CN106636312A - Detection kit for liver cancer related microRNAs - Google Patents
Detection kit for liver cancer related microRNAs Download PDFInfo
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Abstract
The invention relates to a detection kit for liver cancer related microRNAs. The detection kit comprises at least one primary signal amplification probe, at least one secondary signal amplification probe, at least one tertiary signal amplification probe, and a capture probe, aiming at each kind of to-be-detected liver cancer related microRNA, wherein the P1 sequence of the capture probe for the liver cancer related microRNA is selected from SEQ ID NO.1 to SEQ ID NO.12. The capture probes and the signal amplification probes selected by the detection kit can be subjected to hybridization reaction under the homogeneous reaction condition, and no nonspecific binding exists among the probes; the designed probes are good in specificity and high in signal-to-noise ratio during detection. Meanwhile, due to the combined use of the various probes, the identifying kit and a detection method form a system with perfect detection effects.
Description
Technical field
The invention belongs to biology field, is related to medical science and biotechnology, a kind of liver cancer is related particularly to related
MicroRNA detection kits.
Background technology
MicroRNAs (miRNAs) is the endogenic non-coding with adjusting function of a class found in eucaryote
RNA, its size is about 20～25 nucleotides.Ripe miRNAs is through a series of nucleic acid by longer primary transcript
The shearing of enzyme and produce, be subsequently assembled into RNA induction silencing complex (RNA-induced silencing
Complex, RISC), target miRNA is recognized by way of base pair complementarity, and silence is instructed according to the difference of complementarity
Complex is degraded and target miRNA or checks the translation of target miRNA.Recent studies have shown that miRNA is participated in various
Regulation approach, including development, virus defense, hematopoiesis, orga- nogenesis, cell propagation and apoptosis, fat metabolism etc..
Hepatocellular carcinoma (HCC) is my excessively common malignant tumour, and it is multifactor, polygenes and multi-step that it occurs development
Complex process.Recently result of study shows that various miRNA participate in the cell biological program of HCC by adjusting its target gene
Regulation and control, play the function of oncogene and tumor suppressor gene indirectly.Research finds, compared with closing on lung neoplasm and organizing, 77.8% liver cancer
MiR-138 expression in tissue is lowered.The inducible cell-cycle arrests of miR-138 over-expressed in liver cancer tissue, prevent cell
Colony forming, reduces the cell survival rate of tumour cell.Additionally there are some researches show, improving the expression of miR-122 can press down
Liver cancer cell growth processed；MiR-26a is presented expression in HCC and declines, and reduces the PRB of phosphorylation, prevents cell from G1
Phase is excessive to the S phases；MiR-101 is raised can make liver cancer G0/G1 phase cell showed increased, S phase Leukopenias, further real
Test research to show, it passes through to suppress EZH2 gene expressions, to human hepatoma cell proliferation activity with negative regulation effect；And mir-21
Obtain clearly in hepatoma Metastasis and the effect invaded and basic role mechanism.
At present, the detection method for miRNA mainly has Northern Blot, genetic chip, fluorescent quantitation sonde method, base
In the flow cytometry technique of microballoon.It is uncomfortable but NorthernBlot method susceptibilitys are low, time-consuming and the large usage quantity of RNA
Close high throughput analysis；Biochip technology can realize the high throughput analysis of miRNA, i.e., detect multiple simultaneously on one chip
MiRNA, but have the disadvantage that result accuracy is low, poor repeatability is tested expensive；Fluorescent quantitation sonde method detection sensitivity is high,
But testing cost is expensive；And probe is fixed on microballoon and is placed in liquid phase by bead-based flow-cytometry technology, more favorably
In capture miRNA sequence, therefore accuracy is improve, but because miRNA homologys are high, length is shorter, cell or group
Knit that interior content is low, still need to be further improved for its highly sensitive high selectivity detection method.
Hybridization in situ technique is specificity miRNA in the histotomy that a kind of positioning and morphologic detection are preserved or cellular preparations
The method of sequence.But many difficulties are still suffered to the multiple parallel detection of miRNA in currently available technology.Firstly, it is necessary to
The label probe of each target miRNA is prepared respectively；Secondly, it is difficult to while the expression of the various target miRNA of in situ detection.Therefore,
At present the detection to various miRNA of report can only be with different labeling methods, however, can not be very with different labeling methods
Probe is controlled well with the possible crisscrossing of non-specific sequences in cell.
The content of the invention
It is an object of the invention to provide the high liver cancer correlation microRNA detection kits of a kind of high specificity, sensitivity.
Realize that the technical scheme of above-mentioned purpose is as follows.
