CN106636079A - Nasopharyngeal carcinoma microRNA detection kit - Google Patents
Nasopharyngeal carcinoma microRNA detection kit Download PDFInfo
- Publication number
- CN106636079A CN106636079A CN201510733248.3A CN201510733248A CN106636079A CN 106636079 A CN106636079 A CN 106636079A CN 201510733248 A CN201510733248 A CN 201510733248A CN 106636079 A CN106636079 A CN 106636079A
- Authority
- CN
- China
- Prior art keywords
- sequences
- mir
- hsa
- probe
- signal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular biology, and particularly relates to a nasopharyngeal carcinoma microRNA detection kit. A purpose of the present invention is to solve the problems of poor stability, poor reproducibility, low specificity and easy false positive generation of the existing nasopharyngeal carcinoma microRNA detection kit. The invention provides the nasopharyngeal carcinoma microRNA detection kit, which comprises a probe composition, wherein the probe composition comprises a capture probe bound with a target microRNA and a signal amplification composition, and the target microRNA is one or a plurality of microRNA selected from hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-29c-3p, hsa-miR-223-3p, hsa-miR-34b-3p, hsa-miR-34c-5p, hsa-miR-212-3p, hsa-miR-216a-5p, hsa-miR-217, hsa-miR-151a-3p, and hsa-miR-192-5p. The nasopharyngeal carcinoma microRNA detection kit of the present invention has advantages of good stability, good repeatability, high specificity, less false positive generation, and high sensitivity.
Description
Technical field
The invention belongs to biology field, more particularly to a kind of nasopharyngeal carcinoma microRNA detection reagent
Box.
Background technology
MicroRNAs (miRNAs) is that the class found in eucaryote is endogenic with regulation and control work(
The non-coding RNA of energy, its size is about 20～25 nucleotides.Ripe miRNAs is by longer
Primary transcript is produced through a series of shearing of nucleases, is subsequently assembled into RNA inductions
Silencing complex (RNA-induced silencing complex, RISC), by the side of base pair complementarity
Formula recognize target miRNA, and according to the difference of complementarity instruct silencing complex degraded target miRNA or
Person checks the translation of target miRNA.It has recently been demonstrated that miRNA participates in various regulation on the way
Footpath, including development, virus defense, hematopoiesis, orga- nogenesis, cell propagation and apoptosis, fatty generation
Thank etc..Discovered in recent years, the unconventionality expression of part miRNA is close with the occurrence and development of various diseases
Correlation, such as：The unconventionality expression of miRNA is closely related with the occurrence and development of nasopharyngeal carcinoma.Researcher adopts
Biochip technology, by 31 parts of Nasopharyngeal Carcinoma During Biopsy samples and 10 parts of normal nasopharyngeal epithelial cell sample contrasts
207 miRNA are analyzed, the obvious miRNA of 8 differential expressions is as a result found that, is respectively：
MiR-29c, miR-34b, miR-34c, miR-212, miR-216, miR-217, miR-151 and miR-192,
Wherein, in nasopharyngeal carcinoma cell, miR-29c expression concentration only has the 1/5 of normal nasopharyngeal epithelial cell.This
Also there are some researches show outward, miR-17-92 and miR155 is that significantly height is expressed in nasopharyngeal carcinoma, miR-34
Family, miR-143, miR-145 then express downward.
Mainly have for the detection method of nasopharyngeal carcinoma microRNA：It is Northern Blot, genetic chip, glimmering
The quantitative sonde method of light, bead-based flow-cytometry technology.However, said method has susceptibility
It is low, time-consuming, accuracy is low, RNA large usage quantity, testing cost are expensive, poor repeatability the shortcomings of.
At present.The hybridization in situ technique for overcoming disadvantages mentioned above is the tissue that a kind of positioning and morphologic detection are preserved
The method of specificity microRNA sequences in section or cellular preparations.However, current existing original position
In hybridization technique, the kit that the multiple parallel detection for nasopharyngeal carcinoma microRNA is used still is deposited
In many problems, such as：Stability is poor, repeatability is weak, specificity is low, false positive and spirit easily occur
The low shortcoming of sensitivity.
Therefore, a kind of good stability, reproducible, specific height are developed, is not easy false positive occur
And a kind of detection kit of high nasopharyngeal carcinoma microRNA of sensitivity, become people in the art
Member's problem demanding prompt solution.
The content of the invention
In view of this, the invention provides a kind of detection kit of nasopharyngeal carcinoma microRNA, the inspection
Test agent box have good stability, it is reproducible, specific it is high, be not easy to occur false positive and sensitive
The high advantage of degree.
The invention provides a kind of probe compositions, the probe compositions include：With target microRNA
With reference to capture probe and signal amplify composition, the target microRNA is selected from
hsa-miR-17-5p、hsa-miR-20a-5p、hsa-miR-29c-3p、hsa-miR-223-3p、
hsa-miR-34b-3p、hsa-miR-34c-5p、hsa-miR-212-3p、hsa-miR-216a-5p、
One or more in hsa-miR-217, hsa-miR-151a-3p and hsa-miR-192-5p.
Preferably, the base sequence of the capture probe is followed successively by from 5 ' ends to 3 ' ends：With the target
Specific sequence P1, the first spacer sequence, P2 sequences that microRNA is combined, the P1 sequences are
Any one in SEQ ID NO.1～SEQ ID NO.11, the P2 sequences be SEQ ID NO.12～
Any one in SEQ ID NO.23, first spacer sequence is 5～10 T.
