe holds me from 5 ' ends to 3 '
Base composition be followed successively by：The specific sequence P1 that can be combined with marker gene mRNA to be detected, spacer sequence, P2 sequences
Row, the P2 sequences are in the absence of hairpin structure, dimer are not formed between probe interior and probe, in the absence of mispairing, with P1, P4
The sequence of specific binding is not present between marker gene mRNA, the capture for same category of marker gene mRNA is visited
The P2 sequences of pin are identical；
Base composition of the every amplification probe from 5 ' ends to 3 ' ends is followed successively by：Can be complementary with the P2 sequences of respective capture probe
P3 sequences, spacer sequence, the P4 sequences of pairing；The P4 sequences are in the absence of hairpin structure, between probe interior and probe not
Form dimer, in the absence of the sequence that specific binding is not present between mispairing and P1, P2, P3 and total mRNA；
Every label probe has the P5 sequences with corresponding amplification probe P4 sequence complementary pairings, and end modified has fluorescence
Group, the fluorophor of variety classes marker gene are different.
In one of the embodiments, the marker gene mRNA also includes the mRNA for leucocyte marker gene
One kind, the leucocyte marker gene are CD45, using leucocyte marker gene, help to further discriminate between leucocyte and tumour
Cell, exclude interference of the leucocyte to testing result.
In one of the embodiments, in the capture probe of the epithelial cell marker gene：For the spy of EpCAM genes
2 or more than 2 in SEQ ID NO.1~SEQ ID NO.10 of different in nature sequence P1, for the specificity of CDH1 genes
2 or more than 2 in SEQ ID NO.11~SEQ ID NO.20 of sequence P1, for the specific sequence of KRT8 genes
2 or more than 2 in SEQ ID NO.21~SEQ ID NO.30 of P1, for the specific sequence P1 of KRT16 genes
2 or more than 2 in SEQ ID NO.31~SEQ ID NO.40, for KRT17 bases because specific sequence P1 is selected
2 or more than 2 from SEQ ID NO.41~SEQ ID NO.50, it is selected from for KRT18 gene specific sequence P1
2 or more than 2 in SEQ ID NO.51~SEQ ID NO.60, SEQ is selected from for KRT19 gene specific sequence P1
2 or more than 2 in ID NO.61~SEQ ID NO.70, SEQ ID are selected from for KRT20 gene specific sequence P1
2 in NO.71~SEQ ID NO.80 or more than 2；P2 sequences for the capture probe of epithelial cell marker gene are
SEQ ID NO.201；In the amplification probe of the epithelial cell marker gene, P3 sequences are that SEQ ID NO.204, P4 sequences are
SEQ ID NO.207。
In one of the embodiments, in the capture probe of the bone and flesh tumor metastasis correlation interstitial cell marker gene：Pin
To 2 or more than 2 of the specific sequence P1 of MMP2 genes in SEQ ID NO.81~SEQ ID NO.90, for
2 or more than 2 in SEQ ID NO.91~SEQ ID NO.100 of the specific sequence P1 of MMP9 genes, for VIM
2 or more than 2 in SEQ ID NO.101~SEQ ID NO.110 of the specific sequence P1 of gene, for CDH2 bases
2 or more than 2 in SEQ ID NO.111~SEQ ID NO.120 of the specific sequence P1 of cause, for FN1 genes
2 or more than 2 in SEQ ID NO.121~SEQ ID NO.130 of specific sequence P1, for HIF-1a genes
2 or more than 2 in SEQ ID NO.131~SEQ ID NO.140 of specific sequence P1, for SERPINE1 bases
2 or more than 2 in SEQ ID NO.141~SEQ ID NO.150 of the specific sequence P1 of cause, for SURVIVIN
2 or more than 2 in SEQ ID NO.151~SEQ ID NO.160 of the specific sequence P1 of gene, for TWIST1
2 or more than 2 in SEQ ID NO.161~SEQ ID NO.170 of the specific sequence P1 of gene, for AKT2 bases
2 or more than 2 in SEQ ID NO.171~SEQ ID NO.180 of the specific sequence P1 of cause, for SNAIL bases
2 or more than 2 in SEQ ID NO.181~SEQ ID NO.190 of the specific sequence P1 of cause；Turn for osteosarcoma
The P2 sequences that the capture probe of interstitial cell marker gene is closed in phase shift are SEQ ID NO.202；Between the bone and flesh tumor metastasis correlation
In the amplification probe of cell plastid marker gene, P3 sequences are that SEQ ID NO.205, P4 sequence are SEQ ID NO.208.
In one of the embodiments, in the capture probe of the leucocyte marker gene：For the special of CD45 genes
2 or more than 2 in SEQ ID NO.191~SEQ ID NO.200 of property sequence P1；For leucocyte marker gene
The P2 sequences of capture probe are SEQ ID NO.203；In the amplification probe of the leucocyte marker gene, P3 sequences are SEQ ID
NO.206, P4 sequence are SEQ ID NO.209.
In one of the embodiments, the spacer sequence is 5-10 T.
In one of the embodiments, the fluorophor is selected from：FAM、TET、JOE、HEX、Cy3、TAMRA、ROX、
Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488, and for the glimmering of variety classes marker gene
Light group is different.
Main advantages of the present invention are：
(1) different from other types cancer, osteosarcoma is the malignant tumour developed by interstitial cell system, therefore, is used
In carrying out parting and predicting that the mesenchymal cell markers of its metastatic potential can not be used to assess bone to other cancer types CTCs
Sarcoma CTCs transfer ability.The present invention is ground by carrying out many experiments to osteosarcoma primary tumor cell line and metastatic cell strain
Study carefully, with reference to the testing result of clinical sample, in research before, again by comparing and statistical analysis, having filtered out can be with
High specific assesses the mesenchymal cell markers of osteosarcoma CTCs transfer abilities.These bone and flesh tumor metastasis correlation interstitial cell marks
Thing is not expressed on the osteosarcoma CTCs for do not possess metastatic potential or low expression, but the table on metastatic bone sarcoma CTCs
Do not possess the osteosarcoma CTCs of metastatic potential significantly larger than up to amount.By using bone and flesh tumor metastasis correlation interstitial cell mark of the present invention
Will thing, the parting to osteosarcoma CTCs can be not only realized, more further the osteosarcoma CTCs of mesenchymal cell origin can be shifted
Potential is assessed.
(2) the epithelial cell marker gene interstitial cell marker gene related to bone and flesh tumor metastasis selected by the present invention, it is
Inventor carries out comprehensive assessment by lot of experiments, statistical analysis screening draws the specifically expressing on osteosarcoma circulating tumor cell
Gene.The selection of marker gene of the present invention, in addition to it can realize the detection of single marker gene, more with other marker gene one
Rise and use, so as to comprehensively detect osteosarcoma circulating tumor cell, and assess its transfer ability, solve general interstitial
The defects of cell sign thing cannot be used for assessing the osteosarcoma CTCs transfer abilities of mesenchymal cell origin, while avoid CTCs and exist
False negative result is caused due to the difference of some marker gene expressions between various cancers type, and, further carry
The accuracy of high detection.