A kind of liver cancer correlation microRNA detection kits, include：For every kind of liver cancer correlation microRNA to be detected
There are at least one one stage signal amplifying probe, at least one second signal amplifying probe and at least one three-level signal amplifying probe,
And capture probe, the liver cancer correlation microRNA is selected from::hsa-miR-138-5p、hsa-miR-122-5p、
hsa-miR-101-3p、hsa-miR-21-5p、hsa-miR-326、hsa-miR-297、hsa-miR-520b、hsa-miR-181b-5p、
At least one in hsa-miR-519b-3p, hsa-miR-602, hsa-miR-26a-5p and hsa-miR-199a-5p, wherein：
The capture probe is used for linking objective nucleic acid and one stage signal amplifying probe, and every capture probe is held successively from 5 ' ends to 3 '
It is different for the P2 sequences of different target gene for specific P1 sequences, spacer sequence, P2 sequences；
The one stage signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences, spacer sequence, P3 sequences, P3 sequences with
P2 sequence complementary pairings；
The second signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P5 sequences, spacer sequence, P6 sequences；
The P4 sequences contain the base fragment of at least one and P5 sequence complementary pairings, and each and P5 sequence complementary pairings
Spacer sequence is provided between base fragment；
The three-level signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P8 sequences, spacer sequence, P7 sequences, the P8
The end of sequence 5 ' is also modified with fluorophor, for the color of the fluorophor of different target nucleic acid is different or launch wavelength mutually not
It is identical；
The P6 sequences contain the base fragment of at least one and P7 sequence complementary pairings, and each and P7 sequence complementary pairings
Spacer sequence is provided between base fragment；
The P2 sequences, P3 sequences, P4 sequences, P5 sequences, P6 sequences, P7 sequences, P8 sequences are and do not exist hair clip
Structure, does not form dimer, there is no mispairing between each probe interior and probe, and other nucleic acid of whole detection architecture between
There is no the sequence of specific binding.
At least one of the specific P1 sequences of the capture probe of the liver cancer correlation miRNA to be detected in following：Pin
SEQ ID NO.1 to hsa-miR-138-5p, for the SEQ ID NO.2 of hsa-miR-122-5p, for hsa-miR-101-3p
SEQ ID NO.3, for the SEQ ID NO.4 of hsa-miR-21-5p, for the SEQ ID NO.5 of hsa-miR-326,
For the SEQ ID NO.6 of hsa-miR-297, for the SEQ ID NO.7 of hsa-miR-520b, for hsa-miR-181b-5p
SEQ ID NO.8, for the SEQ ID NO.9 of hsa-miR-519b-3p, for the SEQ ID NO.10 of hsa-miR-602,
For the SEQ ID NO.11 of hsa-miR-26a-5p, for the SEQ ID NO.12 of hsa-miR-199a-5p.
Wherein in one embodiment, the P2 sequences are selected from：SEQ ID NO.13～SEQ ID NO.24；The P5 sequences
Column selection is certainly：SEQ ID NO.37～SEQ ID NO.48, the P7 sequences are selected from SEQ ID NO.61～SEQ ID NO.72；
The spacer sequence in the P4 sequences is 3-10 T；The spacer sequence of the P6 sequences is 2-10
T；P8 sequences are polyT.
Wherein in one embodiment, the polyT is 3-10 T.
Wherein in one embodiment, the P4 sequences are selected from：SEQ ID NO.25～SEQ ID NO.36, the P6 sequences
It is selected from：SEQ ID NO.49～SEQ ID NO.60.
Spacerarm sequence wherein in one embodiment, described in the one stage signal amplifying probe between P4 sequences and P3 sequences
Column selection is from 5-20 T；Spacer sequence described in second signal amplifying probe between P5 sequences and P6 sequences is selected from 5-
10 T；Spacer sequence in three-level signal amplifying probe between P7 sequences and P8 sequences is selected from 3-10 T.
Wherein in one embodiment, 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 different target nucleic acid
Fluorophor it is different.
Main advantages of the present invention are：
(1) the detection capture probe that a kind of liver cancer correlation miRNA is provided of the invention and the detection kit being made up of it, institute
The sandwich structure that the capture stated is adopted is (with target miRNA reverse complementary sequence P1-intervening sequence-and one stage signal
The complementary P2 sequences for combining of amplifying probe P3 sequences), compared with prior art, the design is more simple, with more reality
The property used.And the characteristics of the capture probe designed by the technical program can realize high specificity, sensitivity is high.The present invention
Selected signal amplifying probe, is that inventor carries out comprehensive assessment, statistical analysis, many kinds of parameters through lot of experiments
Optimum organization and draw.
(2) in-situ hybridization method itself has the shortcomings that fluorescence signal sensitivity is low, but the present invention adopts new in situ hybridization
Method, by signal amplification system fluorescence signal intensity is improved.Testing process of the present invention can be completed in 8h, single
The miRNA hybridization probes of copy are combined by signal amplifying system with corresponding fluorescence probe, significantly improve miRNA
The detection sensitivity of in situ hybridization.
(3) probe of the various liver cancer correlation miRNA designed by the present invention, can be hybridized under homogeneous reaction condition
Reaction, and there is no non-specific binding between various probes；Designed probe specific good, signal to noise ratio in the detection
It is high.Meanwhile, being applied in combination for various probes makes identification kit and detection method form the intact system of a Detection results.
(4) present invention use many site-specifics of probe to match, the mode of Cascaded amplification realizing the amplification of signal, and not
It is the method for PCR amplifications, improves detection signal, realize the specificity of detection, it is to avoid reverse transcription PCR and reality
When fluorescent quantitative PCR technique false positive.