Preferably, the signal amplifies composition and is selected from：First signal amplifies composition, secondary signal to be put
Big composition and the 3rd signal amplify any one of composition；It is one that first signal amplifies composition
Level signal amplifying probe, first signal amplifies 3 ' ends of composition and is also modified with the first fluorophor,
First fluorophor is selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas
Any one in Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488；It is described
It is one stage signal amplifying probe and second signal amplifying probe that secondary signal amplifies composition, and described second believes
3 ' the ends for number amplifying composition are also modified with the second fluorophor, second fluorophor selected from FAM,
TET、JOE、HEX、Cy3、TAMRA、ROX、Texas Red、LC RED640、Cy5、LC
Any one in RED705 and Alexa Fluor 488；3rd signal amplifies composition for one-level letter
Number amplifying probe, second signal amplifying probe and three-level signal amplifying probe, the 3rd signal amplification group
3 ' ends of compound are also modified with the 3rd fluorophor, the 3rd fluorophor selected from FAM, TET, JOE,
HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705 and
Any one in Alexa Fluor 488.
Preferably, the base sequence of the one stage signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences
The P3 sequences that row, the second spacer sequence are combined with the P2 sequences reverse complemental；The P4 sequences
For any one in SEQ ID NO.24～SEQ ID NO.35, second spacer sequence is 5～10
Individual T.
Preferably, the base sequence of the second signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P5
Sequence, the 3rd spacer sequence, P6 sequences, the P5 sequences contain one or more with the P4 sequences
The base sequence of row reverse complemental；The P5 sequences are in SEQ ID NO.36～SEQ ID NO.47
Any one, the P6 sequences are any one in SEQ ID NO.48～SEQ ID NO.59, institute
The 3rd spacer sequence is stated for 5～10 T.
Preferably, the base sequence of the big probe of the three-level signal is followed successively by from 5 ' ends to 3 ' ends：P8 sequences,
4th spacer sequence, P7 sequences, the P7 sequences contain one or more it is reverse with the P5 sequences
Complementary base sequence；The P7 sequences are any one in SEQ ID NO.60～SEQ ID NO.71
Bar, the P8 sequences are the polyT sequences of 5 bases, and the 4th spacer sequence is 5～10 T.
Preferably, the P1 sequences, P2 sequences, P3 sequences, P4 sequences, P5 sequences, P6 sequences,
There is no hairpin structure in P7 sequences, P8 interior sequences.
Preferably, first spacer sequence, second spacer sequence, the 3rd spacerarm
Sequence is identical with the quantity of T in the 4th spacer sequence or difference.
Preferably, first fluorophor, second fluorophor and the 3rd fluorophor phase
It is same or different.
Present invention also offers a kind of nasopharyngeal carcinoma microRNA detection kit, the nasopharyngeal carcinoma
MicroRNA detection kits include the probe compositions described in any of the above one.
In sum, the present invention is used as target by selecting in the abnormal microRNA of nasopharyngeal carcinoma,
A kind of kit for amplifying composition containing the capture probe combined with target and signal of system, overcomes existing
The kit existence and stability that the multiple parallel detection for having nasopharyngeal carcinoma microRNA is used is poor, repeated
It is weak, specificity is low, false-positive shortcoming easily occur, a kind of obtained nasopharyngeal carcinoma microRNA of the present invention
Kit has good stability, reproducible, specific height, is not easy false positive and sensitivity occur
High advantage.
Specific embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that institute
The embodiment of description is only a part of embodiment of the invention, rather than the embodiment of whole.Based on this
Embodiment in bright, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
In order to the present invention is described in more detail, with reference to a kind of nasopharyngeal carcinoma that embodiment is provided the present invention
MicroRNA detection kits, are specifically described.
Embodiment 1
Present embodiments provide the preparation method of capture probe, the alkali of the capture probe designed by the present embodiment
Basic sequence from 5 ' end to 3 ' end be followed successively by combined with target microRNA to be detected specific sequence P1,
Spacer sequence, P2 sequences.
Spacer sequence just capture probe P2 sequences can be spaced apart with target microRNA to be detected
Come, by the spacer sequence that suitable length is arranged in probe interior, can reduce sterically hindered, improve miscellaneous
Hand over the efficiency of reaction and the specificity of hybridization reaction.The spacer sequence of capture probe of the present invention is preferably 5
Individual T.
The present embodiment for hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-29c-3p,
hsa-miR-223-3p、hsa-miR-34b-3p、hsa-miR-34c-5p、hsa-miR-212-3p、
Hsa-miR-216a-5p, hsa-miR-217, hsa-miR-151a-3p and hsa-miR-192-5p design is caught
Probe is obtained, 1, table 2 is specifically shown in Table：
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 ') |
| 12 | GTCTATAGTG | 18 | GATGACAGTA |
| 13 | GATTCAGTGA | 19 | AGTACTTGTG |
| 14 | TTGAGTAATG | 20 | AGTCTTGAAG |
| 15 | TGTAATGAGT | 21 | TGATGAATTG |
| 16 | GATTAGTGAT | 22 | ATGACGATAG |
| 17 | GTAGATTAGT | 23 | TTGACGTGAA |
Embodiment 2
The preparation method that signal amplifies component is present embodiments provided, preparation method described in the present embodiment is obtained
Signal amplify component be selected from：First signal amplifies component, secondary signal amplification component and the 3rd signal to be put
Any one of big component；First signal amplifies component includes one stage signal amplifying probe, described the
One signal amplifies 3 ' ends of component and is also modified with fluorophor；The secondary signal amplifies component for one-level letter
Number amplifying probe and second signal amplifying probe, the secondary signal amplify 3 ' ends of component be also modified with it is glimmering
Light group；3rd signal amplify component be one stage signal amplifying probe, second signal amplifying probe and
Three-level signal amplifying probe, the 3rd signal amplifies 3 ' ends of component and is also modified with fluorophor.