(3) authentication method of the present invention uses the multiple capture probe for target mRNA, can mark simultaneously a variety of
Osteosarcoma cell marker gene and CTCs specific genes, and by CTCs partings be I types (epitheliated type), (epithelial-mesenchymal mixes II types
Mould assembly) and type III (interstitial type), further increase the accuracy of testing result.Various probes designed by the present invention, can
Hybridization reaction is carried out under homogeneous reaction condition, and non-specific binding is substantially not present between various probes；Designed
Probe in the detection it is specific it is good, signal to noise ratio is high.Meanwhile being applied in combination for a variety of probes makes identification kit and detection method shape
The system intact into a Detection results.The present invention uses more site-specifics of probe to match, the mode of Cascaded amplification is come
The amplification of signal, rather than the method for PCR amplifications are realized, detection signal is improved, realizes the specificity of detection, avoid inverse
Transcribe the false positive of PCR and Real-Time Fluorescent Quantitative PCR Technique.
(4) RNA in-situ hybridization methods have the shortcomings that fluorescence signal sensitivity is low in itself, but the present invention is using new
RNA in-situ hybridization methods, fluorescence signal intensity is improved by signal amplification system.Testing process of the present invention can be complete in 8h
Into the mRNA hybridization probes of single copy combine with corresponding fluorescence probe by signal amplifying system, significantly improve RNA originals
The detection sensitivity of position hybridization.
Brief description of the drawings
Fig. 1 is the positive osteosarcoma CTC qualification result schematic diagrames of the present invention；
Fig. 2 is the positive osteosarcoma CTC genotyping result schematic diagrames of the present invention.
Embodiment
For the ease of understanding the present invention, the present invention will be described more fully below.The present invention can be with many not
With form realize, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is made to this
The understanding of the disclosure of invention more thorough and comprehensive.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook etc.
People, molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) institute in
The condition stated, or according to the condition proposed by manufacturer.Used various conventional chemical reagent, are commercially available in embodiment
Product.
Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention
The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose of example is applied, is not used in the limitation present invention.Term-used in the present invention and/or " include one or more related institutes
The arbitrary and all combination of list of items.
Osteosarcoma circulating tumor cell authentication method of the present invention, is mainly included the following steps that：
(1) obtain removing the biological fluid samples after red blood cell；
(2) filter, osteosarcoma circulating tumor cell is enriched with filter membrane, by being permeabilized, vitellophag, makes mRNA sudden and violent
Dew；
(3) detection epithelial cell marker gene mRNA, and/or bone and flesh tumor metastasis correlation interstitial cell marker gene mRNA is
No presence, the epithelial cell marker gene are selected from：EpCAM、CDH1、KRT8、KRT16、KRT17、KRT18、KRT19、KRT20
In two or more；The bone and flesh tumor metastasis correlation interstitial cell marker gene is selected from：MMP2、MMP9、VIM、CDH2、
Two or more in FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL.
It is thin that the detection epithelial cell marker gene mRNA, and/or bone and flesh tumor metastasis related bone sarcoma shift related interstitial
Born of the same parents' marker gene mRNA whether there is, and comprise the following steps：
(3.1) the capture probe specific sequence P1 of every kind of marker gene combines with corresponding desired mRNA sequences；Every
The base sequence of capture probe from 5 ' end to 3 ' end be followed successively by combined with marker gene mRNA to be detected specific sequence P1,
Spacer sequence, can be with the P2 sequences of the P3 complementary pairings of the amplification probe of respective flag gene；The P2 is in the absence of hair clip
Structure, dimer, special in the absence of being not present between mispairing, with P1, P4 and total mRNA is not formed between probe interior and probe
Property combine sequence；
(3.2) the P2 sequences of capture probe are combined with the P3 sequence-specifics of amplification probe；The amplification probe base sequence
Row are followed successively by from 5 ' ends to 3 ' ends：Can be with P3 sequences, spacer sequence, the sequences of P 4 of capture probe P2 complementary pairings；The P4
Hairpin structure is respectively not present, do not formed between probe interior and probe dimer, in the absence of mispairing, with P1, P2, P3 and total
The sequence of specific binding is not present between mRNA；
(3.3) the P4 sequences of the amplification probe and the P5 sequence-specific knots of the label probe with fluorophor modification
Close, so as to realize the Cascaded amplification of target mRNA signals；The fluorophor of variety classes marker gene is different；
(3.4) detected by fluorescence detector.
It is thin that the detection epithelial cell marker gene mRNA, and/or bone and flesh tumor metastasis related bone sarcoma shift related interstitial
Born of the same parents' marker gene mRNA whether there is, and can also comprise the following steps：
(3.1) the capture probe specific sequence P1 of every kind of marker gene combines with corresponding desired mRNA sequences；Every
The base sequence of capture probe from 5 ' end to 3 ' end be followed successively by combined with marker gene mRNA to be detected specific sequence P1,
Spacer sequence, can be with the P2 sequences of the P3 complementary pairings of the amplification probe of respective flag gene；The P2 is in the absence of hair clip
Structure, dimer, special in the absence of being not present between mispairing, with P1, P4 and total mRNA is not formed between probe interior and probe
Property combine sequence；
(3.2) the P2 sequences of capture probe are combined with the P3 sequences of the amplification probe with fluorophor mark, so as to real
The amplification of existing target mRNA signals；The amplification probe base sequence is followed successively by from 5 ' ends to 3 ' ends：Can be mutual with capture probe P2
Recruit to P3 sequences, spacer sequence, the sequences of P 4,3 ' ends of every amplification probe are also modified with fluorophor, variety classes
The fluorophor of marker gene is different；The P4 is in the absence of hairpin structure, and dimerization is not formed between probe interior and probe
Body, in the absence of between mispairing and P1, P2, P3 and total mRNA be not present specific binding sequence；
(3.3) detected by fluorescence detector.
Embodiment 1
Osteosarcoma circulating tumor cell identification kit described in the present embodiment, there is two types, has label probe
With without label probe.
Wherein, there is the osteosarcoma circulating tumor cell identification kit A of label probe, mainly include：
First, capture probe
Capture probe is made up of three parts, and 5 ' ends to 3 ' are the sequence with the mRNA complementary pairings of corresponding marker gene successively
Arrange P1, spacer sequence, the P2 sequences with corresponding amplification probe P3 sequence complementary pairings, same category of marker gene its catch
The P2 sequences obtained in probe are identical.The spacerarm is for being spaced apart, passing through by capture probe P2 sequences and target mRNA
The spacer sequence of suitable length is set in probe interior, steric hindrance can be reduced, improve efficiency and the hybridization of hybridization reaction
The specificity of reaction.The spacerarm of capture probe of the present invention is preferably 5-10 T, and the present embodiment is preferably 5 T.Each mark
Gene separately designs 10 capture probes, to improve the specificity of detection.(in use, being directed to every kind of target gene, select 2
Or more than 2 capture probes can be completed to detect, specificity and stability are all fine, refer to embodiment 8), the present embodiment is excellent
Elect as and use 10 capture probes, so that specificity reaches best.1 is shown in Table for the capture probe of corresponding marker gene, no
The P2 sequences of the capture probe of the marker gene of same type are shown in Table 2.