Specific 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 different shapes
Formula is realizing, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is made to the present invention
The understanding of disclosure is more thorough comprehensive.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook et al., point
Son clone：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described in condition, or
According to the condition proposed by manufacturer.Used various conventional chemical reagent, is commercially available prod in embodiment.
Unless otherwise defined, the technology of all of technology used in the present invention and scientific terminology and the technical field for belonging to the present invention
The implication that personnel are generally understood that is identical.The term used in the description of the present invention is intended merely to describe the mesh of specific embodiment
, it is not used in the restriction present invention.Term "and/or" used in the present invention includes appointing for one or more related Listed Items
Anticipate and all of combination.
Embodiment 1
Liver cancer correlation miRNA detection kits
The present embodiment provides a kind of liver cancer correlation miRNA detection kits, includes capture probe and signal amplifying probe,
Signal amplifying probe is included, one stage signal amplifying probe, second signal amplifying probe, three-level signal amplifying probe.Visit above
The characteristics of needle set has high specificity, sensitivity is high.
1st, capture probe
Capture probe is connection target nucleic acid and one stage signal amplifying probe, and the base sequence of every capture probe is from 5 ' ends to 3 ' ends
Be followed successively by combined with target nucleic acid to be detected specific sequence P1, spacer sequence, can be combined with one stage signal amplifying probe
The P2 sequences of P3 complementary pairings, it is different for the P2 sequences of different target gene.
The spacerarm is for capture probe P2 sequences is spaced apart with specific sequence P1, by arranging in probe interior
The spacer sequence of suitable length, can reduce sterically hindered, improve the efficiency of hybridization reaction and the specificity of hybridization reaction.
The spacerarm of capture probe of the present invention is preferably 5-10 T, and the present embodiment is preferably 5 T.
The present embodiment for hsa-miR-138-5p, hsa-miR-122-5p, hsa-miR-101-3p, hsa-miR-21-5p,
hsa-miR-326、hsa-miR-297、hsa-miR-520b、hsa-miR-181b-5p、hsa-miR-519b-3p、hsa-miR-602、
Hsa-miR-26a-5p and hsa-miR-199a-5p design capture probes, it is concrete such as table 1, table 2：
The P1 sequences of the capture probe of table 1
The P2 sequences of the capture probe of table 2
| SEQ ID NO. | P2 sequences (5 ' → 3 ') | SEQ ID NO. | P2 sequences (5 ' → 3 ') |
| 13 | GTCTATAGTG | 19 | GATGACAGTA |
| 14 | GATTCAGTGA | 20 | AGTACTTGTG |
| 15 | TTGAGTAATG | 21 | AGTCTTGAAG |
| 16 | TGTAATGAGT | 22 | TGATGAATTG |
| 17 | GATTAGTGAT | 23 | ATGACGATAG |
| 18 | GTAGATTAGT | 24 | TTGACGTGAA |
2nd, signal amplifying probe
1) one stage signal amplifying probe
Signal amplifies component includes the one stage signal amplifying probe of or more, and every one stage signal amplifying probe is from 5 '
Hold to 3 ' ends and be followed successively by：P3 sequences, spacer sequence, the P4 sequences for combining, P3 sequences are matched with P2 sequences reverse complemental
By the Cascaded amplification for being implemented in combination with echo signal with capture probe P2 sequences.
In the present embodiment, the P3 sequences are the base sequence with P2 sequence reverse complementals.
Spacer sequence between P3 and P4 sequences of the present invention can be selected from 5-20 T, and the spacerarm that the present embodiment is used is
10 T.
The P4 sequences contain the base fragment of or more and the P5 sequence reverse complementals of second signal amplifying probe,
The base fragment of 2～5 and P2 sequence complementary pairings is preferably comprised, in the present embodiment, P4 contains 3 with P5 reverse complementals
Base fragment, is provided with spacer sequence between identical base fragment, the spacer sequence is preferably 3-10 T, this enforcement
It is to be provided with 3 T in example.
The P4 sequences of the one stage signal amplifying probe of table 3
| SEQ ID NO. | P4 sequences (5 ' → 3 ') |
| 25 | GATCTC TTT GATCTC TTT GATCTC |
| 26 | ATATCA TTT ATATCA TTT ATATCA |
| 27 | TATCTC TTT TATCTC TTT TATCTC |
| 28 | CACATC TTT CACATC TTT CACATC |
| 29 | TCACAT TTT TCACAT TTT TCACAT |
| 30 | ACATCA TTT ACATCA TTT ACATCA |
| 31 | CATCGA TTT CATCGA TTT CATCGA |
| 32 | TCAGTC TTT TCAGTC TTT TCAGTC |
| 33 | ACTCTC TTT ACTCTC TTT ACTCTC |
| 34 | ATCATC TTT ATCATC TTT ATCATC |
| 35 | ACATCC TTT ACATCC TTT ACATCC |
| 36 | TCAGTA TTT TCAGTA TTT TCAGTA |
2) second signal amplifying probe
The present invention holds successively from 5 ' ends containing the second signal amplifying probe of or more, the second signal amplifying probe to 3 '
For：P5 sequences, spacer sequence, the P6 sequences combined with P4 sequence reverse complementals, the P4 contains one or more
P5 sequence reverse complemental base sequences；
Spacer sequence between P5 and P6 sequences of the present invention can be selected from 5-10 T, and the spacerarm that the present embodiment is used is