(1), the first signal amplify component, the first signal amplify component include one stage signal amplifying probe and
The fluorophor for amplifying the end of component 3 ' in the first signal is modified, is modified and is amplified the end of component 3 ' in the first signal
Any one of fluorophor in Cy3, Cy5 and Alexa Flour 488.
The base sequence of one stage signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences, spacerarm sequence
The P3 sequences that row and the pairing of P2 sequences reverse complemental are combined, the spacerarm sequence of one stage signal amplifying probe
Row are preferably 10 T.
The P4 sequences of the one stage signal amplifying probe of the present embodiment design, are specifically shown in Table 3.
The P4 sequences of the one stage signal amplifying probe of table 3
| SEQ ID NO. | P4 sequences (5 ' → 3 ') |
| 24 | GATCTC TTTTT GATCTC TTTTT GATCTC |
| 25 | ATATCA TTTTT ATATCA TTTTT ATATCA |
| 26 | TATCTC TTTTT TATCTC TTTTT TATCTC |
| 27 | CACATC TTTTT CACATC TTTTT CACATC |
| 28 | TCACAT TTTTT TCACAT TTTTT TCACAT |
| 29 | ACATCA TTTTT ACATCA TTTTT ACATCA |
| 30 | CATCGA TTTTT CATCGA TTTTT CATCGA |
| 31 | TCAGTC TTTTT TCAGTC TTTTT TCAGTC |
| 32 | ACTCTC TTTTT ACTCTC TTTTT ACTCTC |
| 33 | ATCATC TTTTT ATCATC TTTTT ATCATC |
| 34 | ACATCC TTTTT ACATCC TTTTT ACATCC |
| 35 | TCATCA TTTTT TCATCA TTTTT TCATCA |
(2), secondary signal amplify component, secondary signal amplify component include one stage signal amplifying probe and
Second signal amplifying probe, and modification, in the fluorophor at secondary signal amplification component 3 ' end, modification exists
Secondary signal amplifies the fluorophor at the end of component 3 ' selected from selected from Cy3, Cy5 and Alexa Flour 488
In any one.
The base sequence of one stage signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences, spacerarm sequence
The P3 sequences that row and the pairing of P2 sequences reverse complemental are combined, the spacerarm sequence of one stage signal amplifying probe
Row are preferably 10 T.
The base sequence of second signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P5 sequences, spacerarm
Sequence, P6 sequences, P5 sequences contain the base sequence of or more and P4 sequence reverse complementals, and two
The spacer sequence of level signal amplifying probe is preferably 6 T.
The P5 sequences of the second signal amplifying probe of the present embodiment design, are specifically shown in Table 4.
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 ') |
| 36 | GAGATC | 42 | TCGATG |
| 37 | TGATAT | 43 | GACTGA |
| 38 | GAGATA | 44 | GAGAGT |
| 39 | GATGTG | 45 | GATGAT |
| 40 | ATGTGA | 46 | GGATGT |
| 41 | TGATGT | 47 | TGATGA |
The P6 sequences of the second signal amplifying probe of the present embodiment design, are specifically shown in Table 5.
The P6 sequences of the second signal amplifying probe of table 5
| SEQ ID NO. | P6 sequences (5 ' → 3 ') |
| 48 | TACGATTT TACGATTT TACGA |
| 49 | CATAGTTT CATAG TTT CATAG |
| 50 | TAGCA TTT TAGCA TTT TAGCA |
| 51 | ACGTA TTT ACGTATTT ACGTA |
| 52 | TGAAC TTT TGAAC TTT TGAAC |
| 53 | CATTG TTT CATTGTTT CATTG |
| 54 | TGTCCTTT TGTCC TTT TGTCC |
| 55 | CTACG TTT CTACG TTT CTACG |
| 56 | AGCAG TTT AGCAG TTT AGCAG |
| 57 | ACGCT TTT ACGCTTTT ACGCT |
| 58 | TCTAG TTT TCTAG TTT TCTAG |
| 59 | CTCTA TTT CTCTA TTT CTCTA |
(3), the 3rd signal amplify component, the 3rd signal amplify component include one stage signal amplifying probe,
Second signal amplifying probe and three-level signal amplifying probe, and modification is at the 3rd signal amplification component 3 ' end
Fluorophor, modify the 3rd signal amplify component 3 ' end fluorophor be selected from Cy3, Cy5, TET
And any one in Alexa Flour 488.
The base sequence of one stage signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences, spacerarm sequence
The P3 sequences that row and the pairing of P2 sequences reverse complemental are combined, the spacerarm sequence of one stage signal amplifying probe
Row are preferably 10 T.
The base sequence of second signal amplifying probe is followed successively by from 5 ' ends to 3 ' ends：P5 sequences, spacerarm
Sequence, P6 sequences, P5 sequences contain the base sequence of or more and P4 sequence reverse complementals, and two
The spacer sequence of level signal amplifying probe is preferably 6 T.
The base sequence of the big probe of three-level signal is followed successively by from 5 ' ends to 3 ' ends：P8 sequences, spacer sequence,
P7 sequences, P7 sequences contain the base sequence of or more and P5 sequence reverse complementals, three-level signal
The spacer sequence of amplifying probe is preferably 5 T.
The P7 sequences of the three-level signal amplifying probe of the present embodiment design, are specifically shown in Table 6.
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 ') |
| 60 | TCGTA | 66 | GGACA |
| 61 | CTATG | 67 | CGTAG |
| 62 | TGCTA | 68 | CTGCT |
| 63 | TACGT | 69 | AGCGT |
| 64 | GTTCA | 70 | CTAGA |
| 65 | CAATG | 71 | TAGAG |
In the present embodiment, P8 sequences are the polyT sequences of 5 bases.