The P1 sequences of the target gene capture probe of table 1
The P2 sequences of the marker gene capture probe of table 2
2nd, amplification probe
Amplification probe is to connect the sequence between capture probe and signal detection component, and amplification probe is made up of three parts,
5 ' ends are can be with the P3 sequences of capture probe P2 sequence complementary pairings, and spacer sequence, 3 ' ends are mutually to be recruited with label probe
To P4 sequences (during if do not detected with label probe, then the 3 ' of P4 sequences it is terminal modified have fluorophor, such as kit B), in
Between be that (amplification probe spacerarm of the invention is preferably 5-10 T, the present embodiment for 5 oligonucleotide T spacer sequence
Preferably 5 T).
The P3 sequences of the amplification probe of marker gene are shown in Table 3.Hairpin structure, probe interior is not present in the P4 interior sequences
Dimer is not formed between probe, in the absence of non-specific binding is not present between mispairing, with P1, P2, P3 and total mRNA
Sequence, the preferable base composition of the present embodiment P4 sequences is shown in Table 4.
The P3 sequences of the amplification probe of the target gene of table 3
The P4 sequences of the amplification probe of the target gene of table 4
3rd, label probe
Label probe is made up of two parts, and its 5 ' end is can be with the complementary P5 sequences combined of amplification probe P4 sequences, 3 ' ends
Marked with fluorophor, pass through the Cascaded amplification for being implemented in combination with target mRNA signals with amplification probe P4 sequences.(work as detection
When label probe is used in system, the end of P4 sequences 3 ' of amplification probe marks without fluorophor, and by the 3 ' of label probe
End marks with fluorophor, such as kit A) fluorophor of label probe can be selected from：FAM、TET、JOE、HEX、Cy3、
TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor488, FL1, FL2 of label probe
With the color of the different and selected fluorophor of fluorophor of FL3 selections is different or the mutual not phase of launch wavelength
Together, in order to distinguishing different types of marker gene.The fluorophor mark of the present embodiment label probe is preferably as shown in table 5.
The label probe of table 5
Do not have the osteosarcoma circulating tumor cell identification kit B of label probe described in the present embodiment, mainly include：
First, capture probe：With the capture in the above-mentioned osteosarcoma circulating tumor cell identification kit with label probe
Probe is identical.
2nd, amplification probe：With the amplification in the above-mentioned osteosarcoma circulating tumor cell identification kit with label probe
The sequence of probe is identical, but terminal modified there is fluorophor the 3 ' of P4 sequences.
The P4 sequences 3 ' it is terminal modified have fluorophor, be specially：For epithelial cell marker gene detection probe P4
The fluorophor of sequence modification is FL1, bone and flesh tumor metastasis correlation interstitial cell marker gene detection probe P4 sequence modifications it is glimmering
Light group is FL2, and the fluorophor of leucocyte marker gene detection probe P4 sequence modifications is FL3, and the fluorophor is selected from：
FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor
488, the color of the wherein different and corresponding fluorophor of fluorophor corresponding to FL1, FL2 and FL3 it is different or
Launch wavelength is different, in order to distinguish different types of marker gene.3 ' ends of the P4 sequences of marker gene amplification probe
Fluorophor mark preferably it is as shown in table 6.
The dye marker of the marker gene of table 6
Embodiment 2 detects with the kit A in embodiment 1 to Patients with Osteosarcoma Peripheral Circulation tumour cell
The formula of the various solution is as follows：
Probe mixed liquor, amplification mixed liquor, colour developing mixed liquor in the present embodiment is corresponding using the kit A of embodiment 1
Whole probes in list of genes.
First, sample preprocessing, osteosarcoma CTCs is filtered to filter membrane
1. 600 × g horizontal centrifugal 5min, supernatant is abandoned using liquid preservation blood sample is preserved in Sample preservation pipe, removed
Red blood cell.
2. adding 4ml PBS and 1ml fixatives, it is vortexed and mixes, be stored at room temperature 8min.
3. sample filters：Liquid in Sample preservation pipe is transferred in filter, opens vacuum filtration pumping liquid to the greatest extent；
4ml PBS are added in this preservation pipe, liquid is filtered after washing tube wall.
4. filter membrane is transferred in 24 orifice plates, the formalins of 400 μ l 4% are added, room temperature fixes 1h.
5. removing liquid, 1ml PBS washings are added per hole three times, soak 2min every time.
2nd, it is permeabilized
1. adding 50 μ l permeabilization agent per hole in 24 new orifice plates, filter membrane is taken out from PBS, filter membrane piece EDGE CONTACT is inhaled
Water paper, unnecessary liquid is removed, filter membrane is tipped upside down in permeabilization agent, be i.e. filter membrane iron ring is carved with the one of coding and presses close to liquid downwards
Body.It is incubated at room temperature 5min.
2. removing liquid, 1ml PBS are added per hole and are washed twice, soak 2min every time.Filter membrane is maintained in PBS under
One step experimental implementation.
3rd, vitellophag, exposure mRNA, makes it hybridize with probe
1. prepare the digestive ferment working solution of respective concentration：
| Reagent component | Each sample dosage |
| Digestive ferment | 1.25μl |
| PBS | 48.75μl |
| Cumulative volume | 50μl |
Mix, dispensed into 24 orifice plates 2. digestive ferment working solution is vortexed, per the μ l of hole 50.
3. filter membrane is taken out, back-off is into 24 orifice plates on digestive ferment working solution, and ensureing filter membrane, one side is abundant with liquid downwards
Contact, it is impossible to the presence of bubble.It is stored at room temperature 1h.
4. removing liquid, 1ml PBS washings are added per hole three times, soak 2min every time.Filter membrane is maintained at PBS
In to next step experimental implementation.
4th, probe hybridizes, and probe-specific sequence is combined with desired mRNA sequences
1. probe buffer solution, amplification buffer and colorbuffer are using preceding needing 40 DEG C of water-baths preheating 20min.
2. prepare probe face liquid：
| Reagent component | Each sample dosage |
| Probe mixed liquor | 8μl |
| Probe buffer solution (40 DEG C of preheatings) | 42μl |
| Cumulative volume | 50.0μl |
It is vortexed and mixes, dispense into 24 orifice plates, per the μ l of hole 50.
3. filter membrane is taken out, on back-off to 24 orifice plate middle probe working solutions, ensure that one side fully connects filter membrane with liquid downwards
Touch, it is impossible to the presence of bubble.
4. covering 24 orifice plate lids, 40 ± 1 DEG C are incubated 3 hours.
5. removing liquid, the washing of 1ml cleaning solutions is added per hole three times, soaks 2min every time.Filter membrane is maintained at cleaning solution
In to next step experimental implementation, sample in cleaning solution soak time no more than 30min.
5th, amplified hybridization, the amplification of desired mRNA sequences signal
1. prepare amplifier working solution：
It is vortexed and mixes, dispense into 24 orifice plates, per the μ l of hole 50
2. filter membrane is taken out, back-off on amplifier working solution, ensures that one side fully connects filter membrane with liquid downwards into 24 orifice plates
Touch, it is impossible to the presence of bubble.
3. 24 orifice plate lids are covered, 40 ± 1 DEG C of incubation 30min.
4. removing liquid, the washing of 1ml cleaning solutions is added per hole three times, soaks 2min every time.Filter membrane is maintained at cleaning solution
In to next step experimental implementation, sample in cleaning solution soak time no more than 30min.
6th, develop the color, fluorescence labeling echo signal
1. prepare colour developing working solution
| Reagent component | Each sample dosage |
| Develop the color mixed liquor | 2μl |
| Colorbuffer (40 DEG C of preheatings) | 48μl |
| Cumulative volume | 50.0μl |
Lucifuge, which is vortexed, to be mixed, and is dispensed into 24 orifice plates, per the μ l of hole 50
2. filter membrane is taken out, back-off develops the color on working solution into 24 orifice plates, ensures that one side fully connects filter membrane with liquid downwards
Touch, it is impossible to the presence of bubble.
3. 24 orifice plate lids are covered, 40 ± 1 DEG C of incubation 30min.
4. removing liquid, the washing of 1ml cleaning solutions is added per hole three times, soaks 2min every time.Filter membrane is maintained at cleaning solution
In to next step experimental implementation, sample in cleaning solution soak time no more than 30min.
7th, fluorescence microscope osteosarcoma CTCs
The reference substance of the present invention uses DAPI, and as nucleus fluorophor, it launches blue-fluorescence signal.
1. filter membrane cell is placed on slide up, filter membrane is cut off along iron ring inner ring, adds 10 μ l anti-containing DAPI
Quencher, covers 18mm × 18mm cover glass, direct microscopy or is placed in -20 DEG C of preservations.
2. count CTC opposite sex nuclear volumes by 20 times of object lens.
3. position different in nature nuclear locations according to 10 times of object lens, oil dripping, with oil mirror observation experiment result, and result is photographed to record.
4. and then further according to 10 times of next different in nature nuclear locations of object lens positioning, oil dripping, with oil mirror observation experiment result and regard
Open country photographs to record result.
5. repeating to all different in nature core has been clapped, quantity is consistent with 20 times of object lens count results.
Microscope is as follows using passage：
The excitation wavelength and launch wavelength of the fluorophor of table 7
8th, testing result judges and analyzed
1. positive osteosarcoma CTC standards of perfection
On filter membrane, a small amount of osteosarcoma circulating tumor cell and the leucocyte of residual are enriched with, osteosarcoma circulation is swollen
The positive criterion of oncocyte is (referring to Fig. 1)：
1) there is osteosarcoma circulating tumor cell specific marker's thing, shown as in this kit in Cy3 passages and/or
Red fluorescent point and/or green florescent signal point can be shown under the passages of Alexa Fluor 488.
2) do not have leukocyte specific marker, show as not showing that fluorescence is believed under Cy5 passages in this kit
Number point.
3) nucleus DAPI stained positives.
4) osteosarcoma circulating tumor cell nuclear shape is irregular, and diameter is more than 10 μm, hence it is evident that more than filter sizes, filter membrane hole
Footpath is 7 μm.Leucocyte size is close with filter membrane hole size.
2. positive osteosarcoma CTC parting standards：
This kit uses the multiple capture probe for target mRNA, respectively for a variety of osteosarcoma CTCs specificity bases
Cause, can be further by osteosarcoma CTCs partings by different colours fluorescence signal.It is glimmering that wherein I type (epitheliated type) CTCs carries Cy3
Light group (shown in red fluorescence signal point), III type (interstitial type) CTCs carry the fluorophor (displays of Alexa Fluor 488
For green florescent signal point), at the same express the CTCs of I type and III type specificity gene for II type (epithelial-mesenchymal mixed type, together
When show red fluorescence and green florescent signal point).Referring to Fig. 2.Osteosarcoma CTCs parting standards are as follows：
The positive osteosarcoma CTCs parting standards of table 8
3. using above-mentioned detection method, 20 Patients with Osteosarcoma peripheral blood samples are detected and observed, concrete outcome
For (data are CTC numbers in table)：
The pattern detection result of table 9
| Numbering | Epitheliated type | Epithelial-mesenchymal mixed type | Interstitial type | CTC total amounts |
| 1 | 2 | 4 | 9 | 15 |
| 2 | 1 | 8 | 7 | 16 |
| 3 | 1 | 2 | 4 | 7 |
| 4 | 0 | 5 | 3 | 8 |
| 5 | 2 | 10 | 8 | 20 |
| 6 | 1 | 6 | 4 | 11 |
| 7 | 3 | 11 | 7 | 21 |
| 8 | 0 | 2 | 2 | 4 |
| 9 | 2 | 3 | 5 | 10 |
| 10 | 1 | 7 | 4 | 12 |
| 11 | 1 | 2 | 8 | 11 |
| 12 | 1 | 9 | 5 | 15 |
| 13 | 2 | 10 | 1 | 13 |
| 14 | 1 | 2 | 5 | 8 |
| 15 | 0 | 3 | 6 | 9 |
| 16 | 1 | 7 | 4 | 12 |
| 17 | 1 | 1 | 3 | 5 |
| 18 | 2 | 3 | 3 | 8 |
| 19 | 2 | 16 | 4 | 22 |
| 20 | 1 | 4 | 6 | 11 |
Embodiment 3 detects with the kit A in embodiment 1 to osteosarcoma cell line
First, the selection of cell line
Kit epithelial cell marker gene of the present invention is selected from：EpCAM、CDH1、KRT8、KRT16、KRT17、KRT18、
Two or more in KRT19, KRT20, bone and flesh tumor metastasis correlation interstitial cell marker gene is selected from：MMP2、MMP9、
Two or more in VIM, CDH2, FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL, in vain
Cell sign gene is CD45.Epithelial cell marker gene of the present invention, bone and flesh tumor metastasis correlation interstitial cell marker gene and white
Various marker gene in cell are inventors by lots of comparing experiments and statistical analysis screening draw in osteosarcoma CTCs
The gene of upper specifically expressing, parting can be carried out to the osteosarcoma CTCs of mesenchymal cell origin simultaneously and transfer ability is assessed, had
Good sensitivity, specificity and accuracy.
The present embodiment is tested from osteosarcoma primary tumor cell line SW1353 and metastatic cell strain MG63, negative right
Photo cell is selected good strains in the field for seed with CCRF-HSB-2 lymphoblasts, and those skilled in the art are only it is to be understood that the title of cell line is i.e. commercially available
Arrive.About 1000 SW1353 cells (being determined by cell counter) are taken, sample are divided into 5 parts after being well mixed, numbering 21-
25；About 1000 MG63 cells (being determined by cell counter) are taken, sample are divided into 5 parts after being well mixed, numbering 26-
30；About 1000 CCRF-HSB-2 lymphoblasts (being determined by cell counter) are taken, sample is divided into 5 after being well mixed
Part, numbering 31-35.
2nd, pattern detection
With whole probes (including all marker gene, the epithelial cell marker gene of the kit A in embodiment 1
EpCAM、CDH1、KRT8、KRT16、KRT17、KRT18、KRT19、KRT20；Bone and flesh tumor metastasis correlation interstitial cell marker gene
MMP2, MMP9, VIM, CDH2, FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL), leucocyte mark
Gene C D45, detection process and method detect to sample 21-35 as described in embodiment 2, and reading in each sample has DAPI
50 cells of blue-fluorescence signal, wherein, the cell number in sample is chosen by fluorescence microscope automatically scanning, for mesh
The fluorescence signal intensity of mark detection mark, reads the phosphor dot quantity of the respective color of this 50 cells respectively, and calculates flat
Count, specific testing result is as follows：
The pattern detection result of table 10
The testing result of three kinds of cell lines is compared and analyzed, kit of the present invention can be realized to osteosarcoma cell phase
The detection of marker gene is closed, to osteosarcoma primary tumor cell line SW1353 in sample and metastatic cell strain MG63, CCRF-HSB-
The recall rate of 2 lymphoblasts is 100%, wherein, osteosarcoma correlating markings gene only detects in osteosarcoma cell line
MRNA is expressed, and is not expressed in lymphoblast, it can be seen that, kit of the present invention can detect epithelial cell mark base
Cause, bone and flesh tumor metastasis correlation interstitial cell marker gene and leucocyte marker gene mRNA expressions, meanwhile, epithelial cell mark
The detection of will gene and/or bone and flesh tumor metastasis correlation interstitial cell marker gene has specificity, that is to say, that epithelial cell mark
The detection of will gene and/or bone and flesh tumor metastasis correlation interstitial cell marker gene, can effectively distinguish the osteosarcoma cell in sample
And leucocyte.In addition, the high expression of epithelial cell marker gene (count by average fluorescence signal in primary tumor cell line SW1353
38), the expression quantity of bone and flesh tumor metastasis correlation interstitial cell marker gene is then very low (average fluorescence signal points are 5)；And shift
Property cell line MG63 in the medium expression of epithelial cell marker gene (average fluorescence signal points are 12), between bone and flesh tumor metastasis correlation
The expression quantity of cell plastid marker gene is then significantly larger than primary tumor cell line SW1353 (average fluorescence signal points are 36), explanation
The bone and flesh tumor metastasis correlation mesenchymal cell markers gene that kit of the present invention is selected has specificity well, can specificity
Assess the transfer ability of the osteosarcoma cell of mesenchymal cell origin in ground.
The selection of the variety classes cell sign gene of embodiment 4
First, design (selection of target detection marker gene value volume and range of product) prepared by kit
Kit epithelial cell marker gene of the present invention is selected from：EpCAM、CDH1、KRT8、KRT16、KRT17、KRT18、
KRT19 and KRT20；Bone and flesh tumor metastasis correlation interstitial cell marker gene is：MMP2、MMP9、VIM、CDH2、FN1、HIF-1a、
SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL, corresponding selection is done according to experimental group.
The present embodiment is by taking the selection of bone and flesh tumor metastasis correlation interstitial cell marker gene value volume and range of product as an example, referring to implementation
Group 1-3, chooses a kind of, two kinds, six kinds and a kind of ten marker gene respectively, contrasts its Detection results, and epithelial cell mark
Gene and leucocyte marker gene are as shown in table 11 using whole target genes, specific design.
The composition sum of the capture probe of every group of corresponding marker gene of the present embodiment, amplification probe and label probe
Amount, detection method etc. are as described in the kit A of embodiment 1 and embodiment 2.
The selection of the bone and flesh tumor metastasis correlation interstitial cell marker gene gene of table 11
2nd, pattern detection
The present embodiment is tested from osteosarcoma metastatic cell strain MG63, U2OS and Saos2, those skilled in the art
Only it is to be understood that the title of cell line can be by being commercially available.Take about 1000 MG63 cells (being determined by cell counter) mixed
Sample is divided into 5 parts after closing uniformly, numbering 36-40；About 1000 U2OS cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 41-45；About 1000 Saos2 cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 46-50.
The kit prepared using the marker gene of table 11, detection process and method are entered to sample 36-50 as described in embodiment 2
Row detection, reads 50 cells for having DAPI blue-fluorescence signals in each sample, wherein, the cell number in sample passes through fluorescence
Microscope automatically scanning is chosen, and for the fluorescence signal intensity of target detection mark, it is thin to read this 50 osteosarcoma respectively
The phosphor dot quantity of the respective color of born of the same parents, and averagely counting is calculated, specific testing result is following, and (data are cell number in table 12
Mesh, data are mean fluorecence points in table 13)：
Table 12 uses the comparison of varying number bone and flesh tumor metastasis correlation interstitial cell marker gene Detection results
The comparison that table 13 is counted using varying number bone and flesh tumor metastasis correlation interstitial cell marker gene mean fluorecence
The experimental result that contrast experiment organizes 1-4 is understood, when bone and flesh tumor metastasis correlation interstitial cell marker gene is chosen respectively
A kind of, two kinds, six kinds and ten one kind, when epithelial cell marker gene uses full gene, epithelial cell marker gene detection knot
Fruit is stable, and bone and flesh tumor metastasis correlation interstitial cell marker gene testing result is variant：Because different osteosarcoma cells shift
The difference of related interstitial cell marker gene expression, when only being detected from a kind of marker gene, can be caused a certain degree of
Missing inspection, using 2 kinds or two or more when, Detection results reach stable, and average green florescent signal point is from experimental group 1 to experiment
Group 4 gradually increases with the increase of bone and flesh tumor metastasis correlation interstitial cell marker gene quantity, and Detection results are more excellent, wherein,
During from whole bone and flesh tumor metastasis correlation interstitial cell marker gene, detection signal is most strong most stable, and effect is optimal.Meanwhile
Because epithelial cell marker gene is from 8 kinds of whole genes, its Detection results is stable, and the average red of 4 experimental groups is glimmering
Optical signal point does not have difference, illustrates that the increase of bone and flesh tumor metastasis correlation interstitial cell marker gene has no effect on epithelial cell mark
The testing result of gene, it follows that being substantially not present between all kinds probe designed by kit of the present invention non-specific
Property combine, specificity is good.The choice experiment result interstitial related to bone and flesh tumor metastasis of epithelial cell marker gene gene dosage is thin
Born of the same parents' marker gene is consistent.It is other be directed to the epithelial cell marker gene using varying number and species, bone and flesh tumor metastasis it is related between
The kit of cell plastid marker gene, its result is still reliable and stable, and specific data are omitted.
The selection of the kit target detection marker gene of embodiment 5
First, design (selection of target detection marker gene value volume and range of product) prepared by kit
Kit epithelial cell marker gene of the present invention is selected from：EpCAM、CDH1、KRT8、KRT16、KRT17、KRT18、
KRT19、KRT20；Bone and flesh tumor metastasis correlation interstitial cell marker gene is selected from：MMP2、MMP9、VIM、CDH2、FN1、HIF-1a、
SERPINE1、SURVIVIN、TWIST1、AKT2、SNAIL。
Contrived experiment group 5-8, two kinds, four kinds, ten kinds and 19 kinds of marker gene is chosen respectively, contrast its detection effect
Fruit, leucocyte use marker gene CD45, and specific design is as shown in table 14.
The composition sum of the capture probe of every group of corresponding marker gene of the present embodiment, amplification probe and label probe
Amount, detection method etc. are as described in the kit A of embodiment 1 and embodiment 2.
The selection of the kit marker gene gene of table 14
2nd, pattern detection
The present embodiment is tested from osteosarcoma metastatic cell strain MG63, U2OS and Saos2, those skilled in the art
Only it is to be understood that the title of cell line can be by being commercially available.Take about 1000 MG63 cells (being determined by cell counter) mixed
Sample is divided into 5 parts after closing uniformly, numbering 51-55；About 1000 U2OS cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 56-60；About 1000 Saos2 cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 61-65.
The kit prepared using above-mentioned design, detection process and method are examined to sample 51-65 as described in embodiment 2
Survey, read 50 cells for there are DAPI blue-fluorescence signals in each sample, wherein, the cell number in sample passes through fluorescence microscopy
Mirror automatically scanning is chosen, and for the fluorescence signal intensity of target detection mark, reads this 50 osteosarcoma cells respectively
The phosphor dot quantity of respective color, and calculate averagely counting, specific testing result is following, and (data are cell number in table 15, table
Data are counted for mean fluorecence in 16)：
The kit of table 15 uses the comparison of varying number marker gene Detection results
The kit of table 16 uses the comparison of varying number marker gene average fluorescence signal points Detection results
The testing result that contrast experiment organizes 5-8 understands, can be due to different bone and flesh during using 2 kinds of marker gene (experimental groups 5)
The differential expression of gene on oncocyte and shine into false negative result, carry out detection timeliness using 4 kinds and more than 4 kinds marker gene
Fruit reaches stable, and the fluorescence signal point that 2 a variety of class mark gene pairs are answered on cell from experimental group 5 to experimental group 8 with mark
The increase of gene dosage and gradually increase, Detection results are more excellent, wherein, from whole marker gene when, detection signal is most strong
Most stable, effect is optimal.Other kits being directed to using varying number and species, its result is still reliable and stable, specific number
According to omission.
Detection of the kit of the different interval arm of embodiment 6 to osteosarcoma circulating tumor cell mRNA in situ hybridizations
First, design (selection of spacerarm) prepared by kit
By taking epithelial cell marker gene (totally 8 genes) as an example, respectively from different spacerarms, specific design such as table 17
It is shown.Remaining capture probe, the base composition of amplification probe and label probe and quantity used all with the kit of embodiment 1
A is identical, the difference is that only spacerarm difference, detection method etc. as described in Example 2.Corresponding capture probe, amplification are visited
The spacerarm of pin is identical.
The spacerarm of table 17 and its length
2nd, pattern detection
The present embodiment is tested from osteosarcoma metastatic cell strain MG63, U2OS and Saos2, those skilled in the art
Only it is to be understood that the title of cell line can be by being commercially available.Take about 1000 MG63 cells (being determined by cell counter) mixed
Sample is divided into 5 parts after closing uniformly, numbering 66-70；About 1000 U2OS cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 71-75；About 1000 Saos2 cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 76-80.
The kit prepared using above-mentioned design, detection process and method are examined to sample 66-80 as described in embodiment 2
Survey, read 50 cells for there are DAPI blue-fluorescence signals in each sample, wherein, the cell number in sample passes through fluorescence microscopy
Mirror automatically scanning is chosen, and specific testing result is following (data are cell number in table)：
The epithelial cell marker gene detection probe of table 18 uses the Comparison of experiment results of different interval arm
Understand that the Detection results of 4 groups of experimental designs do not have difference by the testing result for contrasting 4 experimental groups, therefore, this
The design of 4 kinds of spacerarms is equivalent.Bone and flesh tumor metastasis correlation interstitial cell marker gene and the detection of leucocyte marker gene
Probe is consistent with epithelial cell marker gene using different spaced walls testing results, and specific data are omitted.It is other to be directed to capture
The kit of different intervening sequences inside probe, amplification probe and label probe, its result is still reliable and stable, specific data
Omit.
Embodiment 7：The utilization of label probe
First, design (signal detection component) prepared by kit
Kit signal detection component of the present invention has two kinds of selections, 1) the 3 ' of amplification probe P4 sequences terminal modified have fluorescent base
Group；2) amplification probe 3 ' holds P4 sequences to be combined with the P5 sequences of label probe by base pair complementarity, while label probe
3 ' ends carry fluorophor.Both signal detection components can realize that signal amplifies, and detect normal signal.Wherein, use
The label probe of fluorophor modification, detection signal is more stable, and effect is more excellent.
By taking two kinds of signal detection components of the detection epithelial cell marker gene of the kit as an example, specific design such as table
Shown in 19.The composition of i.e. described kit is：
Experimental group 11：Respective capture probe and amplification probe composition and quantity are with the kit A of embodiment 1, but amplification probe
3 ' terminal modified have fluorophor Cy3；There is no label probe, it is specific such as the kit B of embodiment 1；
Experimental group 12：Respective capture probe, amplification probe and label probe composition and quantity expand with the kit A of embodiment 1
Increase probe and do not have fluorophor, but be configured with label probe, the P5 sequences 3 ' of label probe are terminal modified fluorophor Cy3.
The signal detection component of table 19
2nd, pattern detection
The present embodiment is tested from osteosarcoma metastatic cell strain MG63, U2OS and Saos2, those skilled in the art
Only it is to be understood that the title of cell line can be by being commercially available.Take about 1000 MG63 cells (being determined by cell counter) mixed
Sample is divided into 5 parts after closing uniformly, numbering 81-85；About 1000 U20S cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 86-90；About 1000 Saos2 cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after uniformly, numbering 91-95.
The kit prepared using above-mentioned design, detection process and method are examined to sample 81-95 as described in embodiment 2
Survey, read 50 cells for there are DAPI blue-fluorescence signals in each sample, wherein, the cell number in sample passes through fluorescence microscopy
Mirror automatically scanning is chosen, and for the fluorescence signal intensity of target detection mark, reads this 50 osteosarcoma cells respectively
The phosphor dot quantity of respective color, and calculate averagely counting, specific testing result is following, and (data are cell number in table 20, table
Data are counted for mean fluorecence in 21)：
The epithelial cell marker gene of table 20 is compared using the testing result of unlike signal detection probe
The epithelial cell marker gene of table 21 is compared using unlike signal detection probe mean fluorecence points testing result
The testing result of two groups of designs is subjected to statistical analysis, it was demonstrated that detection of two groups of designs to cell number in sample
As a result there is no difference, therefore, both detections of signal detection component to signal are equivalent.Wherein, repaiied using fluorophor
The label probe (i.e. experimental group 14) of decorations, it detects that epithelial cell marker gene fluorescence points are more, and signal is more stable, effect
Fruit is more excellent.Other are directed to the utilization testing result and epithelial cell of bone and flesh tumor metastasis correlation interstitial cell marker gene label probe
Marker gene testing result is consistent, and specific data are omitted.
The quantity selection of the capture probe of the marker gene of embodiment 8
First, design (selection of capture probe quantity) prepared by kit
Osteosarcoma circulating tumor cell identification kit of the present invention, separately designed for different types of each marker gene
10 capture probes, and the P2 sequences in the capture probe of same kind of marker gene are identical., can be with actual use
For every kind of marker gene, at least 2 capture probes corresponding to selection can be completed to detect, and specificity and stability are attained by
Demand.
To investigate influence of the selection of capture probe quantity to kit Detection results, with epithelial cell marker gene
Exemplified by EpCAM capture probe quantity selection, referring to experimental group 15-17, the capture probe of 1,2 and 10 is chosen respectively,
Contrast its Detection results.In the contrast experiment, epithelial cell marker gene is used only EpCAM (referring to table 22), and osteosarcoma
Shift related interstitial cell marker gene and leucocyte marker gene and use whole bases as listed by the kit A of embodiment 1
Cause and probe.
The selection of the epithelial cell marker gene EpCAM of table 22 capture probe
2nd, sample detection
The present embodiment is tested from osteosarcoma metastatic cell strain MG63, U20S and Saos2, those skilled in the art
Only it is to be understood that the title of cell line can be by being commercially available.Take about 1000 MG63 cells (being determined by cell counter) mixed
Sample is divided into 5 parts after closing uniformly, numbering 96-100；About 1000 U2OS cells (being determined by cell counter) are taken, are mixed
Sample is divided into 5 parts after closing uniformly, numbering 101-105；About 1000 Saos2 cells (being determined by cell counter) are taken,
Sample is divided into 5 parts after well mixed, numbering 106-110.
The kit prepared using above-mentioned design, detection process and method are carried out to sample 96-110 as described in embodiment 2
Detection, reads 50 cells for having DAPI blue-fluorescence signals in each sample, wherein, the cell number in sample is shown by fluorescence
Micro mirror automatically scanning is chosen, and for the fluorescence signal intensity of target detection mark, reads this 50 osteosarcoma cells respectively
Respective color phosphor dot quantity, and calculate averagely counting, specific testing result it is following (data are cell number in table 23,
Data are counted for mean fluorecence in table 24)：
The epithelial cell marker gene EpCAM of table 23 is compared using the testing result of varying number capture probe
The epithelial cell marker gene EpCAM of table 24 is compared using varying number capture probe mean fluorecence points testing result
By three groups of Experimental comparisons, when only selecting epithelial cell marker gene EpCAM, 1,2 and 10 is used
Capture probe can complete to detect, when capture probe use 2 or more when, its specificity and stability it is all fine.Wherein,
When using all capture probes of 10, the fluorescence signal points that epithelial gene detects are more, and signal is stronger more stable, inspection
Survey best results.
It is other to be directed to epithelial cell marker gene, bone and flesh tumor metastasis correlation interstitial cell marker gene and leucocyte mark base
The kit using varying number capture probe of cause, its result is still reliable and stable, and specific data are omitted.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of osteosarcoma circulating tumor cell identification kit, it is characterised in that including the mRNA for every kind of marker gene
Capture probe and signal amplifying system, the marker gene includes following two class：Selected from EpCAM, CDH1, KRT8,
At least two epithelial cell marker gene in KRT16, KRT17, KRT18, KRT19, KRT20, selected from MMP2, MMP9,
At least two osteosarcoma in VIM, CDH2, FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL turns
Interstitial cell marker gene is closed in phase shift；The signal amplifying system includes the end modified amplification probe for having fluorophor, or
Include amplification probe and the end modified label probe for having fluorophor, the mutual not phase of fluorophor of variety classes target gene
Together；Wherein,
The capture probe is used to connect marker gene mRNA and amplification probe, base of the every capture probe from 5 ' ends to 3 ' ends
Composition is followed successively by：The specific sequence P1 that can be combined with marker gene mRNA to be detected, spacer sequence, P2 sequences, it is described
P2 sequences are in the absence of hairpin structure, dimer are not formed between probe interior and probe, in the absence of mispairing, with P1, P4 and mark
The sequence of specific binding is not present between gene mRNA, for the P2 of same category of marker gene mRNA capture probe
Sequence is identical；
Base composition of the every amplification probe from 5 ' ends to 3 ' ends is followed successively by：Can be with the P2 sequence complementary pairings of respective capture probe
P3 sequences, spacer sequence, P4 sequences；The P4 sequences are in the absence of hairpin structure, are not formed between probe interior and probe
Dimer, in the absence of between mispairing and P1, P2, P3 and total mRNA be not present specific binding sequence；
Every label probe has the P5 sequences with corresponding amplification probe P4 sequence complementary pairings.
2. osteosarcoma circulating tumor cell identification kit according to claim 1, it is characterised in that the marker gene
MRNA also include one kind of the mRNA for leucocyte marker gene, the leucocyte marker gene is CD45.
3. osteosarcoma circulating tumor cell identification kit according to claim 1, it is characterised in that the epithelial cell
In the capture probe of marker gene：SEQ ID NO.1~SEQ ID NO.10 are selected from for the specific sequence P1 of EpCAM genes
In 2 or more than 2, for CDH1 genes specific sequence P1 in SEQ ID NO.11~SEQ ID NO.20
2 or more than 2, for 2 in SEQ ID NO.21~SEQ ID NO.30 of specific sequence P1 of KRT8 genes
Or more than 2, for 2 or 2 in SEQ ID NO.31~SEQ ID NO.40 of specific sequence P1 of KRT16 genes
More than bar, for 2 or 2 because of specific sequence P1 in SEQ ID NO.41~SEQ ID NO.50 of KRT17 bases
More than, for KRT18 gene specific sequence P1 2 or 2 in SEQ ID NO.51~SEQ ID NO.60 with
On, for KRT19 2 or more than 2 in SEQ ID NO.61~SEQ ID NO.70 of gene specific sequence P1,
For KRT20 2 or more than 2 in SEQ ID NO.71~SEQ ID NO.80 of gene specific sequence P1.
4. osteosarcoma circulating tumor cell identification kit according to claim 3, it is characterised in that for epithelial cell
The P2 sequences of the capture probe of marker gene are SEQ ID NO.201；In the amplification probe of the epithelial cell marker gene, P3
Sequence is that SEQ ID NO.204, P4 sequence are SEQ ID NO.207.
5. osteosarcoma circulating tumor cell identification kit according to claim 1, it is characterised in that the osteosarcoma turns
Phase shift is closed in the capture probe of interstitial cell marker gene：SEQ ID NO.81 are selected from for the specific sequence P1 of MMP2 genes
2 in~SEQ ID NO.90 or more than 2, for MMP9 genes specific sequence P1 be selected from SEQ ID NO.91~
2 in SEQ ID NO.100 or more than 2, SEQ ID NO.101~SEQ are selected from for the specific sequence P1 of VIM genes
2 in ID NO.110 or more than 2, SEQ ID NO.111~SEQ ID are selected from for the specific sequence P1 of CDH2 genes
2 in NO.120 or more than 2, SEQ ID NO.121~SEQ ID are selected from for the specific sequence P1 of FN1 genes
2 in NO.130 or more than 2, SEQ ID NO.131~SEQ ID are selected from for the specific sequence P1 of HIF-1a genes
2 in NO.140 or more than 2, SEQ ID NO.141~SEQ are selected from for the specific sequence P1 of SERPINE1 genes
2 in ID NO.150 or more than 2, for SURVIVIN genes specific sequence P1 be selected from SEQ ID NO.151~
2 in SEQ ID NO.160 or more than 2, for TWIST1 genes specific sequence P1 be selected from SEQ ID NO.161~
2 in SEQ ID NO.170 or more than 2, for AKT2 genes specific sequence P1 be selected from SEQ ID NO.171~
2 in SEQ ID NO.180 or more than 2, for SNAIL genes specific sequence P1 be selected from SEQ ID NO.181~
2 in SEQ ID NO.190 or more than 2.
6. osteosarcoma circulating tumor cell identification kit according to claim 5, it is characterised in that turn for osteosarcoma
The P2 sequences that the capture probe of interstitial cell marker gene is closed in phase shift are SEQ ID NO.202；Between the bone and flesh tumor metastasis correlation
In the amplification probe of cell plastid marker gene, P3 sequences are that SEQ ID NO.205, P4 sequence are SEQ ID NO.208.
7. osteosarcoma circulating tumor cell identification kit according to claim 2, it is characterised in that the leucocyte mark
In the capture probe of will gene：SEQ ID NO.191~SEQ ID NO.200 are selected from for the specific sequence P1 of CD45 genes
In 2 or more than 2.
8. osteosarcoma circulating tumor cell identification kit according to claim 7, it is characterised in that for leucocyte mark
The P2 sequences of the capture probe of will gene are SEQ ID NO.203；In the amplification probe of the leucocyte marker gene, P3 sequences
It is SEQ ID NO.209 for SEQ ID NO.206, P4 sequence.
9. the osteosarcoma circulating tumor cell identification kit according to claim any one of 1-8, it is characterised in that described
Marker gene includes：EpCAM, CDH1, KRT8, KRT16, KRT17, KRT18, KRT19, KRT20 epithelial cell mark base
Cause, MMP2, MMP9, VIM, CDH2, FN1, HIF-1a, SERPINE1, SURVIVIN, TWIST1, AKT2, SNAIL osteosarcoma
Shift related interstitial cell marker gene；CD45 leucocyte marker gene；
In the capture probe of the epithelial cell marker gene：There are SEQ ID NO.1 for the specific sequence P1 of EpCAM genes
~SEQ ID NO.10, there are SEQ ID NO.11~SEQ ID NO.20 for the specific sequence P1 of CDH1 genes, for
The specific sequence P1 of KRT8 genes has SEQ ID NO.21~SEQ ID NO.30, for the specific sequence of KRT16 genes
P1 has SEQ ID NO.31~SEQ ID NO.40, for KRT17 bases because specific sequence P1 has SEQ ID NO.41~SEQ
ID NO.50, there are SEQ ID NO.51~SEQ ID NO.60 for KRT18 gene specific sequence P1, for KRT19's
Gene specific sequence P1 has SEQ ID NO.61~SEQ ID NO.70, has SEQ for KRT20 gene specific sequence P1
ID NO.71~SEQ ID NO.80；
In the capture probe of the bone and flesh tumor metastasis correlation interstitial cell marker gene：For the specific sequence P1 of MMP2 genes
There are SEQ ID NO.81~SEQ ID NO.90, there are SEQ ID NO.91~SEQ ID for the specific sequence P1 of MMP9 genes
NO.100, there are SEQ ID NO.101~SEQ ID NO.110 for the specific sequence P1 of VIM genes, for CDH2 genes
Specific sequence P1 has SEQ ID NO.111~SEQ ID NO.120, has SEQ ID for the specific sequence P1 of FN1 genes
NO.121~SEQ ID NO.130, there are SEQ ID NO.131~SEQ ID for the specific sequence P1 of HIF-1a genes
NO.140, there are SEQ ID NO.141~SEQ ID NO.150 for the specific sequence P1 of SERPINE1 genes, for
The specific sequence P1 of SURVIVIN genes has SEQ ID NO.151~SEQ ID NO.160, for the special of TWIST1 genes
Property sequence P1 has SEQ ID NO.161~SEQ ID NO.170, has SEQ ID for the specific sequence P1 of AKT2 genes
NO.171~SEQ ID NO.180, there are SEQ ID NO.181~SEQ ID for the specific sequence P1 of SNAIL genes
NO.190；
There are SEQ ID NO.191~SEQ ID NO.200 for the specific sequence P1 of CD45 genes.
10. the osteosarcoma circulating tumor cell identification kit according to claim any one of 1-8, it is characterised in that institute
It is 5-10 T to state spacer sequence；Fluorophor in the fluorescence signal amplification system is selected from：FAM、TET、JOE、HEX、
Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488 and Alexa Fluor
750, and it is different for the fluorophor of variety classes target gene.
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| CN113881768A (en) * | 2021-06-15 | 2022-01-04 | 上海长征医院 | Gene for osteosarcoma typing and osteosarcoma prognosis evaluation and application thereof |
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| CN110456034A (en) * | 2018-05-07 | 2019-11-15 | 上海市第十人民医院 | A kind of detection method of circulating tumor cell |
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| CN113881768A (en) * | 2021-06-15 | 2022-01-04 | 上海长征医院 | Gene for osteosarcoma typing and osteosarcoma prognosis evaluation and application thereof |
| CN113881768B (en) * | 2021-06-15 | 2023-10-03 | 上海长征医院 | Gene for osteosarcoma typing and assessing osteosarcoma prognosis and application thereof |
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Application publication date: 20171215 |