6 T.
The P5 sequences of the second signal amplifying probe of table 4
| SEQ ID NO. | P5 sequences (5 ' → 3 ') | SEQ ID NO. | P5 sequences (5 ' → 3 ') |
| 37 | GAGATC | 43 | TCGATG |
| 38 | TGATAT | 44 | GACTGA |
| 39 | GAGATA | 45 | GAGAGT |
| 40 | GATGTG | 46 | GATGAT |
| 41 | ATGTGA | 47 | GGATGT |
| 42 | TGATGT | 48 | TACTGA |
The P6 sequences contain the base sequence of or more and the P7 sequence reverse complementals of three-level signal amplifying probe, preferably contain
Have base fragments of the 2-5 with P7 sequence complementary pairings, in the present embodiment, the P6 sequences contain three it is reverse with P7 sequences
Complementary fragment, is provided with spacer sequence between identical base sequence, spacer sequence is preferably 2-10 T, in the present embodiment
To there is 2 T.
The P6 sequences of the second signal amplifying probe of table 5
| SEQ ID NO. | P6 sequences (5 ' → 3 ') |
| 49 | CATGA TT CATGA TT CATGA |
| 50 | ACGAC TT ACGAC TT ACGAC |
| 51 | GACTC TT GACTC TT GACTC |
| 52 | CAGTT TT CAGTT TT CAGTT |
| 53 | ACATG TT ACATG TT ACATG |
| 54 | AGCAT TT AGCAT TT AGCAT |
| 55 | CTAGA TT CTAGA TT CTAGA |
| 56 | ACGTG TT ACGTG TT ACGTG |
| 57 | GACGA TT GACGA TT GACGA |
| 58 | GCTGA TT GCTGA TT GCTGA |
| 59 | TCTGA TT TCTGATT TCTGA |
| 60 | GTATC TT GTATC TT GTATC |
3) three-level signal amplifying probe
Three-level signal amplifying probe of the present invention is followed successively by from 5 ' ends to 3 ' ends：P8 sequences, spacer sequence, P7 sequences,
The P6 sequences contain one or more with P7 sequence reverse complementary sequences；The end of P8 sequences 5 ' is also modified with fluorophor；
Spacer sequence between P7 and P8 sequences of the present invention can be selected from 3-10 T, and the spacerarm that the present embodiment is used is 5
T。
The P7 sequences of the three-level signal amplifying probe of table 6
| SEQ ID NO. | P7 sequences (5 ' → 3 ') | SEQ ID NO. | P7 sequences (5 ' → 3 ') |
| 61 | TCATG | 67 | TCTAG |
| 62 | GTCGT | 68 | CACGT |
| 63 | GAGTC | 69 | TCGTC |
| 64 | AACTG | 70 | TCAGC |
| 65 | CATGT | 71 | TCAGA |
| 66 | ATGCT | 72 | GATAC |
In the present embodiment, P8 sequences are the polyT sequences of 5 bases, and with fluorophor mark, fluorophor can at its 5 ' end
To be selected from：FAM、TET、JOE、HEX、Cy3、TAMRA、ROX、Texas Red、LC RED640、Cy5、
LC RED705 and Alexa Fluor 488, i.e., selected fluorescent base different for the fluorophor of different target nucleic acid
The color of group is different or launch wavelength is different, in order to distinguish different types of target nucleic acid.
Signal amplifying probe designed by the present invention, the particular probe reverse complemental situation about matching completely limited except above-mentioned condition with
Outward, P3, P4, P5, P6, P7, P8 sequence does not form dimerization to there is no hairpin structure between probe interior and probe
Body, there is no mispairing, the sequence that there is no specific binding and other nucleic acid of whole detection architecture between.
A kind of liver cancer correlation miRNA detection kits of embodiment 2
The invention provides a kind of liver cancer correlation miRNA detection kits, the kit can detect that target miRNA includes：
hsa-miR-138-5p、hsa-miR-122-5p、hsa-miR-101-3p、hsa-miR-21-5p、hsa-miR-326、hsa-miR-297、
Hsa-miR-520b, hsa-miR-181b-5p, hsa-miR-519b-3p, hsa-miR-602, hsa-miR-26a-5p and
The expressions such as hsa-miR-199a-5p, in actually detected, can according to specific needs, using corresponding P1～P8 sequences,
Composition detection kit, you can realize detection.
The component of the present embodiment detection kit is included：Capture probe, signal amplifying probe and fluorophor, concrete probe
Component is shown in Table 7.
Detection kit described in the present embodiment, when in use, is randomly divided into 3 groups and is detected.
Detection kit of the table 7 for objectives gene (form numeral is SEQ ID NO.)
Embodiment 3 is detected with the kit in embodiment 2 to sample
The present embodiment is detected using the kit of embodiment 2 to HCC.
HCC source is：Hepatoma cell strain HuH-7, those skilled in the art, according to cell line title, you can existing
The cell line of correlation is obtained in product.
The formula of the various solution is as follows：
Signal amplifying probe mixed liquor in the present embodiment is using the whole probes in the respective list of embodiment 3.
First, sample preprocessing, CTCs is filtered to filter membrane
1. liquid is preserved used in Sample preservation pipe and preserve blood sample, 600 × g horizontal centrifugal 5min abandon supernatant.
2. 4mL PBS and 1mL fixatives are added, is vortexed and is mixed, be stored at room temperature 8min.
3. sample is filtered：Liquid in Sample preservation pipe is transferred in filter, vacuum filtration pumping liquid to the greatest extent is opened；In this guarantor
Deposit addition 4mL PBS in pipe, suction filtration liquid after washing tube wall.
4. filter membrane is transferred in 24 orifice plates, adds the formalins of 400 μ L 4%, room temperature to fix 1h.
5. liquid is removed, adds 1mL PBS to wash per hole three times, 2min is soaked every time.
2nd, it is permeabilized
1. the saturating agents of 50 μ L are added per hole in 24 new orifice plates, filter membrane are taken out from PBS, filter membrane piece EDGE CONTACT blotting paper,
Unnecessary liquid is removed, filter membrane is tipped upside down in agent, i.e., filter membrane iron ring is carved with the one of coding and faces down liquid.
Incubation at room temperature 5min.
2. liquid is removed, adds 1ml PBS to wash twice per hole, 2min is soaked every time.Filter membrane is maintained in PBS to next step
Experimental implementation.
3rd, vitellophag, exposes miRNA so as to probe hybridization
1. the digestive ferment working solution of respective concentration is prepared：
| Reagent component | Each sample consumption |
| Digestive ferment | 1.25μL |
| PBS | 48.75μL |
| Cumulative volume | 50μL |
2. digestive ferment working solution is vortexed and mixes, and dispenses into 24 orifice plates, per the μ l of hole 50.
3. filter membrane is taken out, in back-off to 24 orifice plates on digestive ferment working solution, it is ensured that downwards one side is fully contacted filter membrane with liquid,
Can not be with the presence of bubble.It is stored at room temperature 1h.
4. liquid is removed, adds 1ml PBS to wash per hole three times, 2min is soaked every time.Filter membrane is maintained in PBS
To next step experimental implementation.
4th, probe hybridization, probe-specific sequence is combined with target miRNA sequence
1. capture probe mixed liquor, probe buffer solution preheat 20min using the 40 DEG C of water-baths of front need.
2. capture probe working solution is prepared：
| Reagent component | Each sample consumption |
| Capture 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, in back-off to 24 orifice plates on capture probe working solution, it is ensured that downwards one side is fully contacted filter membrane with liquid,
Can not be with the presence of bubble.
4. 24 orifice plate lids are covered, and 40 ± 1 DEG C are incubated 3 hours.
5. liquid is removed, adds 1ml RI cleaning solutions to wash per hole three times, 2min is soaked every time.Filter membrane is maintained in cleaning solution
To next step experimental implementation, sample soak time no more than 30min in cleaning solution.
5th, desired mRNA sequences signal amplifies
1. probe buffer solution preheats 20min using the 40 DEG C of water-baths of front need.
2. probe face liquid is prepared：
| Reagent component | Each sample consumption |
| Signal amplifying 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, it is ensured that downwards one side is fully contacted filter membrane with liquid, no
Can be with the presence of bubble.
4. 24 orifice plate lids are covered, and 40 ± 1 DEG C are incubated 3 hours.
5. liquid is removed, adds 1ml cleaning solutions to wash per hole three times, 2min is soaked every time.Filter membrane is maintained in cleaning solution under
One step experimental implementation, sample soak time no more than 30min in cleaning solution.
6th, develop the color, fluorescence labeling echo signal
1. colorbuffer (40 DEG C of preheatings) lucifuge is vortexed and mixes, dispenses into 24 orifice plates, per the μ l of hole 50.
2. filter membrane is taken out, in back-off to 24 orifice plates on colorbuffer, it is ensured that downwards one side is fully contacted filter membrane with liquid, no
Can be with the presence of bubble.
3. 24 orifice plate lids, 40 ± 1 DEG C of incubation 30min are covered.
4. liquid is removed, adds 1ml cleaning solutions to wash per hole three times, 2min is soaked every time.Filter membrane is maintained in cleaning solution under
One step experimental implementation, sample soak time no more than 30min in cleaning solution.
7th, fluorescence microscope CTCs
The reference substance of the present invention uses DAPI as nucleus fluorophor, its transmitting blue-fluorescence signal.
1. filter membrane cell is faced up and be placed on slide, cut off filter membrane along iron ring inner ring, plus the anti-quenchers of 10 μ L, cover
The cover glass of 18mm × 18mm, direct microscopy or is placed in -20 DEG C of preservations.
2. CTC opposite sex nuclear volumes are counted by 20 times of object lens.
3. according to the different in nature nuclear locations of 10 times of object lens positioning, oil dripping, with oil mirror observation experiment result, and Taking Pictures recording result.
4. and then further according to the next opposite sex nuclear locations of 10 times of object lens positioning, oil dripping, is taken pictures note with oil mirror observation experiment result and the visual field
Record result.
5. repeat to all of 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 8
8th, testing result judges and analyzes
1. masculine liver cancer cellular identification standard
On filter membrane, HCC is enriched with, the positive criterion of HCC is：
1) with corresponding target miRNA specific marker's things, show as being shown under corresponding fluorescence channel in this kit
Fluorescence signal point.
2) nucleus DAPI stained positives.
3) HCC nuclear shape is irregular, with diameter greater than 10 μm, hence it is evident that more than filter sizes, and filter sizes are 7 μm.In vain
Cell size is close with filter membrane hole size.
2. above-mentioned detection method is used, each sample is detected and observed, wherein, for nuclear DAPI dyeing, make
Indicate whether to detect fluorescence with "-" or "+"；For the fluorescence signal intensity of target detection miRNA, each sample is read respectively
The miRNA phosphor dot quantity of the respective color of 10 HCCs in this, and averagely counting is calculated, concrete outcome is：
The pattern detection result of table 9 (fluorescence signal points)
The stability of the kit of embodiment 4
1st, stabilization of kit detection
The present invention provides a kind of kit of liver cancer correlation miRNA detections, and described kit is directed to different target detections
MiRNA, chooses the capture probe of varying number, constitutes corresponding probe mixed liquor, so as to realize that varying number liver cancer is related
The parallel detection of miRNA.
The kit that the present embodiment will be constituted using probe groups Group1 of embodiment 2,15 to three groups of different cell line sources
Hsa-miR-138-5p, hsa-miR-122-5p, hsa-miR-101-3p in kind of sample (5 samples of every kind of cell line),
Hsa-miR-21-5p expressions are detected, so as to assess the stability of kit of the present invention.
2nd, with regard to detecting samples sources
The present embodiment use liver cancer 3 in different cell line sources HCC as detection object, so as to verify that its is effective
Property and stability (repeatability), concrete cell line and sample as shown in table 10, those skilled in the art, according to cell line name
Claim, you can the cell line of correlation is obtained in existing product.Experimental procedure reference implementation example 3.
The cell line of table 10 and detection sample
| Catalogue number(Cat.No.) | Hepatoma cell strain | Experimental group |
| 16～sample of sample 20 | HuH-7 | Group4 |
| 21～sample of sample 25 | HepG2 | Group5 |
| 26～sample of sample 30 | QGY-7701 | Group6 |
3rd, testing result
Using mentioned reagent box, each sample is detected and observed, wherein, for nuclear DAPI dyeing, using "-"
Or "+" indicates whether to detect fluorescence；For the fluorescence signal intensity of target detection miRNA marker, each sample is read respectively
The miRNA phosphor dot quantity of the respective color of 10 HCCs in this, and averagely counting is calculated, concrete outcome is：
The pattern detection result of table 11 (fluorescence signal points)
From above-mentioned testing result, on the one hand, the sample in different cell line sources, its testing result is different, therefore, this
It is bright being that by different miRNA expressions detections, it is effective；On the other hand, 5 of same cell strain source
MiRNA in sample, its hsa-miR-138-5p, hsa-miR-122-5p, hsa-miR-101-3p, hsa-miR-21-5p etc. 4
Fluorescence points testing result it is close (± 3), be specifically shown in Group4 (sample 16～20), Group5 (sample 21～25) or
Group6 (sample 26～30), therefore, this kit is reproducible；As can be seen here, kit of the present invention be it is effective, surely
Qualitative reliable, other are directed to the kit of difference miRNA species, and its result is still reliable and stable, and concrete data are omitted.
The target miRNA quantity of embodiment 5 is selected
1st, design (selection of capture probe quantity) prepared by kit
The present invention provides a kind of kit of target miRNA detections, and described kit is directed to different target detections
MiRNA, chooses the capture probe of varying number, corresponding probe mixed liquor is constituted, so as to realize varying number miRNA's
Parallel detection.
The present embodiment is respectively directed to a kind, 3 kinds, 5 kinds, 7 kinds of miRNA, and from capture probe, signal amplifying probe is selected
One stage signal method probe, two grades of amplifying probes and three-level signal amplifying probe, constitute detection kit, to same cell line
The sample in HuH-7 sources is detected that contrast its Detection results, concrete composition such as table 12, the probe is selected from embodiment 1～2,
Experimental procedure reference implementation example 3.
Table 12 selects the capture probe (form numeral is SEQ ID NO.) of varying number target miRNA
2nd, using mentioned reagent box, each sample is detected and is observed, wherein, for nuclear DAPI dyeing, using "-"
Or "+" indicates whether to detect fluorescence；For the fluorescence signal intensity of target detection miRNA marker, each sample is read respectively
The miRNA phosphor dot quantity of the respective color of 10 HCCs in this, and averagely counting is calculated, concrete outcome is：
The pattern detection result of table 13 (fluorescence signal points)
By above-mentioned 4 groups of Experimental comparisons, this kit can be directed to the target miRNA of varying number and be detected, make
With 1,3 and 5,7 for different miRNA capture probes can complete detection, specificity and stability all very
It is good.