Embodiment 3
Present embodiments provide a kind of preparation method of nasopharyngeal carcinoma microRNA detection kit, the nose
Pharynx cancer microRNA detection kit includes：Probe components obtained in embodiment 1 and embodiment 2 are obtained
Signal amplify component.
A kind of composition of nasopharyngeal carcinoma microRNA detection kit of the present embodiment design, is specifically shown in Table 7.
A kind of nasopharyngeal carcinoma microRNA detection kit of table 7 (form numeral is SEQ ID NO.)
Embodiment 4
Present embodiments provide a kind of nasopharyngeal carcinoma microRNA detection kit obtained in Application Example 3
Nasopharyngeal carcinoma cell is detected.The source of the nasopharyngeal carcinoma cell that the present embodiment is used is：Nasopharyngeal carcinoma is thin
Born of the same parents strain CNE-2Z (is purchased from ATCC).
Table 8 provides the formula of the various solution that the present embodiment is used.
The solution formula of table 8
Step one：Sample preprocessing, by CNE-2Z cells filter membrane is transferred to
1st, CNE-2Z freeze-stored cell pipes are taken out from liquid nitrogen and is recovered, after cell in pipe melts,
Cell quantity 1 × 107, 600 × g horizontal centrifugal 5min abandon supernatant, preserve used in Sample preservation pipe
Liquid preserves blood sample.
2nd, 4mL PBS and 1mL fixatives are added, is vortexed and mixes, be stored at room temperature 8min.
3rd, sample is filtered：Liquid in Sample preservation pipe is transferred in filter, vacuum filtration pump is opened
Take out most liquid；4mL PBS, suction filtration liquid after washing tube wall are added in this preservation pipe.
4th, filter membrane is transferred in 24 orifice plates, adds the formalins of 400 μ L 4%, room temperature to fix 1h.
5th, liquid is removed, adds 1mL PBS to wash per hole three times, 2min is soaked every time.
Step 2：It is permeabilized
1st, the saturating agents of 50 μ L are added per hole in one piece of 24 new orifice plate, filter membrane are taken out from PBS,
Filter membrane piece EDGE CONTACT blotting paper, removes unnecessary liquid, 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.
2nd, liquid is removed, adds 1ml PBS to wash twice per hole, 2min is soaked every time.Filter membrane is protected
Hold in PBS to next step experimental implementation.
Step 3：Vitellophag, exposes miRNA so as to capture probe hybridization
1st, digestive ferment working solution is prepared, takes the μ L of digestive ferment 1.25 and PBS48.75 μ L, digestive ferment work is obtained
Make the μ L of liquid 50.
2nd, digestive ferment working solution is vortexed and mixes, and dispenses into 24 orifice plates, per the μ l of hole 50.
3rd, filter membrane is taken out, in back-off to 24 orifice plates on digestive ferment working solution, it is ensured that the downward one side of filter membrane
It is fully contacted with liquid, it is impossible to the presence of bubble.It is stored at room temperature 1h.
4th, liquid is removed, adds 1ml PBS to wash per hole three times, 2min is soaked every time.Filter membrane is kept
To next step experimental implementation in PBS.
Step 4：Probe hybridizes, and probe-specific sequence is combined with target miRNA sequence
1st, capture probe mixed liquor, probe buffer solution preheat 20min using the 40 DEG C of water-baths of front need.
2nd, capture probe working solution is prepared, takes the μ L of the capture probe mixed liquor 8 and μ L of probe buffer solution 42, made
Obtain the μ L of capture probe working solution 50.0.Capture probe working solution is vortexed and mixes, and dispenses into 24 orifice plates, often
The μ l of hole 50.
3rd, filter membrane is taken out, in back-off to 24 orifice plates on capture probe working solution, it is ensured that filter membrane is to next
Face is fully contacted with liquid, it is impossible to the presence of bubble.
4th, 24 orifice plate lids are covered, 40 ± 1 DEG C are incubated 3 hours.
5th, liquid is removed, adds 1ml cleaning solutions to wash per hole three times, 2min is soaked every time.Filter membrane is protected
Hold to next step experimental implementation in cleaning solution, sample soak time no more than 30min in cleaning solution.
Step 5, desired mRNA sequences signal amplify
1st, probe buffer solution preheats 20min using the 40 DEG C of water-baths of front need.
2nd, probe face liquid, the μ L of the number of winning the confidence amplifying probe mixed liquor 8 and the μ L systems of probe buffer solution 42 are prepared
Obtain the μ l of probe face liquid 50.It is vortexed and mixes, dispense into 24 orifice plates, per the μ l of hole 50.
3rd, filter membrane is taken out, on back-off to 24 orifice plate middle probe working solutions, it is ensured that filter membrane downwards one side with
Liquid is fully contacted, it is impossible to the presence of bubble.
4th, 24 orifice plate lids are covered, 40 ± 1 DEG C are incubated 3 hours.
5th, liquid is removed, adds 1ml cleaning solutions to wash per hole three times, 2min is soaked every time.By filter membrane
It is maintained in cleaning solution to next step experimental implementation, soak time is no more than in cleaning solution for sample
30min。
Step 6, colour developing, fluorescence labeling echo signal
1st, colorbuffer (40 DEG C of preheatings) lucifuge vortex mixed, dispenses into 24 orifice plates, per the μ l of hole 50.
2nd, filter membrane is taken out, in back-off to 24 orifice plates on colorbuffer, it is ensured that filter membrane downwards one side with
Liquid is fully contacted, it is impossible to the presence of bubble.
