CN107843731A - The kit of pancreatic cancer cell mark in a kind of detection peripheral blood - Google Patents
The kit of pancreatic cancer cell mark in a kind of detection peripheral blood Download PDFInfo
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- CN107843731A CN107843731A CN201710825555.3A CN201710825555A CN107843731A CN 107843731 A CN107843731 A CN 107843731A CN 201710825555 A CN201710825555 A CN 201710825555A CN 107843731 A CN107843731 A CN 107843731A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- Urology & Nephrology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
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- Hospice & Palliative Care (AREA)
- Gastroenterology & Hepatology (AREA)
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- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The present invention provides a kind of kit for detecting pancreatic cancer cell in peripheral blood, including the magnetic bead for being combined with pancreas anticancrin and corresponding buffer reagent, the kit uses magnetic bead sorting, the biological activity of tumour cell in the method detection Pancreas cancer patients blood circulation of RT PCR equimolecular biology, determine the gene of AD-targeted drugs, by the expression for detecting EpCAM, KRT19 and MUC16, determine the development of cancer of pancreas and lapse to, theoretical foundation and experimental evidence are provided to the individualized treatment of cancer of pancreas.Kit detection sample size provided by the invention is small, small to patient's injury, and detection sensitivity is high, and recall rate is up to more than 80%.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of cancer of pancreas detection kit, and in particular to a kind of accuracy is high
Detection peripheral blood in pancreatic cancer cell mark kit.
Background technology
1.1 cancers of pancreas are summarized
Cancer of pancreas (pancreatic carcinoma) is a kind of common malignant tumor of digestive tract, about 90% be originating from
The duct adenocarcinoma (pancreatic ductal adenocarcinoma, PDAC) of ductal epithelium, its morbidity and mortality is near
Substantially rise over year, 90% Pancreas cancer patients life cycle is no more than 1 year, and life cycle, less than 5%, was prognosis more than 5 years
One of worst malignant tumour.The undesirable influence factor for the treatment of of pancreatic cancer effect has its early symptom not to be true to type, complicated disease
Shortage of reason physiological mechanisms, early diagnosis and prognostic marker etc..Although the diagnosis of tumour and treatment means are continuous in recent years
Improve and progressive, the prognosis of many tumours has been improved, but is counted according to American Cancer Society, and the prognosis of Patients with Pancreatic Cancer improves
It is least obvious.The reason for cancer of pancreas high mortality, is：1) although Imaging Method plays weight in cancer of pancreas detects and treats
Act on, however, to small lesion, pancreatitis, pancreatic intraepithelial neoplasia and other benign lesions and cancer of pancreas remain difficult to differentiate,
Only 7% cancer of pancreas diagnoses in the early stage.2) advanced pancreatic carcinoma causes Most patients poor prognosis without effective treatment means, outside
5 years relative survival rates 22% of patient of the resectable local lesion of section's operation, and the progressive stage metastatic lesion survival rate not cut off
Only 1-2%.3) the low survival rate of cancer of pancreas is its invasive biological phenotype, shows as early stage local diffusion and transfer.
From the perspective of economics, biological markers are likely to become more cost effective disease early detection and examined
Disconnected method, mark most classical as cancer of pancreas the sugar antigen CA199 of clinical practice at present, because it is at cancer of pancreas initial stage
Normally, also raised in benign lesion such as pancreatitis or other tumours so that its sensitivity and specificity Shortcomings.In view of mesh
Before there is no single biological markers to reach the requirement of cancer of pancreas Newly diagnosed, there is an urgent need to research and develop new cancer of pancreas phase
The biological markers of pass, healthy individuals and Early pancreatic carcinoma or precursor lesion are distinguished, so as to reduce the incidence of disease of disease and dead
Die rate.Circulating tumor cell (circulating tumor cells, CTCs) refer to it is spontaneous or because of operation of diagnosis and treatment by solid tumor or
Transfer stove discharges into the tumour cell of Peripheral Circulation, its can aid in early diagnosis and good pernicious judgement, instruct by stages and
Molecule parting, auxiliary treatment medication screening and curative effect monitor in real time, micrometastasis and prognosis etc. are prompted, because peripheral blood sample easily obtains
And wound it is small the features such as show CTC clinical advantage.
