CN107385017A - A kind of molecular beacon probe and kit for detecting circulating tumor cell - Google Patents

A kind of molecular beacon probe and kit for detecting circulating tumor cell Download PDF

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Publication number
CN107385017A
CN107385017A CN201710449406.1A CN201710449406A CN107385017A CN 107385017 A CN107385017 A CN 107385017A CN 201710449406 A CN201710449406 A CN 201710449406A CN 107385017 A CN107385017 A CN 107385017A
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molecular beacon
probe
beacon probe
circulating tumor
tumor cell
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孙喜元
王季武
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Cheng Mei Bio Tech Ltd Suzhou
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Cheng Mei Bio Tech Ltd Suzhou
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a kind of molecular beacon probe for detecting circulating tumor cell, the base sequence of the molecular beacon probe is：Probe E790：5'CCCTACCCTGCAGCTCATCATGCAGCTCATGCCGGGTAGGG 3', the 5' ends of the molecular beacon probe are marked with fluorophor, and 3' ends are marked with quenching group.Molecular beacon probe and kit of the present invention can detect to circulating tumor cell, and detection sensitivity is high, accuracy in detection is high.

Description

A kind of molecular beacon probe and kit for detecting circulating tumor cell
Technical field
The present invention relates to the detection technique field of molecular biology, and in particular to a kind of molecule for detecting circulating tumor cell Beacon probe and kit.
Background technology
Circulating tumor cell (circulating tumor cells, CTCs) is spontaneous or because of operation of diagnosis and treatment, by solid tumor It is that malignant tumor patient postoperative recurrence or DISTANT METASTASES IN occurs or metastatic lesion discharges into the tumour cell of Peripheral Circulation Important symbol.EGFR is EGF-R ELISA.EGFR is located at cell membrane surface, by coming with ligand binding in active cell Kinase signal pathway.The physiology courses such as growth, propagation and differentiation of the EGFR signal paths to cell play an important role.It is many With the presence of mutant egf R genes in tumour, it has now been found that many kinds of EGFR genetic mutation types.Synthesized using EGFR genetic mutation Detection of the probe to tumour cell in tumor tissues and the detection to tumour cell in the circulatory system have great importance.
Diagnosing tumor method traditional at present relies primarily on tumor tissues and the morphological feature of tumour cell is judged, Need to be sampled detection to the tumor tissues with certain volume, can not realize that detecting it when tumour cell quantity is seldom deposits That is, the early detection of tumour can not realized；In addition, the detection sensitivity and accuracy in detection of existing detection method need into one Step improves.
The content of the invention
The technical problem to be solved in the present invention is the defects of overcoming prior art, there is provided a kind of detection circulating tumor cell Molecular beacon probe and kit.
To solve above technical problem, the technical solution adopted by the present invention is：
A kind of molecular beacon probe for detecting circulating tumor cell, the base sequence of the molecular beacon probe are：
Probe E790：
5'-CCCTACCCTGCAGCTCATCATGCAGCTCATGCCGGGTAGGG-3', the 5' of the molecular beacon probe End is marked with fluorophor, and 3' ends are marked with quenching group.
Preferably, the fluorophor be selected from FAM, Fluorescein, JOE, TET, HEX, Cyanine 3, ROX, Texas Red, Cyanine 5 or Cyanine 5.5, the quenching group are selected from BHQ-1, BHQ-2, BHQ-2 or DABCYL.
A kind of kit, including the above-mentioned molecular beacon probe stated.
Preferably, mentioned reagent box also includes hybridization solution, and hybridization solution described in per 100ml includes following components：25mL 50* Denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon sperm DNA, 10g dextran sulfate.
Preferably, when being diluted using above-mentioned hybridization solution to molecular beacon, the concentration of the molecular beacon probe is 5pmol/ul。
The present invention also provides a method detected using above-mentioned molecular beacon probe to circulating tumor cell, this method It is used detection sample be blood sample, using EGFR genetic mutation as molecular labeling, preferably using EGFR genetic mutation for divide Son mark, is detected with corresponding molecular beacon probe.Used cell fixer composition is during detection：Per 1000mL In, there is 40% formaldehyde 120ml, sodium dihydrogen phosphate (NaH2PO4；H2O) 4g, disodium hydrogen phosphate (Na2HPO4) 13g, surplus are Distilled water.Detection method comprises the following steps：
(1) molecular beacon probe is designed according to EGFR gene T790M mutant nucleotide sequence；The molecular beacon probe base sequence For：
5'-CCCTACCCTGCAGCTCATCATGCAGCTCATGCCGGGTAGGG-3'；
(2) peripheral blood is taken, separates leucocyte,
(3) leucocyte is fixed, then hybridized with molecular beacon probe；
(4) in fluorescence microscopy Microscopic observation result.
The beneficial effect that the present invention is reached：
Molecular beacon probe and kit of the present invention can detect to circulating tumor cell, and detection sensitivity height, Accuracy in detection is high.
Embodiment
With reference to specific embodiment, the invention will be further described.Following examples are only used for clearly illustrating Technical scheme, and can not be limited the scope of the invention with this.
Embodiment 1
Molecular beacon probe is designed according to EGFR gene T790M mutant nucleotide sequence, the molecular beacon probe base sequence is：
5'FAM-CCCTACCCTGCAGCTCATCATGCAGCTCATGCCGGGTAGGG-BHQ1 3'.The molecular beacon is By the distinguished sequence of the neck ring structure of base composition, wherein that 5' ends mark is FAM, and 3' ends mark is BHQ1, fluorophor Excitation wavelength be 494nm, launch wavelength 515nm.
Then peripheral blood 2ml is taken, leucocyte is separated with erythrocyte cracked liquid and counted, culture NCI-H1975 adenocarcinomas of lung are thin Born of the same parents' (carrying EGFR gene T790M mutation) simultaneously count.The tumour cell of peripheral white blood cells and culture is proportionally mixed, point Wei not simple peripheral white blood cells, simple lung adenocarcinoma cell, 1:100 ratio adds lung adenocarcinoma cell.By cell with 10% Formalin (i.e. 1 part of formalin adds 9 parts of water to be formulated) 250ul fixers fix 5 minutes, and 2 are washed with 1xPBS 500ul Secondary, last 3000rpm centrifuges 5 points, discards supernatant, the molecular beacon probe through thermal denaturation is diluted to 1xDNA hybridization solutions 5pmol/ul, 200ul is added cell is suspended, 42 DEG C are incubated 15 minutes.Cell is added dropwise in slide, directly under fluorescence microscope Observe and take a picture, calculate the ratio of fluorescence developing cell.As a result the leucocyte unstressed configuration dyeing of lung adenocarcinoma cell is not added with, merely Lung adenocarcinoma cell all has fluorescent staining, when adding carrying EGFR gene T790M mutated tumor cells, it is observed that cell The ratio of fluorescent staining cell and the cell of unstressed configuration dyeing is 1:100.
Include following components per the above-mentioned DNA hybridization liquid of 100ml：25mL 50*denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon sperm DNA, 10g dextran sulfate.
Embodiment 2
Circulating tumor cell (CTCs) enrichment procedure step：
Strict implement blood taking criterion is taken a blood sample and uses ACD anti-coagulants anti-freezings, and patient's median basilic vein blood is gathered on an empty stomach in early morning 5ml.Take 4ml whole bloods to add 10 times of concentration PBS, BSA buffer solutions and carry out erythrocyte splittings, add magnetic particle washing lotion, every part 100ul, centrifuge 20min.3ml metal salts, PBS mixed liquors are added, 300r/min centrifuges 5min at room temperature, and solution is divided into 3 layers, 2 layers of solution are drawn into 15ml centrifuge tube, add 10 times of PBS, BSA buffer solutions concentrated to 14ml, 1000r/ after mixing Min, room temperature centrifugation 5min, abandons supernatant to 300ul.2ml centrifuge tubes are transferred to, 2~3min on magnetic frame is placed in, liquid is moved to 1.5ml centrifuge tubes, room temperature 2000r/min, 3min is centrifuged, abandon supernatant to 100ul, produce circulating tumor cell (CTCs) suspension Liquid.
The circulating tumor cell of above-mentioned enrichment extracts DNA through cell DNA extracts kit, enters performing PCR using this DNA as template EGFR gene fragment is expanded, EGFR gene T790M mutation be present is confirmed whether with DNA sequencing to the EGFR gene of amplification.
It will confirm to suspend containing EGFR gene T790M sudden change samples (40) circulating tumor cell (CTCs) through DNA sequencing Liquid is positive as goldstandard；And confirm not containing EGFR gene T790M sudden change samples (40) circulating tumor cell through DNA sequencing (CTCs) suspension is negative as goldstandard.With molecular beacon probe Probe E790 and fluorescence labeling oligonucleotide probe Probe E790c are tested.
Molecular beacon probe experimental procedure:
By circulating tumor cell (CTCs) suspension with 10% formalin (i.e. 1 part of formalin add 9 parts of water prepare and Into) 250ul fixers fix 5 minutes, are washed 2 times with 1xPBS 500ul, last 3000rpm centrifuges 5 points, discards supernatant, will be through heat The molecular beacon probe of denaturation is diluted to 5pmol/ul with 1xDNA hybridization solutions, adds 200ul cell is suspended, and 42 DEG C are incubated 15 points Clock.The read tablet under fluorescence microscope.Fluorescence labeling oligonucleotide probe
Probe E790c：5'-FAM-TGCAGCTCATCATGCAGCTCATGCC-3
Common fluorescent labeled oligonucleotide probe experimental procedure:
(1) by circulating tumor cell (CTCs) sample smear with being dried at room temperature after (- 20 DEG C) fixations of ice acetone.
(2) it is fluorescence labeling oligonucleotide probe is dilute with hybridization solution (2xSSC, 50% formamide, 10% dextran sulfate) Release to final concentration of 5pmol/ul, 75 DEG C of denaturation 5min, the position of the sample on slide is added dropwise, is capped slide and closes, then It is incubated 30~45 minutes at 50 DEG C；
(3) by slide successively with washing lotion A (2xSSC, 50% formamide), washing lotion B (10mM PBS pH8.0,0.1% Nonidet P-40) respectively cleaning 2 times.
(4) read tablet under fluorescence microscope.
Molecular beacon is identical with the criterion of common fluorescent labeled oligonucleotide probe experimental result to be：In 20 times of things More visual field observations are carried out to sample under mirror and the cell to sending fluorescence in every visual field is counted and added up, judge to mark by following It is accurate：
Sample feminine gender (-)：40 different visuals field of Continuous Observation, find no the cell for sending fluorescence；The sample positive (+)： 40 different visuals field of Continuous Observation, the cell number for sending fluorescence are more than 1/40 visuals field, carry out experimental result judgement.
Judged by above-mentioned criterion, experimental result such as following table：
Key sample (goldstandard) Molecular beacon probe Fluorogenic oligonucleotide probe Sample number
+ + + 34
+ + - 6
- - - 37
- - + 3
Note：In above-mentioned list, it is known that sample (goldstandard)：It is known that this 40 works of EGFR gene (T790M) mutation are not present For negative goldstandard, it is known that 40 positive goldstandards of conduct of EGFR gene (T790M) sudden change sample be present；“-”：Represent that feminine gender is It is mutated in the absence of EGFR gene (T790M)；“+”：Represent positive and EGFR gene (T790M) mutation be present.
Negative, positive as goldstandard using known sample, the positive rate of molecular beacon is (34+6)/(34+6) X100%=100%, the positive rate of fluorescence labeling oligonucleotide probe is 34/ (34+6) X100%=85%；Molecule is believed Target false positive recall rate is 0/ (37+3) X100%=0%, and the false positive recall rate of fluorescence labeling oligonucleotide probe is 3/ (37+3) X100%=7.5%.From experimental result, molecular beacon probe is better than fluorogenic oligonucleotide probe.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, some improvement and deformation can also be made, these are improved and deformation Also it should be regarded as protection scope of the present invention.

Claims (5)

  1. A kind of 1. molecular beacon probe for detecting circulating tumor cell, it is characterised in that the base sequence of the molecular beacon probe It is classified as：Probe E790：5'-CCCTACCCTGCAGCTCATCATGCAGCTCATGCCGGGTAGGG-3', the molecular beacon The 5' ends of probe are marked with fluorophor, and 3' ends are marked with quenching group.
  2. 2. a kind of molecular beacon probe for detecting circulating tumor cell according to claim 1, it is characterised in that described glimmering Light group be selected from FAM, Fluorescein, JOE, TET, HEX, Cyanine 3, ROX, Texas Red, Cyanine 5 or Cyanine 5.5, the quenching group are selected from BHQ-1, BHQ-2, BHQ-2 or DABCYL.
  3. 3. a kind of kit, it is characterised in that including the molecular beacon probe described in claim 1 or 2.
  4. A kind of 4. kit according to claim 3, it is characterised in that also including hybridization solution, hybridization solution described in per 100ml Including following components：25mL 50*denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon Smart DNA, 10g dextran sulfate.
  5. 5. a kind of kit according to claim 4, the concentration of the molecular beacon probe is 5pmol/ul.
CN201710449406.1A 2017-06-14 2017-06-14 A kind of molecular beacon probe and kit for detecting circulating tumor cell Pending CN107385017A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN110669839A (en) * 2018-07-03 2020-01-10 北京福安华生物科技有限公司 Artificial simulated molecular beacon and kit for detecting EGFR gene T790M mutation

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110669839A (en) * 2018-07-03 2020-01-10 北京福安华生物科技有限公司 Artificial simulated molecular beacon and kit for detecting EGFR gene T790M mutation
CN110669839B (en) * 2018-07-03 2022-04-15 北京福安华生物科技有限公司 Artificial simulated molecular beacon and kit for detecting EGFR gene T790M mutation

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Application publication date: 20171124