3rd, 24 orifice plate lids, 40 ± 1 DEG C of incubation 30min are covered.
4th, liquid is removed, adds 1ml cleaning solutions to wash per hole three times, 2min is soaked every time.Filter membrane is protected
Hold to next step experimental implementation in cleaning solution, sample soak time no more than 30min in cleaning solution.
Step 7, fluorescence microscope CNE-2Z cells
The reference substance of the present invention uses DAPI as nucleus fluorophor, its transmitting blue-fluorescence signal.
1st, filter membrane cell is faced up and is placed on slide, cut off filter membrane along iron ring inner ring, plus 10 μ L
Anti- fluorescent quenching mounting liquid (Antifade Mounting Medium) (being purchased from the green skies, article No. P0126),
Cover the cover glass of 18mm × 18mm, direct microscopy or be placed in -20 DEG C of preservations.
2nd, different in nature nuclear volume in CNE-2Z cells is counted by 20 times of object lens.
3rd, according to the different in nature nuclear locations of 10 times of object lens positioning, oil dripping with oil mirror observation experiment result, and is clapped
According to record result.
4 and then further according to the next opposite sex nuclear locations of 10 times of object lens positioning, oil dripping is real with oil mirror observation
Test result and visual field Taking Pictures recording result.
5th, repeat to all of different in nature core has been clapped, quantity is consistent with 20 times of object lens count results.It is aobvious
Micro mirror uses passage such as table 9：
The excitation wavelength and launch wavelength of the fluorophor of table 9
Step 8：Testing result and discriminatory analysis
1st, positive CNE-2Z standards of perfection.On filter membrane, nasopharyngeal carcinoma cell, nasopharyngeal carcinoma cell are enriched with
Positive criterion is：1) with corresponding target miRNA specific marker's things, in this kit
Show as showing fluorescence signal point under corresponding fluorescence channel.2) nucleus DAPI stained positives.3)
Nasopharyngeal carcinoma cell nuclear shape is irregular, with diameter greater than 10 μm, hence it is evident that more than filter sizes, filter sizes are
7μm.Leucocyte size is close with filter membrane hole size.
2nd, using above-mentioned detection method, each sample is detected and is observed, wherein, for nucleus
DAPI dyeing, indicate whether to detect fluorescence using "-" or "+"；For target detection miRNA
Fluorescence signal intensity, reads respectively the miRNA of the respective color of 10 nasopharyngeal carcinoma cells in each sample
Phosphor dot quantity, and averagely counting is calculated, concrete testing result is shown in Table 10：
The pattern detection result of table 10
Draw from the present embodiment, a kind of nasopharyngeal carcinoma microRNA kit can be right obtained in embodiment 3
Target miRNA is accurately detected.
Embodiment 5
Present embodiments provide the detection of the stability for kit.
A kind of nasopharyngeal carcinoma miRNA detection kit that the present invention is provided, the nasopharyngeal carcinoma miRNA detection
Kit is directed to different target miRNA, chooses the capture probe of varying number, constitutes corresponding probe
Mixed liquor, so as to realize the parallel detection of varying number miRNA.
The present embodiment detects nasopharyngeal carcinoma miRNA constituted using probe groups Group1 of embodiment 3
Kit, 15 kinds of sample (every kind of cell lines 5 to three groups of different cell line sources (being purchased from ATCC)
Individual sample) in hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-29c-3p, hsa-miR-223-3p
Expression is detected, so as to assess the stability of kit of the present invention.Concrete packet refers to table 11.
The cell line of table 11 and detection sample
| Catalogue number(Cat.No.) | Human nasopharyngeal epithelioma 1 | Experimental group |
| 16～sample of sample 20 | CNE-2Z | Group4 |
| 21～sample of sample 25 | NPC | Group5 |
| 26～sample of sample 30 | 5-8F | Group6 |
Probe described in the present embodiment is selected from embodiment 1～3, experimental procedure reference implementation example 4.
Testing result：Using mentioned reagent box, each sample is detected and observed, wherein, for
Nuclear DAPI dyeing, indicates whether to detect fluorescence using "-" or "+"；For target detection
The fluorescence signal intensity of miRNA marker, reads respectively the phase of 10 nasopharyngeal carcinoma cells in each sample
The miRNA phosphor dot quantity of color is answered, and calculates averagely counting, concrete pattern detection result refers to table
12：
The pattern detection result of table 12
From the testing result of table 12, on the one hand, the sample in different cell line sources, its testing result is not
Together, therefore, the present invention is that by the detection of different miRNA expressions, it was demonstrated that the present invention one
Plant the advantage of microRNA detection kit good stabilities；On the other hand, the 5 of same cell strain source
Individual sample, its hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-29c-3p, hsa-miR-223-3p
4 miRNA 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 has repetition
Property good and good specific advantage；As can be seen here, kit of the present invention has reproducible, special
The high advantage of property.
Embodiment 6
Present embodiments provide the detection reagent with more specificity for varying number target miRNA
Box.
1st, design (selection of capture probe quantity) prepared by kit
The present embodiment provides a kind of kit of nasopharyngeal carcinoma microRNA detection, and described kit is directed to
Different target points miRNAs, can choose the capture probe of varying number, constitute corresponding probe mixed liquor,
So as to realize the parallel detection of varying number miRNA.
The present embodiment is respectively directed to a kind, 3 kinds, 5 kinds, 7 kinds of miRNA, from capture probe, signal
Amplify the signal of compositional selecting the 3rd and amplify component, detection kit is constituted, to same cell line CNE-2Z
The sample in source detected, contrasts its Detection results.The concrete composition of kit refers to table 13, described
Probe is selected from embodiment 1～3, experimental procedure reference implementation example 4.