Other are directed to the kit of the use varying number capture probe of marker gene, and its result is still reliable and stable, concrete data
Omit.
The selection of the label probe of embodiment 6
1st, the design (selection of signal amplifying probe combination) that prepared by kit
The present invention provides a kind of kit of liver cancer correlation miRNA detections, and described kit is directed to different target detections
MiRNA, chooses unlike signal probe combinations, corresponding probe mixed liquor is constituted, so as to realize the parallel detection of miRNA.
The kit that the present embodiment will be constituted using the signal amplifying probes at different levels for being provided of embodiment 1, to same cell line
(HuH-7) hsa-miR-138-5p, hsa-miR-101-3p, hsa-miR-326 and hsa-miR-181b-5p in 5 samples in source
Expression detected, so as to assess P2 provided by the present invention, P3, P4, P5, P6, P7 signal amplifying probe it is suitable
The property used.Specific kit forms are as follows：
The unlike signal amplifying probe of table 14 combination (form numeral is SEQ ID NO.)
2nd, using mentioned reagent box, (detection method described in Application Example 3) and observation are detected to each sample, wherein, pin
Nuclear DAPI is dyeed, indicates whether to detect fluorescence using "-" or "+"；For target detection miRNA marker
Fluorescence signal intensity, the miRNA phosphor dot quantity of the respective color of 10 HCCs in each sample is read respectively, and
Averagely counting is calculated, concrete outcome is：
The testing result of the unlike signal amplifying probe of table 15 combination
By above-mentioned 3 groups of Experimental comparisons, the kit constituted using unlike signal probe combinations is detected to miRNA,
Its testing result is consistent, provided by the present invention in addition to the miRNA and its amplifying probe of the present embodiment detection are combined
The combination of other miRNA and other signal amplifying probes, can equally realize the Detection results of the present embodiment, concrete test knot
Fruit omits.It follows that the signal amplifying probe that the present invention is provided can need arbitrarily to be arranged in pairs or groups according to practical operation, and
MiRNA detections of the present invention can be realized, its testing result is consistent, illustrates signal amplifying probe pair provided by the present invention
There is good applicability in liver cancer correlation miRNA detections.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not in above-described embodiment
The all possible combination of each technical characteristic be all described, if however, there is no contradiction in the combination of these technical characteristics,
All it is considered to be the scope of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and in detail, but can not be because
This and be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art,
On the premise of without departing from present inventive concept, some deformations and improvement can also be made, these belong to protection scope of the present invention.
Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of liver cancer correlation microRNA detection kits, it is characterised in that include：For every kind of liver cancer to be detected
Related microRNA has at least one one stage signal amplifying probe, at least one second signal amplifying probe and at least one 3
Level signal amplifying probe, and capture probe, the liver cancer correlation microRNA is selected from::hsa-miR-138-5p、
hsa-miR-122-5p、hsa-miR-101-3p、hsa-miR-21-5p、hsa-miR-326、hsa-miR-297、hsa-miR-520b、
In hsa-miR-181b-5p, hsa-miR-519b-3p, hsa-miR-602, hsa-miR-26a-5p and hsa-miR-199a-5p
At least one, wherein：
The capture probe is used for linking objective nucleic acid and one stage signal amplifying probe, and every capture probe is held successively from 5 ' ends to 3 '
It is different for the P2 sequences of different target gene for specific P1 sequences, spacer sequence, P2 sequences；
The one stage signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences, spacer sequence, P3 sequences, P3 sequences
With P2 sequence complementary pairings；
The second signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P5 sequences, spacer sequence, P6 sequences；
The P4 sequences contain the base fragment of at least one and P5 sequence complementary pairings, and each and P5 sequence complementary pairings
Base fragment between be provided with spacer sequence；
The three-level signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P8 sequences, spacer sequence, P7 sequences, the P8
The end of sequence 5 ' is also modified with fluorophor, for the color of the fluorophor of different target nucleic acid is different or launch wavelength mutually not
It is identical；
The P6 sequences contain the base fragment of at least one and P7 sequence complementary pairings, and each and P7 sequence complementary pairings
Base fragment between be provided with spacer sequence；
The P2 sequences, P3 sequences, P4 sequences, P5 sequences, P6 sequences, P7 sequences, P8 sequences are and do not exist hairpin structure,
Dimer is not formed between each probe interior and probe, there is no mispairing, do not existed and other nucleic acid of whole detection architecture between
The sequence of specific binding.
2. liver cancer correlation microRNA detection kits according to claim 1, it is characterised in that described to be detected
The specific P1 sequences of the capture probe of liver cancer correlation miRNA are selected from following at least one：For the SEQ of hsa-miR-138-5p
ID NO.1, for the SEQ ID NO.2 of hsa-miR-122-5p, for the SEQ ID NO.3 of hsa-miR-101-3p, pin
SEQ ID NO.4 to hsa-miR-21-5p, for the SEQ ID NO.5 of hsa-miR-326, for hsa-miR-297's
SEQ ID NO.6, for the SEQ ID NO.7 of hsa-miR-520b, for the SEQ ID NO.8 of hsa-miR-181b-5p,
For the SEQ ID NO.9 of hsa-miR-519b-3p, for the SEQ ID NO.10 of hsa-miR-602, for
The SEQ ID NO.11 of hsa-miR-26a-5p, for the SEQ ID NO.12 of hsa-miR-199a-5p.
3. liver cancer correlation microRNA detection kits according to claim 2, it is characterised in that the P2 sequences are selected from：
SEQ ID NO.13～SEQ ID NO.24；The P5 sequences are selected from：SEQ ID NO.37～SEQ ID NO.48, the P7
Sequence is selected from SEQ ID NO.61～SEQ ID NO.72；The spacer sequence in the P4 sequences is 3-10 T；
The spacer sequence of the P6 sequences is 2-10 T；P8 sequences are polyT.
4. liver cancer correlation microRNA detection kits according to claim 3, it is characterised in that the polyT is
3-10 T.
5. liver cancer correlation microRNA detection kits according to claim 1, it is characterised in that the P4 sequences
It is selected from：SEQ ID NO.25～SEQ ID NO.36, the P6 sequences are selected from：SEQ ID NO.49～SEQ ID NO.60.
6. liver cancer correlation microRNA detection kits according to any one of claim 1-5, it is characterised in that described
Spacer sequence described in one stage signal amplifying probe between P4 sequences and P3 sequences is selected from 5-20 T；Second signal is put
Spacer sequence described in big probe between P5 sequences and P6 sequences is selected from 5-10 T；P7 in three-level signal amplifying probe
Spacer sequence between sequence and P8 sequences is selected from 3-10 T.
7. liver cancer correlation microRNA detection kits according to any one of claim 1-5, it is characterised in that described
Fluorophor is selected from：FAM、TET、JOE、HEX、Cy3、TAMRA、ROX、Texas Red、LC RED640、
Cy5, LC RED705 and Alexa Fluor 488, and it is different for the fluorophor of different target nucleic acid.
8. liver cancer correlation microRNA detection kits according to any one of claim 1-5, it is characterised in that described
P2 sequences are：For the SEQ ID NO.13 of hsa-miR-138-5p, for the SEQ ID NO.14 of hsa-miR-122-5p,
For the SEQ ID NO.15 of hsa-miR-101-3p, for the SEQ ID NO.16 of hsa-miR-21-5p, for
The SEQ ID NO.17 of hsa-miR-326, for the SEQ ID NO.18 of hsa-miR-297, for the SEQ of hsa-miR-520b
ID NO.19, for the SEQ ID NO.20 of hsa-miR-181b-5p, for the SEQ ID NO.21 of hsa-miR-519b-3p,
For the SEQ ID NO.22 of hsa-miR-602, for the SEQ ID NO.23 of hsa-miR-26a-5p, for
The SEQ ID NO.24 of hsa-miR-199a-5p.
9. liver cancer correlation microRNA detection kits according to any one of claim 1-5, it is characterised in that described
P5 sequences are：For the SEQ ID NO.37 of hsa-miR-138-5p, for the SEQ ID NO.38 of hsa-miR-122-5p,
For the SEQ ID NO.39 of hsa-miR-101-3p, for the SEQ ID NO.40 of hsa-miR-21-5p, for
The SEQ ID NO.41 of hsa-miR-326, for the SEQ ID NO.42 of hsa-miR-297, for the SEQ of hsa-miR-520b
ID NO.43, for the SEQ ID NO.44 of hsa-miR-181b-5p, for the SEQ ID NO.45 of hsa-miR-519b-3p,
For the SEQ ID NO.46 of hsa-miR-602, for the SEQ ID NO.47 of hsa-miR-26a-5p, for
The SEQ ID NO.48 of hsa-miR-199a-5p.
10. liver cancer correlation microRNA detection kits according to any one of claim 1-5, it is characterised in that institute
Stating P7 sequences is：For the SEQ ID NO.61 of hsa-miR-138-5p, for the SEQ ID NO.62 of hsa-miR-122-5p,
For the SEQ ID NO.63 of hsa-miR-101-3p, for the SEQ ID NO.64 of hsa-miR-21-5p, for
The SEQ ID NO.65 of hsa-miR-326, for the SEQ ID NO.66 of hsa-miR-297, for the SEQ of hsa-miR-520b
ID NO.67, for the SEQ ID NO.68 of hsa-miR-181b-5p, for the SEQ ID NO.69 of hsa-miR-519b-3p,
For the SEQ ID NO.70 of hsa-miR-602, for the SEQ ID NO.71 of hsa-miR-26a-5p, for
The SEQ ID NO.72 of hsa-miR-199a-5p.
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| CN108192970A (en) * | 2018-01-24 | 2018-06-22 | 苏州大学 | A kind of diagnosing cancer of liver marker and its application |
| CN113227375A (en) * | 2018-12-20 | 2021-08-06 | 原住民股份有限公司 | Synthetic microRNA mimetics |
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