1.2 influence the correlative factor of cancer of pancreas prognosis
For many years, operative treatment is still unique effective means of radical cure cancer of pancreas, and it has head in treatment of pancreatic cancer
Status, only radical excision tumor tissues are wanted, patient is obtained longer life span.And cancer of pancreas onset is hidden,
It has been late period when 85% patient makes a definite diagnosis, has missed opportunity of operation.To unresectable cancer of pancreas, after clarifying a diagnosis, even if row
NACT, life span are usually no more than 6 months.Most of research reports show, the prognosis of cancer of pancreas and age, sex,
Family history of cancer and tumor locus are without substantial connection.The relevance of diabetes and cancer of pancreas has dispute always, there is research table
Bright, diabetic history can increase the risk of cancer of pancreas, and diabetes can also reduce the postoperative life of ductal adenocarcinoma of pancreas patient
Deposit rate.Pancreas cancer patients prognosis is invaded to pancreas week, DISTANT METASTASES IN is related, and these are also the Main Basiss of cancer of pancreas clinical stages
(cancer of pancreas is shown in Table 1 by stages：The 8th edition cancer of pancreas Staging System of AJCC).Cancer of pancreas early symptom unobvious, some patientss have benefited from
Early detection, it is able to radical excision and significantly improves survival rate, therefore realizes that the early diagnosis of cancer of pancreas can be carried effectively
The life cycle of high cancer of pancreas.
The 8th edition cancer of pancreas Staging System of the AJCC of table 1
1.3 clinical conventional cancer of pancreas detection techniques
At present clinically for cancer of pancreas serology tumor markers detection mainly have CA199, CA242, CEA and
MUC16 etc..Wherein CA199 is one of the most frequently used pancreatopathy cancer serum marker detection, and CA199 is a kind of mucin type carbohydrate
Protein tumor mark, ascendant trend in Infusion in Patients with Digestive serum be present, therefore frequently as the mark of tumor in digestive tract
Thing, while be also the most strong mark of current cancer of pancreas sensitiveness.Also there are some researches show CA199 is with patient's cancer staging in just
The expression of correlation, i.e. CA199 is in progress and raised by stages with cancer of pancreas, and its level also has certain related to patient's prognosis in addition
Property.But it is only 57.69% in the positive rate of I phase cancers of pancreas, the discovery for Early pancreatic carcinoma helps little.And due to
CA199 is also raised in obstructive jaundice, pancreatitis and other a variety of non-pancreatic neoplasms, false positive easily occurs, therefore not
Can be separately as the diagnostic criteria for differentiating cancer of pancreas.MUC16 is one kind sugar of coelomic epithelium cell expression during cancer embryonic development
Proteantigen, it is often used as the gynecological tumor mark such as oophoroma.Research shows, in Pancreas cancer patients MUC16 also apparently higher than
Normal level, while in respectively by stages, MUC16 expressions and positive rate are in rising trend, and ductal adenocarcinoma of pancreas can be used as pre-
Bad independentpredictor afterwards.But these factors the defects of having itself, can not be separately as diagnosis of pancreatic cancer mark.
Therefore it is clinical to need a kind of wound small, help to early diagnose and can detect in real time the effective detection method of tumour progression.
The mechanism of 1.4 pancreas metastasis of cancer
For a long time, " being what determines which organ shifts " this problem perplexs people always, and 1889
Year, Stephen Paget observe that patient with breast cancer is more biased towards liver metastasis, and Pagett is it is thought that uncommon because of it
His organ such as spleen also can equally receive identical influence, because spleen and liver have identical CBF, this discovery
Pagett is promoted to propose " seed-soil " theory, he assumes some tumour cells, that is, seed, is selectively cloned into remote
End organ, that is, soil, its provide the environment of a suitable growth of tumour cell " when plant starts to sow, its seed
Can sow to any direction but its can fall grown in suitable soil " therefore some organs must provide it is suitable suitable
The environment of clutch official's transspecific is so as to causing the selectivity of organ.It is false that the process of pancreas carcinogenesis transfer has also confirmed this
Say, the survival rate of cancer of pancreas is extremely low, and DISTANT METASTASES IN easily occurs, and wherein hepatic metastases is most commonly seen.Hematogenous metastasis is pancreas carcinogenesis
The main path of transfer, tumour cell enter blood from primary carcinoma tissue loss, with flow propagation to distant organs and form transfer stove.