The each sequence content of the kit of table 13 (form numeral is SEQ ID NO.)
2nd, using mentioned reagent box, the sample in same cell line CNE-2Z sources is detected and is observed,
Wherein, dye for nuclear DAPI, indicate whether to detect fluorescence using "-" or "+"；For
The fluorescence signal intensity of target detection miRNA marker, reads respectively 10 nasopharyngeal carcinoma in each sample
The miRNA phosphor dot quantity of the respective color of cell, 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
Detected, using 1,3 and 5,7 can be complete for different miRNA capture probes
Into detection, with good stability.
Embodiment 7
A kind of nasopharyngeal carcinoma miRNA detection kit that the present invention is provided, in order to assess the examination of present invention offer
The specificity of agent box and it is not easy false positive occur, the detection reagent that the present embodiment will be provided using the present invention
Box, to 3 kinds of different tumor cell line (human nasopharyngeal carcinoma cell line CNE-2Z, human lung carcinoma cell line SPC-A1
And human stomach cancer cell line Hs746T, purchased from ATCC)) and normal nasopharyngeal cell strain NP69 carry out
Detection.The present embodiment with detect each cell line (every kind of cell line detect 5 samples) hsa-miR-17-5p,
hsa-miR-20a-5p、hsa-miR-29c-3p、hsa-miR-223-3p、hsa-miR-34c-5p、
As a example by hsa-miR-216a-5p and hsa-miR-151a-3p expressions are detected.Concrete test arrangement
Such as table 14,15：
The kit of table 14 design (form numeral is SEQ ID NO.)
The cell line of table 15 and detection sample
Probe described in the present embodiment is selected from embodiment 1～3, experimental procedure reference implementation example 4.
Testing result：Using mentioned reagent box, each sample is detected and observed, wherein, for
Nuclear DAPI dyeing, indicates whether to detect fluorescence using "-" or "+"；For target detection
The fluorescence signal intensity of miRNA marker, reads respectively the phase of 10 nasopharyngeal carcinoma cells in each sample
The miRNA phosphor dot quantity of color is answered, and calculates averagely counting, concrete pattern detection the results are shown in Table 16：
The pattern detection result of table 16
From the testing result of table 16, on the one hand, hsa-miR-17-5p, hsa-miR-20a-5p,
Hsa-miR-29c-3p, hsa-miR-223-3p, hsa-miR-34c-5p, hsa-miR-216a-5p and
Expressions of the hsa-miR-151a-3p in human nasopharyngeal epithelioma 1 and nasopharyngeal epithelial cells strain is different, and
Significant difference (refers to the testing result of the present embodiment experimental group 11 and experimental group 12)；On the other hand,
hsa-miR-17-5p、hsa-miR-20a-5p、hsa-miR-29c-3p、hsa-miR-223-3p、
Hsa-miR-34c-5p, hsa-miR-216a-5p and hsa-miR-151a-3p are in various cancers cell line
Expression is also not quite similar, and the expression significant difference of part miRNA (refers to the present embodiment reality
Test group 11,13,14).Can clearly draw herein, the kit that the present invention is provided has specificity
It is high and be not easy false-positive advantage occur.
Embodiment 8
The present invention provides a kind of detection of nasopharyngeal carcinoma miRNA detection kit sensitivity.
As a example by the present embodiment is to detect human nasopharyngeal carcinoma cell line CNE-2Z, described human nasopharyngeal carcinoma cell line
CNE-2Z mother liquors cell concentration is 1 × 107/ mL, by human nasopharyngeal carcinoma cell line CNE-2Z mother liquors 3 are diluted to
Individual gradient, is respectively 1 × 103/ mL, 100/mL and 10/mL.
The present embodiment and detection hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-29c-3p and
As a example by hsa-miR-151a-3p, each cell concentration gradient detects 5 samples, and each sample takes 1mL and enters
Row detection.Specific test arrangement such as table 17,18：
The kit of table 17 design (form numeral is SEQ ID NO.)
The cell concentration of table 18 and detection sample
Probe described in the present embodiment is selected from embodiment 1～3, experimental procedure reference implementation example 4.
Testing result：Using mentioned reagent box, each sample is detected and observed, wherein, for
Nuclear DAPI dyeing, indicates whether to detect fluorescence using "-" or "+".Detected by comparing
Target miRNA cell numbers and the cell number for detecting, evaluate the sensitivity of kit provided by the present invention.Tool
Body pattern detection the results are shown in Table 19 (data represent number of cells in table)：
The Detection results of the different cell concentrations of table 19
According to above-mentioned testing result find, kit provided by the present invention to hsa-miR-17-5p,
The detection sensitivity of hsa-miR-20a-5p, hsa-miR-29c-3p and hsa-miR-151a-3p is high, and detects
As a result it is consistent with actual conditions.Meanwhile, detect low-density people's nasopharynx using kit provided by the present invention
JEG-3 CNE-2Z (about 10 cell/mL), detects target miRNA cell numbers and detects
Cell number at least 90%, even up to 100%.100 cell/mL are generally at least needed with prior art
Can be only achieved Detection results to compare, kit sensitivity obtained in the present invention can be apparent from apparently higher than now
There is technology.For the detection of other different miRNA and its various combinations provided by the present invention, also can
The Detection results of the present embodiment are realized, concrete testing result is omitted.It can be seen that, it is provided by the present invention to be directed to
The detection kit detection sensitivity of nasopharyngeal carcinoma correlation miRNA is high.
Can clearly be deduced by above experiment content, the combination of other miRNA that the present invention is provided is also same
Sample can realize the effect of the present embodiment, and specific experiment data are omitted.
In sum, a kind of detection kit of nasopharyngeal carcinoma microRNA that the present invention is provided has stable
Property it is good, reproducible, specific it is high, be not easy the high advantage of false positive and sensitivity occur.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, some improvement and profit can also be made
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of probe compositions, it is characterised in that the probe compositions include：With target microRNA
With reference to capture probe and signal amplify composition, the target microRNA is selected from
hsa-miR-17-5p、hsa-miR-20a-5p、hsa-miR-29c-3p、hsa-miR-223-3p、
hsa-miR-34b-3p、hsa-miR-34c-5p、hsa-miR-212-3p、hsa-miR-216a-5p、
One or more in hsa-miR-217, hsa-miR-151a-3p and hsa-miR-192-5p.
2. probe compositions according to claim 1, it is characterised in that the alkali of the capture probe
Basic sequence is followed successively by from 5 ' ends to 3 ' ends：The specific sequence P1 that combined with the target microRNA,
First spacer sequence, P2 sequences, the P1 sequences are in SEQ ID NO.1～SEQ ID NO.11
Any one, the P2 sequences are any one in SEQ ID NO.12～SEQ ID NO.22, institute
The first spacer sequence is stated for 5～10 T.
3. probe compositions according to claim 2, it is characterised in that the signal amplifies combination
Thing is selected from：First signal amplifies composition, secondary signal and amplifies composition and the 3rd signal amplification composition
Any one；First signal amplifies composition for one stage signal amplifying probe, first signal
3 ' the ends for amplifying composition are also modified with the first fluorophor, first fluorophor selected from FAM,
TET、JOE、HEX、Cy3、TAMRA、ROX、Texas Red、LC RED640、Cy5、LC
Any one in RED705 and Alexa Fluor 488；The secondary signal amplifies composition for one-level letter
Number amplifying probe and second signal amplifying probe, the secondary signal is amplified 3 ' ends of composition and is also modified with
Second fluorophor, second fluorophor selected from FAM, TET, JOE, HEX, Cy3, TAMRA,
Appointing in ROX, Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488
Meaning is a kind of；It is one stage signal amplifying probe, second signal amplifying probe that 3rd signal amplifies composition
With three-level signal amplifying probe, 3 ' ends of the 3rd signal amplification composition are also modified with the 3rd fluorescent base
Group, the 3rd fluorophor selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX,
Any one in Texas Red, LC RED640, Cy5, LC RED705 and Alexa Fluor 488.
4. probe compositions according to claim 3, it is characterised in that the one stage signal amplifies
The base sequence of probe is followed successively by from 5 ' ends to 3 ' ends：P4 sequences, the second spacer sequence and the P2
The P3 sequences that sequence reverse complemental is combined；The P4 sequences are SEQ ID NO.24～SEQ ID NO.35
In any one, second spacer sequence be 5～10 T.
5. probe compositions according to claim 4, it is characterised in that the second signal amplifies
The base sequence of probe is followed successively by from 5 ' ends to 3 ' ends：P5 sequences, the 3rd spacer sequence, P6 sequences,
The P5 sequences contain the base sequence of or more and the P4 sequences reverse complemental；The P5 sequences
Any one be classified as in SEQ ID NO.36～SEQ ID NO.47, the P6 sequences are SEQ ID
Any one in NO.48～SEQ ID NO.59, the 3rd spacer sequence is 5～10 T.
6. probe compositions according to claim 5, it is characterised in that the big spy of the three-level signal
The base sequence of pin is followed successively by from 5 ' ends to 3 ' ends：P8 sequences, the 4th spacer sequence, P7 sequences, institute
State the base sequence that P7 sequences contain or more and the P5 sequences reverse complemental；The P7 sequences
For any one in SEQ ID NO.60～SEQ ID NO.71, the P8 sequences are 5 bases
PolyT sequences, the 4th spacer sequence is 5～10 T.
7. probe compositions according to claim 6, it is characterised in that the P1 sequences, P2
Sequence, P3 sequences, P4 sequences, P5 sequences, P6 sequences, P7 sequences and P8 interior sequences are not present
Hairpin structure.
8. probe compositions according to claim 7, it is characterised in that the first spacerarm sequence
T in row, second spacer sequence, the 3rd spacer sequence and the 4th spacer sequence
Quantity can be with identical or difference.
9. probe compositions according to claim 8, it is characterised in that first fluorophor,
Second fluorophor is identical with the 3rd fluorophor or difference.