1.5 circulating tumor cell
Circulating tumor cell refers to that primary tumo(u)r or transfer stove come off into the tumour cell of blood, be suggested for the first time be
1869, the ability that the clonal growth in the microenvironment that tissue is supported forms transfer stove may be possessed by partly entering the CTC of blood, because
This CTC number and characterization of molecules can be used as a kind of " liquid biopsy " means in real time noninvasive in real time to provide on prognosis, treatment
The clinical information such as selection and validity.CTC researchs now turn into quite active field, and numerous studies indicate CTC and disliked
Property tumour in presence and its clinical meaning, these research prompting CTC be prognosis mala independent predictor, CTC detection sun
Property patient progression free survival phase and Overall survival be significantly shorter than the negative patient of CTC detections, and effective cytoreductive art or
CTC numbers decline after chemicotherapy, prompt CTC to aid in detecting therapeutic effect so as to make further treatment guidance；Separately
Outside, the hereditary information of primary tissue can be also carried in CTC, by the change of molecular level can be provided for individualized treatment according to
According to.
In ductal adenocarcinoma of pancreas, EpCAM, KRT19 and MUC16 high expression are related to the poor prognosis of cancer of pancreas.
In summary, the quantity of circulating tumor cell in Pancreas cancer patients blood is detected for diagnosis of pancreatic cancer and is assessed pre-
There is great theoretical and practical significance afterwards.
The content of the invention
It is an object of the invention to provide a kind of kit that can accurately detect pancreas carcinoma marker, is primarily referred to as a kind of logical
The detection kit and compound detection technique for the pancreatic cancer cell crossed in collection Pancreas cancer patients peripheral blood, specifically
The detection technique and kit of a kind of high detection pancreatic cancer cell (malignant cell) of accuracy, can particularly detect warp
The detection kit of the free pancreatic cancer cell of remaining in blood after Radiotherapy chemotherapy is treated is crossed, the testing result is controlled cancer of pancreas
The selection and prognosis for the treatment of scheme have great importance.
The present invention invention thinking be：Using Beads enrichment, the method detection cancer of pancreas of RT-PCR equimolecular biology is suffered from
The biological activity of tumour cell in person's blood circulation, by detecting EpCAM, KRT19 and MUC16 expression, it is determined that
The development of cancer of pancreas and lapse to, theoretical foundation and experimental evidence are provided to the individualized treatment of cancer of pancreas.
To achieve the above object, the present invention takes following technical scheme：
The present invention provides a kind of kit for detecting pancreas carcinoma marker in peripheral blood, and the kit is by following component group
Into：
It is combined with the magnetic bead of pancreas anticancrin：
It is combined with oligonucleotides Oligo (dT) magnetic bead；
Buffer A；100-200mL
Buffer B：100-200mL；
Cracking/combination buffer 150-250mL；
10 × buffer solution 4.0-5.0mL；
DNTPs, 5mM each 4.0-5.0mL
Reverse transcriptase, 10000U/ μ L 2.0-3.0mL
RNase inhibitor, 40U/ μ L 0.4-0.6mL
2 × heat start PCR mixed liquor 2-3mL
RNase-free water 1-2mL；
10 μm of ol/L 3.0-5.0mL of pancreatic tumour mark primer；
Wherein described pancreatic tumour mark is the one or more in EpCAM, KRT19, MUC16；
The EpCAM primer sequences are：
Sense primer such as SEQ ID NO：Shown in 1：5’-TGAGCGAGTGAGAACCTA-3’；
Anti-sense primer such as SEQ ID NO：Shown in 2：5’-CACAACAATTCCAGCAAC-3’；
The KRT19 primer sequences are：
Sense primer such as SEQ ID NO：Shown in 3：5’-CTGACACCATTCCTCCCT-3’；
Anti-sense primer such as SEQ ID NO：Shown in 4：5’-CCGACGACTGGCGATA-3’；
The MUC16 primer sequences are：
Sense primer such as SEQ ID NO：Shown in 5：5’-TCCCTGGATGCTGCTA-3’；
Anti-sense primer such as SEQ ID NO：Shown in 6：5’-TGCTGAGGTGGCTATG-3’；
The pancreas anticancrin is anti-EpCAM monoclonal antibody and Monoclonal anti-CK20 antibody.
Wherein, HotStarTaq DNA Polymerase 5U/ μ L are included in 2 × heat start PCR mixed liquor.
Wherein, the formula of the buffer A is：10mM Tris-HCl,pH7.5；0.15M LiCl；1mM EDTA；
0.1%LiDS.
Wherein, the formula of the buffer B is：10mM Tris-HCl,pH7.5；0.15M LiCl；1mM EDTA.
Wherein, the cracking/combination buffer formula：100mM Tris-HCl,pH7.5；500mM LiCl；10mM
EDTA,pH8；1%LiDS；5mM dithiothreitol.
The tumor markers for being used for pancreatic cancer cell in enrichment cycles blood in the present invention is EpCAM, CK20；And identify pancreas
The mark of cancer is EpCAM, KRT19 and MUC16.
Primer is prepared：
Primer is each first to be diluted to 10 μM (i.e. 10 μm of ol/L), is then by primer mixing and water adding to each primer concentration
2 μM, each final concentration of 0.2 μM of primer in multi-PRC reaction system.
The magnetic bead after 25 μ L antibody labelings is used during use per 1mL blood.
Reaction result is analyzed：
PCR primer thing is analyzed using the biological analysers of Agilent 2100：
The beneficial effects of the present invention are：
The present invention provides a kind of kit for detecting pancreas carcinoma marker in peripheral blood, the detection sample of kit requirement
Measure small, patient injured small, detection sensitivity is high, to the detected level of cancer cell can as little as 2 cellular levels, i.e., contain 2 in sample
Individual free cancer cell can detect, and can determine detection for pancreatic cancer cell still according to the content of pancreatic tumour mark
Pancreas cancer stem cell, up to 80%, the personalized treatment for whether recurring and recurring after being treated to Pancreas cancer patients provides recall rate
Theoretical foundation and experimental evidence.
Brief description of the drawings
Fig. 1 is that multiplex PCR verifies expression figure of each factor in ductal adenocarcinoma of pancreas peripheral blood DNA.
Fig. 2 is the dose-effect relationship figure of EpCAM, KRT19 and Muc16 in kit provided by the invention.
Fig. 3 is the expression knot of EpCAM, KRT19 and Muc16 in Pancreas cancer patients CTC in kit provided by the invention
Fruit.
Embodiment
Agents useful for same and instrument in the present invention：
1. anti-EpCAM monoclonal antibody and Monoclonal anti-CK20 antibody are purchased from Abcom companies.
2. the magnetic bead for binding antibody selects invitrogen companies of the U.S.M-
450Tosylactivated, labeling of monoclonal antibody amount are every μ L magnetic beads of 100 μ g antibody labelings 500, labeling method and according to it
Specification is operated.
3. the magnetic bead for being combined with oligonucleotides Oligo (dT) selects invitrogen companies of the U.S.Oligo
(dT)25Magnetic bead.
4.10 × buffer solution：Reacted for the Sensiscript Reverse Transcriptase of QIAGEN companies of the U.S.
Buffer solution (10x Buffer RT).
5.2 × heat start PCR mixed liquor：For QIAGEN companies of U.S. Multiplex PCR Master Mix, wherein containing
It is concentration to have HotStarTaq DNA Polymerase specifications：5U/μL.
The biological analysers of 6.Agilent 2100 are purchased from Agilent Technologies (China) Co., Ltd.
Not specified reagent and instrument are laboratory routine articles for use.
Embodiment 1：With reference to the preparation of the magnetic bead of pancreas anticancrin：
The antibody used is anti-EpCAM monoclonal antibody and Monoclonal anti-CK20 antibody, after marking above-mentioned 2 kinds of antibody respectively
Above-mentioned 2 kinds of labeled magnetic beads are used in mixed way.
Labeling method：Magnetic bead is produced using invitrogen companies of the U.S.M-450
Tosylactivated.The method of mark is carried out in strict accordance with product description.
Mark ratio is：Every μ L magnetic beads of 100 μ g antibody labelings 500, mixed in equal amounts after mark.
Embodiment 2：The separation of pancreatic cancer cell：
2.1 sample treatment：
Pancreas cancer patients peripheral blood 5mL, EDTA anti-freezing is taken, 4 degree of preservations, is used within 48 hours.
The processing of 2.2 magnetic beads：
The magnetic bead for being combined with pancreas anticancrin (antibody labeling magnetic bead, for the mixing magnetic bead of two kinds of antibody of mark, and is pressed
1mL samples add the ratio of 25 μ L magnetic beads to prepare antibody labeling magnetic bead) cleaned repeatedly three times with PBS, magnetic bead is isolated, it is standby on ice
With；
Concrete operations are：Draw the magnetic bead that 125 μ L have been marked to add in 1.5mL centrifuge tube, with pipettor blowing gently
It is placed on after persorption even (attention can not use DL instrument) on enrichment with magnetic bead device and stands 1min, magnetic bead is attached on centrifugation tube wall,
Supernatant discarding；Then 1mL PBS are added, are placed on 1min on enrichment with magnetic bead device, magnetic bead is attached on centrifugation tube wall, discards
Clearly；Cleaned altogether using PBS 3 times using same procedure, to remove preservative；Remove and 200 μ L PBS are added after supernatant, it is standby on ice
With.
Subsequent step Beads enrichment is also separated using enrichment with magnetic bead device.
The sorting of 2.3 pancreatic cancer cells：
Sample and antibody labeling magnetic bead are added in 15mL conical centrifuge tubes, reaction tube is placed on to 4 DEG C of test tube and rotated
On blender, 30min is incubated with 10rpm speed, separates magnetic bead, supernatant discarding；Add 1mL PBS to wash magnetic bead, remove supernatant
Liquid with remove should not cell, add after 1mL PBS mix magnetic bead, be transferred in new 1.5mL centrifuge tubes, it is standby.
2.4 pancreatic cancer cells crack：
PBS is removed, the μ L of cracking/combination buffer 200 are added in the cell for combining antibody labeling magnetic bead and are mixed, 55 DEG C
The cell that water-bath (or metal bath) reaction 5min makes to combine on magnetic bead cracks, and mRNA is discharged into supernatant, centrifuge tube is put into
5min is stood on magnet, supernatant is transferred in new 1.5mL centrifuge tubes, antibody labeling magnetic bead is discarded.
Pancreatic cancer cell selection step terminates, and sample mRNA is used for further experiment or -20 DEG C of storage mRNA are most long 1 week,
Long-term -70 DEG C of storages of storage use.
Embodiment 3：The detection of pancreatic cancer marker thing：
3.1mRNA purifying
Magnetic beads of the 40 μ L with oligonucleotides Oligo (dT) is mixed and is added in 1.5mL centrifuge tubes, 500 μ L is added and splits
Solution/combination buffer is rinsed 2 times, separates magnetic bead, adds supernatant, is mixed, and is incubated at room temperature 10min, mRNA is combined with magnetic bead；
Then magnetic bead is separated, 2 magnetic beads is cleaned using 500 μ L buffer As, separates magnetic bead, then 2 magnetic are cleaned with 500 μ L buffer Bs
Pearl, magnetic bead is separated, then cleaned once with 100 μ L RNase-free water, separate magnetic bead, be resuspended with 29.5 μ L RNase-free water
Magnetic bead, 55 DEG C of water-baths (or metal bath) are middle to be incubated 5min, places 2min on ice, obtains mRNA/ magnetic bead mix.
MRNA/ magnetic bead mix can not store, and need to carry out reverse transcription immediately.
3.2 reverse transcription：
Reverse transcription step is as follows：
Reverse transcription process：
37 DEG C of 60min, 93 DEG C of 5min, 4 DEG C of preservations.
Reverse transcription continues PCR or is stored in -20 DEG C (most long to preserve 2 weeks) after terminating.
Reaction substitutes sample using RNase-free water simultaneously and is used as negative control.
3.3PCR
Primer sequence：
Positive control is：Pancreatic carcinoma BxPC-3 is collected into extraction RNA reverse transcription translations and obtains positive control
cDNA；
Negative control is：High purity water.
Reaction system
PCR:Program
95℃15min；94 DEG C of 30s, 58 DEG C of 90s, 72 DEG C of 60s, 35 circulations；60℃30min；4℃∞
PCR primer is placed on ice or -20 DEG C preserve.
3.5 interpretation of result
Experimental configuration is analyzed PCR primer using the analysis of Agilent biological analysers.
1) sample of all patients must have the band (internal standard) of Actin genes；
2) RT negative control can not have the band more than 80 nucleotides；
If 3) product is more than 1kb, illustrate by contaminating genomic DNA (introne)；
4) any one band can be determined that result for the positive in 3 positive bands in PCR primer in this experiment
For the positive；
5) in PCR results, 3 PCR primers in addition to Actin are present in tumour cell, and EpCAM PCR primer
It is that epithelial cell is relevant with the source of tumour cell；
6) it is outside the band that must occur every time in addition to Actin, other 3 bands can go out at random in PCR results
It is existing.But using this method to detect, minimum PCR detections should be 2 cells, i.e., simply by the presence of 2 and 2 in sample
Tumour cell or tumor stem cell more than individual, can detect any one in 3 PCR primers.
Multiplex PCR determines three kinds of identification of M arker reliability first, and Fig. 1 is shown three in BxPC-3 pancreatic carcinomas
Kind marker qualification result.After pancreatic carcinoma extraction RNA reverse transcriptions cDNA, EpCAM, KRT19 and MUC16 are respectively
PCR results, internal reference selection Actin.Secondly by the sensitivity of positive control assay kit, as shown in Fig. 2 Fig. 2 is
The dose-effect relationship result of tri- kinds of labels of EpCAM, KRT19 and MUC16, it is seen that when cancer cell number is in 2 and more than 2,
Kit detection pancreatic cancer cell provided by the invention can be used.And with increasing for cancer cell number, the amount of PCR primer
Also increase, there is obvious dose-effect relationship.This has great application value for Pancreas cancer patients prognosis evaluation.Fig. 3 shows three
Kind Marker is to the sensitivity of patient periphery blood specimen, and BxPC-3 pancreatic carcinomas are positive control, and high purity water is negative right
According to, choose the preoperative and postoperative peripheral blood of patient, be enriched with CTC according to above-mentioned experiment flow extracts RNA afterwards, reverse transcription into cDNA,
Subsequent multiplex PCR verifies the result of three kinds of labels.As a result show and free pancreas has been detected in patient's preoperative and postoperative peripheral blood
Gland cancer CTC.
From above-described embodiment as can be seen that the kit test method of pancreatic cancer cell is simple in present invention detection peripheral blood
Just, and detection sample size is small, small to patient's injury, and detection sensitivity is high, is containing 2 pancreatic cancer cells in peripheral blood sample
It can detect, Detection accuracy can reach 80%, and testing result is credible.
Kit provided by the invention is controlled Pancreas cancer patients mainly for the monitoring after Pancreas cancer patients treatment for doctor
Whether recurred after treatment and treatment effectiveness evaluation provides reliable experimental data.
SEQUENCE LISTING
<110>Beijing ox cow genome Technology Co., Ltd.
Peking University
Beijing Inst of Tumor Prevention and Treatment
<120>The kit of pancreatic cancer cell mark in a kind of detection peripheral blood
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
tgagcgagtg agaaccta 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
cacaacaatt ccagcaac 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
ctgacaccat tcctccct 18
<210> 4
<211> 16
<212> DNA
<213>Artificial sequence
<400> 4
ccgacgactg gcgata 16
<210> 5
<211> 16
<212> DNA
<213>Artificial sequence
<400> 5
tccctggatg ctgcta 16
<210> 6
<211> 16
<212> DNA
<213>Artificial sequence
<400> 6
tgctgaggtg gctatg 16
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence
<400> 7
gaaatcgtgc gtgacatta 19
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<400> 8
aggcagctcg tagctctt 18
Claims (5)
1. a kind of kit for detecting pancreatic cancer cell mark in peripheral blood, it is characterised in that the kit is by such as the following group
It is grouped into：
It is combined with the magnetic bead of pancreas anticancrin：
It is combined with oligonucleotides Oligo (dT) magnetic bead；
Buffer A；100-200mL
Buffer B：100-200mL；
Cracking/combination buffer 150-250mL；
10 × buffer solution 4.0-5.0mL；
DNTPs, 5mM each 4.0-5.0mL
Reverse transcriptase, 10000U/ μ L 2.0-3.0mL
RNase inhibitor, 40U/ μ L 0.4-0.6mL
2 × heat start PCR mixed liquor 2-3mL
RNase-free water 1-2mL；
10 μm of ol/L 3.0-5.0mL of pancreatic tumour mark primer；
Wherein described pancreatic tumour mark is the one or more in EpCAM, KRT19, MUC16；
The EpCAM primer sequences are：
Sense primer such as SEQ ID NO：Shown in 1；
Anti-sense primer such as SEQ ID NO：Shown in 2；
The KRT19 primer sequences are：
Sense primer such as SEQ ID NO：Shown in 3；
Anti-sense primer such as SEQ ID NO：Shown in 4；
The MUC16 primer sequences are：
Sense primer such as SEQ ID NO：Shown in 5；
Anti-sense primer such as SEQ ID NO：Shown in 6；
The pancreas anticancrin is anti-EpCAM monoclonal antibody, Monoclonal anti-CK20 antibody.
2. kit as claimed in claim 1, it is characterised in that included in 2 × heat start PCR mixed liquor
HotStarTaq DNA Polymerase 5U/μL。
3. kit as claimed in claim 1, it is characterised in that the formula of the buffer A is：10mM Tris-HCl,
pH7.5；0.15M LiCl；1mM EDTA；0.1%LiDS.
4. kit as claimed in claim 1, it is characterised in that the formula of the buffer B is：10mM Tris-HCl,
pH7.5；0.15M LiCl；1mM EDTA.
5. kit as claimed in claim 1, it is characterised in that the cracking/combination buffer formula：100mM Tris-
HCl,pH7.5；500mM LiCl；10mM EDTA,pH8；1%LiDS；5mM dithiothreitol.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7507528B2 (en) * | 2001-09-06 | 2009-03-24 | Adnagen Ag | Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells |
| CN102109525A (en) * | 2011-01-26 | 2011-06-29 | 牛刚 | Kit for detecting free breast cancer cell marker in blood |
| CN102565404A (en) * | 2010-12-24 | 2012-07-11 | 牛刚 | Kit for detecting free colorectal cancer cell markers in blood |
| CN102892900A (en) * | 2010-03-02 | 2013-01-23 | 汉堡大学医院埃佩多夫公司 | Method for isolating target cells |
| CN104619862A (en) * | 2012-06-15 | 2015-05-13 | 得克萨斯系统大学董事会 | High throughput sequencing of multiple transcripts of a single cell |
| CN105849262A (en) * | 2013-09-30 | 2016-08-10 | 第三共株式会社 | Anti-LPS 011 antibody |
-
2017
- 2017-09-14 CN CN201710825555.3A patent/CN107843731B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7507528B2 (en) * | 2001-09-06 | 2009-03-24 | Adnagen Ag | Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells |
| CN102892900A (en) * | 2010-03-02 | 2013-01-23 | 汉堡大学医院埃佩多夫公司 | Method for isolating target cells |
| CN102565404A (en) * | 2010-12-24 | 2012-07-11 | 牛刚 | Kit for detecting free colorectal cancer cell markers in blood |
| CN102109525A (en) * | 2011-01-26 | 2011-06-29 | 牛刚 | Kit for detecting free breast cancer cell marker in blood |
| CN104619862A (en) * | 2012-06-15 | 2015-05-13 | 得克萨斯系统大学董事会 | High throughput sequencing of multiple transcripts of a single cell |
| CN105849262A (en) * | 2013-09-30 | 2016-08-10 | 第三共株式会社 | Anti-LPS 011 antibody |
Non-Patent Citations (2)
| Title |
|---|
| ANDREIA DE ALBUQUERQUE，ET AL: "Multimarker gene analysis of circulating tumor cells in pancreatic cancer patients:a feasibility study", 《ONCOLOGY》 * |
| DHANYA HARIDAS，ET AL: "Pathobiological Implications of MUC16 Expression in Pancreatic Cancer", 《PLOS ONE》 * |
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