10. a kind of nasopharyngeal carcinoma microRNA detection kit, it is characterised in that the nasopharyngeal carcinoma
MicroRNA detection kits include the probe compositions in claim 1 to 9 described in any one.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510733248.3A CN106636079B (en) | 2015-10-30 | 2015-10-30 | Nasopharyngeal carcinoma microRNA detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510733248.3A CN106636079B (en) | 2015-10-30 | 2015-10-30 | Nasopharyngeal carcinoma microRNA detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106636079A true CN106636079A (en) | 2017-05-10 |
| CN106636079B CN106636079B (en) | 2019-12-24 |
Family
ID=58810641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510733248.3A Active CN106636079B (en) | 2015-10-30 | 2015-10-30 | Nasopharyngeal carcinoma microRNA detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636079B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475388A (en) * | 2017-08-22 | 2017-12-15 | 深圳市恩普电子技术有限公司 | Applications and nasopharyngeal carcinoma detection kit of the related miRNA of nasopharyngeal carcinoma as biomarker |
| CN109136358A (en) * | 2017-06-27 | 2019-01-04 | 华中科技大学同济医学院生殖医学中心 | The reagent of remaining sperm and miRNA are in application wherein in antidiastole NOA patient's testis |
| CN109536612A (en) * | 2018-12-24 | 2019-03-29 | 朱伟 | One kind blood plasma miRNA marker relevant to nasopharyngeal carcinoma auxiliary diagnosis and its application |
| CN109593852A (en) * | 2018-12-24 | 2019-04-09 | 朱伟 | One kind serum miRNA marker relevant to nasopharyngeal carcinoma auxiliary diagnosis and its application |
| CN113583964A (en) * | 2021-04-02 | 2021-11-02 | 四川农业大学 | Method for regulating and controlling ovarian granulosa cells by using miR-212 and target gene thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104031993A (en) * | 2014-05-27 | 2014-09-10 | 益善生物技术股份有限公司 | Circulating tumor cell identification kit and circulating tumor cell identification method |
| CN104232741A (en) * | 2013-06-07 | 2014-12-24 | 中南大学 | Application of miR-17, miR-20a, miR-29c and miR-223 as nasopharyngeal carcinoma molecular markers |
-
2015
- 2015-10-30 CN CN201510733248.3A patent/CN106636079B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232741A (en) * | 2013-06-07 | 2014-12-24 | 中南大学 | Application of miR-17, miR-20a, miR-29c and miR-223 as nasopharyngeal carcinoma molecular markers |
| CN104031993A (en) * | 2014-05-27 | 2014-09-10 | 益善生物技术股份有限公司 | Circulating tumor cell identification kit and circulating tumor cell identification method |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136358A (en) * | 2017-06-27 | 2019-01-04 | 华中科技大学同济医学院生殖医学中心 | The reagent of remaining sperm and miRNA are in application wherein in antidiastole NOA patient's testis |
| CN109136358B (en) * | 2017-06-27 | 2021-08-03 | 华中科技大学同济医学院生殖医学中心 | Reagent for identifying and diagnosing residual sperms in testis of NOA patient and application of miRNA in reagent |
| CN107475388A (en) * | 2017-08-22 | 2017-12-15 | 深圳市恩普电子技术有限公司 | Applications and nasopharyngeal carcinoma detection kit of the related miRNA of nasopharyngeal carcinoma as biomarker |
| CN107475388B (en) * | 2017-08-22 | 2020-05-19 | 深圳市恩普电子技术有限公司 | Application of nasopharyngeal carcinoma related miRNA as biomarker and nasopharyngeal carcinoma detection kit |
| CN109536612A (en) * | 2018-12-24 | 2019-03-29 | 朱伟 | One kind blood plasma miRNA marker relevant to nasopharyngeal carcinoma auxiliary diagnosis and its application |
| CN109593852A (en) * | 2018-12-24 | 2019-04-09 | 朱伟 | One kind serum miRNA marker relevant to nasopharyngeal carcinoma auxiliary diagnosis and its application |
| CN109536612B (en) * | 2018-12-24 | 2022-02-11 | 朱伟 | Plasma miRNA marker related to nasopharyngeal carcinoma auxiliary diagnosis and application thereof |
| CN109593852B (en) * | 2018-12-24 | 2022-03-08 | 朱伟 | Serum miRNA marker related to nasopharyngeal carcinoma auxiliary diagnosis and application thereof |
| CN113583964A (en) * | 2021-04-02 | 2021-11-02 | 四川农业大学 | Method for regulating and controlling ovarian granulosa cells by using miR-212 and target gene thereof |
| CN113583964B (en) * | 2021-04-02 | 2023-09-08 | 四川农业大学 | Method for regulating and controlling ovarian granule cells by utilizing miR-212 and target genes thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106636079B (en) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636079A (en) | Nasopharyngeal carcinoma microRNA detection kit | |
| CN104031993B (en) | Circulating tumor cell identification kit and method | |
| CN106636309B (en) | Probe combination for detecting esophageal cancer related marker and kit thereof | |
| CN106636318B (en) | Nucleic acid signal amplification detection kit | |
| CN107354239B (en) | Multiplex fluorescence quantitative PCR method and kit for detecting EB virus | |
| CN106636310A (en) | Prostate cancer-related microRNA detection kit | |
| CN106636317A (en) | Lung cancer related mciroRNA detection kit | |
| CN106636308B (en) | Probe combination for detecting skin cancer related marker and kit thereof | |
| CN106755572A (en) | I types DHV and duck plague virus double fluorescent quantitative PCR method | |
| CN106636312B (en) | Liver cancer related microRNA detection kit | |
| CN106636316B (en) | Ovarian cancer related microRNA detection kit | |
| CN107475356A (en) | Osteosarcoma circulating tumor cell identification kit | |
| CN106995837A (en) | Early diagnosing mammary cancer kit | |
| WO2017096714A1 (en) | Circulating tumor cell typing identification kit | |
| CN106868100A (en) | Colorectal cancer circulating tumor cell identification kit | |
| CN106636077A (en) | Bile duct cancer microRNA detection kit | |
| CN106048080A (en) | Primer and method for rapidly screening HPV (human papillomavirus) sub-types | |
| CN106636314B (en) | Breast cancer related microRNA detection kit | |
| CN106868104A (en) | Lung cancer circulating tumor cell Classification Identification kit | |
| CN107299129A (en) | Circle nucleic acid as breast cancer biomarker application | |
| CN106636311A (en) | Detection kit for colorectal cancer related microRNAs | |
| Peng et al. | Detection of epstein-barr virus infection in gastric carcinomas using quantum dot-based fluorescence in-situ hybridization | |
| CN114381551A (en) | Real-time fluorescent RAA primer, probe and kit for detecting iridovirus of micropterus salmoides | |
| CN106636078B (en) | Probe combination for detecting myeloma related marker and kit thereof | |
| CN104388541B (en) | The purposes of miR 1914* and miR 1915 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |