CN109988825A - Construct the method and its application of the full transcript amplified production of cell - Google Patents
Construct the method and its application of the full transcript amplified production of cell Download PDFInfo
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- WXCXUHSOUPDCQV-UHFFFAOYSA-N enzalutamide Chemical compound C1=C(F)C(C(=O)NC)=CC=C1N1C(C)(C)C(=O)N(C=2C=C(C(C#N)=CC=2)C(F)(F)F)C1=S WXCXUHSOUPDCQV-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention proposes a kind of methods for constructing the full transcript amplified production of cell.This method comprises: cell sample is carried out magnetic capture processing, to obtain cell to be processed；And the cell to be processed is subjected to unicellular full transcript and is expanded, to obtain the full transcript amplified production of the cell to be processed, wherein, before carrying out unicellular full transcript amplification, it further comprise that the cell to be processed is subjected to cracking processing, cracking processing is by PBS being added into the cell to be processed and lysate carries out, based on 1~100 cell to be processed, the dosage of the PBS is 7 microlitres, the dosage of the lysate is 4 microlitres, and the lysate includes 100mM Tris-HCl, 500mM KCl, 25mM MgCl2, 10%NP40,0.1M DTT, RNase inhibitor (40U/ μ l), 0.5 μM of UP1 primer, 2.5mM dNTP and nuclease-free water.This method combines magnetic capture technology with unicellular full transcript amplification technique is ingenious, the additional amount of strict control cell to be processed and lysate and PBS, can stablize and obtain few cells (such as circulating tumor cell) full transcript amplified production.
Description
Technical field
The present invention relates to field of biotechnology, in particular it relates to construct the side of the full transcript amplified production of cell
Method and its application, more particularly it relates to construct the method for the full transcript amplified production of cell, detection cell in it is predetermined because
Son method, detection cell in predetermined factor system and detection prostate cancer circulating tumor cell in AR-V7 kit.
Background technique
Prostate cancer is one of most common malignant tumour of urinary system, and it is pernicious to occupy male for its disease incidence in the world
The 2nd of tumour, the death rate occupies the 5th.Nearly ten years, with China human mortality aging and living-pattern preservation, prostate
The morbidity and mortality of cancer rise year by year trend in apparent.
Androgen receptor (AR) plays an important role in the generation, development of prostate cancer, by adjusting downstream gene
Expression, promote the progress and transfer of prostate cancer.The treatment method of advanced prostate cancer is mainly endocrine therapy, i.e., male to swash
Element deprives treatment (ADT).But in the therapeutic process of prostate cancer, drug resistance is usually generated to it, and develops into castration resistance
Property prostate cancer (CRPC).Although androgen levels are suppressed in CRPC, AR signal path is still played in CRPC to pass
Important role.AR-V7 (androgen receptor splice variant 7) is AR-Vs (androgen receptor
One of splice variants), structure is similar to AR, but lacks ligand binding domain, can not depend on androgen and continue
Ground activation.
Antonarakis et al. has inquired into the expression of AR-V7 and CRPC patient in circulating tumor cell (CTC) and has targeted to AR
Correlation between the miscellaneous Shandong amine of the drug grace of therapy or abiraterone therapeutic response.Result of study shows, En Zalu amine treatment group
It is positive that AR-V7 in circulating tumor cell occur in 39% patient and the patient of abiraterone group 19%.The miscellaneous Shandong amine group AR-V7 sun of grace
Patient is compared with negative patient for property, and PSA (prostate specific antigen) reactivity is substantially reduced (0%VS 53%, P=0.004),
PSA Progression free survival time (1.4 months VS 6 months, P < 0.001), clinical or iconography Progression free survival time (2.1
Month VS 6.1 months) and Overall survival be all obviously shortened；Also there is similar result (Antonarakis in abiraterone group
ES,Lu C,Wang H,et al.AR-V7and resistance to enzalutamide and abiraterone in
prostate cancer[J].N Engl J Med.2014,371(11):1028-1038.).This illustrates that AR-V7 takes part in grace
The development of miscellaneous Shandong amine and abiraterone drug resistance.Existing numerous studies prove at present, to prostate cancer circulating tumor cell AR-V7
Detection the drug resistance of AR targeted therapies in CRPC patient can be assessed.
Therefore, the AR-V7 in circulating tumor cell in patients with prostate cancer (CTC) how is quickly and accurately detected, is referred to
Lead the critical issue of patients with prostate cancer medication.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.
In the first aspect of the present invention, the invention proposes a kind of methods for constructing the full transcript amplified production of cell.Root
According to the embodiment of the present invention, which comprises cell sample is carried out magnetic capture processing, to obtain cell to be processed；
And the cell to be processed is subjected to unicellular full transcript and is expanded, expand to obtain the full transcript of the cell to be processed
Increase production object, wherein further comprise carrying out the cell to be processed at cracking before carrying out unicellular full transcript amplification
Reason, cracking processing are by PBS being added into the cell to be processed and lysate carries out, based on 1~100 wait locate
Cell is managed, the dosage of the PBS is 7 microlitres, and the dosage of the lysate is 4 microlitres, and the lysate includes 100mM Tris-
HCl、500mM KCl、25mM MgCl2, 10%NP40,0.1M DTT, RNase inhibitor (40U/ μ l), 0.5 μM of UP1 primer,
2.5mM dNTP and nuclease-free water.According to the method for the embodiment of the present invention, by magnetic capture technology and unicellular full transcription
This amplification technique is ingenious to be combined, the additional amount of strict control cell to be processed and lysate and PBS, and it is micro thin can to stablize acquisition
The full transcript amplified production of born of the same parents' (such as circulating tumor cell).
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, the cell sample derives from peripheral blood.
According to an embodiment of the invention, the peripheral blood is blood of cancer patients.
According to an embodiment of the invention, the cell to be processed is circulating tumor cell.
According to an embodiment of the invention, the cracking processing includes inhale to the cell to be processed with pipettor beating weight
Outstanding processing at least 5 times.And then it is more thorough to the cracking of cell to be processed processing.
According to an embodiment of the invention, the magnetic capture processing carries out in the following way: 1) by 5mL peripheral blood
First mix with 100 μ L magnetic beads, the magnetic bead advances with PBS and carries out the first resuspension processing, described first mix be
30min is carried out under conditions of 5rpm；2) first mix products are stood into 3min on magnetic frame, to remove on first
Clearly；3) precipitating obtained by step 2) is subjected to the second resuspension processing, the second resuspension processing is carried out in 5ml PBS；4)
Processing product is resuspended by second and stands 1min on magnetic frame, to remove the second supernatant；5) step 3) and 4) twice is repeated；6)
Precipitating obtained by step 5) is subjected to third resuspension processing, the third resuspension processing is carried out in 1ml PBS；7) by third
Processing product is resuspended and stands 1 minute on magnetic frame, to obtain the circulating tumor cell；Wherein, the magnetic bead is coated with
Identify the antibody of the circulating tumor cell.According to a particular embodiment of the invention, the antibody include EpCAM, Her2 extremely
It is one of few.And then the circulating tumor cell in blood of cancer patients obtains further under aforesaid operations sequence and operating condition
Ground efficiently concentrating.
According to an embodiment of the invention, the unicellular full transcript amplification obtains in the following way: 1) will follow
Ring tumour cell carries out cracking 5 minutes under conditions of 24 DEG C, then cracks 3 minutes under conditions of 95 DEG C, is cooled to 4 DEG C；
2) pyrolysis product that step 1) obtains is stood on magnetic frame 1 minute, draws third supernatant；3) by the obtained third of step 2)
Supernatant is mixed with 2 μ l gDNA Wipeout Buffer, and the mixing is carried out 10 minutes under conditions of 42 DEG C；4) will
The obtained product of step 3) is mixed with 6 μ l Quantiscript RT mix, it is described mixing be under conditions of 42 DEG C into
Row 60 minutes；5) step 4) products therefrom is incubated for 3 minutes under conditions of 95 DEG C, puts cold on ice set；6) in step 5) gained
Product is mixed with the ligation mix of 10 μ l, and the mixing is carried out 30 minutes under the conditions of 24 DEG C；It 7) will be obtained by step 6)
Product stands 5 minutes under the conditions of 95 DEG C, puts cold on ice set；8) by the REPLI-g of step 7) products therefrom and 30 μ l
SensiPhi amplification mix is mixed, and the mixing is to carry out 2h under the conditions of 30 DEG C；9) by step 8) institute
Product is obtained to be incubated for 5 minutes under conditions of 65 DEG C；10) step 9) products therefrom is subjected to purification process, to obtain described follow
The full transcript amplified production of ring tumour cell.And then the circulating tumor cell being enriched with thoroughly is split under above-mentioned cracking condition
It solves, mRNA is sufficiently discharged in circulating tumor cell, according further to above-mentioned RNA reverse transcription condition and operation, to single
Cell or limited sample carry out highly homogeneous full transcript amplification (WTA), amplification individual cells (1 to 100 that can be uniform
Cell) or purifying total serum IgE, cover full transcript profile, it is ensured that resulting RNA really reflects intracorporal gene expression profile.It utilizes
Unicellular transcript profile amplification technique under aforesaid operations mode carries out full transcript amplification to the CTC of capture, and it is predetermined that CTC can be improved
The sensibility of factors check.
In the second aspect of the present invention, the invention proposes a kind of methods of predetermined factor in detection cell.According to this hair
Bright embodiment, which comprises method as described above constructs the full transcript amplified production of cell；And based on institute
It states full transcript amplified production and carries out the PCR amplification for being directed to predetermined factor, to detect predetermined factor in the cell.Utilize root
According to the above method of the embodiment of the present invention, can efficient detection go out the predetermined factor in few cells, detection efficiency significantly improves.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, the cell is prostate cancer circulating tumor cell.
According to an embodiment of the invention, the predetermined factor is AR-V7.The above method of embodiment according to the present invention can
AR-V7 positive cell in 5ml blood containing 2 or more is detected.
According to an embodiment of the invention, the PCR amplification is ddPCR amplification, ddPCR primer has NO:1~2 SEQ ID
Shown in nucleotide sequence.
CAGCAGAAATGATTGCACTATTGAT(SEQ ID NO:1)。
CTGGTCATTTTGAGATGCTTGCAAT(SEQ ID NO:2)。
According to an embodiment of the invention, the probe of ddPCR amplification has nucleotide sequence shown in SEQ ID NO:3.
AATTCCGAAGGAAAAATTGTCCATCTT(SEQ ID NO:3)。
According to an embodiment of the invention, the reaction condition of ddPCR amplification is
In turn, under above-mentioned ddPCR amplification condition, the detection efficiency of AR-V7 is further increased, is implemented according to the present invention
The accuracy of the method for example and confidence level further increase.
According to an embodiment of the invention, the primer of the PCR amplification has nucleotides sequence shown in SEQ ID NO:1 or 4
Column；
CAGCAGAAATGATTGCACTATTGAT(SEQ ID NO:1)。
CCAGGTTTCTCCAGACTATCCAC(SEQ ID NO:4)。
According to an embodiment of the invention, the reaction condition of PCR amplification is
In turn, under the conditions of above-mentioned PCR amplification, the detection efficiency of AR-V7 is further increased, according to embodiments of the present invention
Method accuracy and confidence level further increase.
In the third aspect of the present invention, the invention proposes a kind of systems of predetermined factor in detection cell.According to this hair
Bright embodiment, the system comprises: the building full transcript amplified production device of cell, the full transcript amplification of building cell
Product device is for constructing the full transcript amplified production of cell and PCR amplification device, the PCR amplification device and the structure
It builds the full transcript amplified production device of cell to be connected, for carrying out based on the full transcript amplified production for predetermined factor
PCR amplification, to detect predetermined factor in cell.
According to a particular embodiment of the invention, the full transcript amplified production device of building cell further comprises:
Magnetic capture unit, the magnetic capture unit are used to capture the cell to be processed in cell sample；
Unit is cracked, the cracking unit is connected with the magnetic capture unit, for carrying out the cell to be processed
Cracking processing, cracking processing are by PBS being added into the cell to be processed and lysate carries out, based on 1~100
A cell to be processed, the dosage of the PBS are 7 microlitres, and the dosage of the lysate is 4 microlitres, and the lysate includes 100mM
Tris-HCl、500mM KCl、25mM MgCl2, 10%NP40,0.1M DTT, RNase inhibitor (40U/ μ l), 0.5 μM of UP1
Primer, 2.5mM dNTP and nuclease-free water；
Unicellular full transcript amplification unit, the unicellular full transcript amplification unit are connected with the cracking unit,
It is expanded for carrying out unicellular full transcript to cracking processing product, to obtain the full transcript amplified production of cell.
Using above system according to an embodiment of the present invention, can efficient detection go out the predetermined factor in few cells, detect
Efficiency significantly improves.
In the fourth aspect of the present invention, the invention proposes AR-V7 in a kind of detection prostate cancer circulating tumor cell
Kit.According to an embodiment of the invention, the kit includes: reagent, the reagent includes nucleic acid, and the nucleic acid has
Nucleotide sequence shown in NO:1~3 SEQ ID.It, can be real in micro-example using kit according to an embodiment of the present invention
The efficient detection of existing AR-V7.
According to an embodiment of the invention, mentioned reagent box can further include following additional technical feature at least it
One:
According to an embodiment of the invention, the reagent further has the core of nucleotide sequence shown in SEQ ID NO:4
Acid.Using kit according to an embodiment of the present invention, the efficient detection of AR-V7 can be realized in micro-example.
Detailed description of the invention
Fig. 1 is the flow diagram of CTC capture in the method for predetermined factor in detection cell according to an embodiment of the present invention；
Fig. 2 is turned entirely in the method for predetermined factor to the CTC of capture in detection cell according to an embodiment of the present invention
Record the flow diagram of this amplification and AR-V7 detection.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
The embodiments described below utilizes AdnaTest ProstateCancerSelect kit (Qiagen, 395032)
CTC capture is carried out to patients with prostate cancer blood, utilizes the kit REPLI-g WTASingle Cell of unicellular RNA amplification
Kit (Qiagen, 150063) cracks the CTC of capture, and obtained RNA carries out reverse transcription and cDNA amplification, designs AR-V7
DdPCR primer carry out AR-V7 detection.It realizes and the AR-V7 positive cell in 5ml blood containing 2 or more is examined
Out.Detailed process can refer to Fig. 1 and Fig. 2.
Embodiment
1, CTC is captured
CTC capture is carried out using AdnaTest ProstateCancerSelect kit (Qiagen, 395032).
1) ProstateSelect Beads 1 is resuspended with pipettor, it should not vortex oscillation.
2) magnetic bead (each 100 μ L of sample) is added in 1.5ml centrifuge tube.More than 10 samples need to increase by one
1.5ml centrifuge tube.
3) centrifuge tube is placed on magnetic frame 1 minute, removes supernatant with pipettor.
4) addition 1ml PBS inhales to beat repeatedly is resuspended magnetic bead.
5) centrifuge tube is placed on magnetic frame 1 minute, removes supernatant with pipettor.
6) step 4) -5 is repeated) it (is total to three times) twice.
7) Beads is suspended in PBS to initial volume (each 100 μ l of sample).
8) 5ml blood is drawn into 15ml reaction tube, and 100 μ L magnetic beads are added.
9) it is slowly centrifuged (blending instrument about 5rpm) at room temperature 30 minutes.
10) reaction tube is placed on magnetic frame 3 minutes, removes supernatant with pipettor, does not touch magnetic bead.
11) 5ml PBS is added, jiggles with the Bead/Cell compound that suspends again.
12) reaction tube is placed on magnetic frame 1 minute, removes supernatant with pipettor, does not touch magnetic bead.
13) step 11) -12 is repeated) (altogether three times).
14) 1ml PBS is added and magnetic bead (jog mixing) is resuspended, be transferred in 1.5ml centrifuge tube.
15) centrifuge tube is placed on magnetic frame 1 minute, completely removes supernatant with pipettor.
2, RNA reverse transcription and amplification
RNA reverse transcription and amplification are carried out using REPLI-g WTA Single Cell Kit (Qiagen, 150063).
1) the Lysis Buffer of 7 μ l PBS and 4 μ l are added in centrifuge tube, plays resuspension at least 5 times with pipettor suction.
2) it is incubated for 5 minutes for 24 DEG C, subsequent 95 DEG C are incubated for 3 minutes, are cooled to 4 DEG C.
3) centrifuge tube is placed on magnetic frame 1 minute, with pipettor transfer supernatant into new centrifuge tube.
4) 2 μ l gDNA Wipeout Buffer are added, mix centrifugation, 42 DEG C are incubated for 10 minutes.
5) Quantiscript RT mix is configured according to table 1.
Table 1:Quantiscript RT mix
| Ingredient | Volume |
| RT/Polymerase Buffer | 4μl |
| Oligo dT Primer | 1μl |
| Quantiscript RT Enzyme Mix | 1μl |
| Total volume | 6μl |
6) the Quantiscript RT mix of 6 μ l of fresh configuration is added in the cell sample of each cracking, mixes gently
After be centrifuged, 42 DEG C be incubated for 60 minutes.
7) it is incubated for 3 minutes for 95 DEG C, puts cold on ice set.
8) ligation mix is configured according to table 2.
Table 2:ligation mix
| Ingredient | Volume |
| Ligase Buffer | 8μl |
| Ligase Mix | 2μl |
| Total volume | 10μl |
9) in 7) reaction solution of step each sample be added fresh configuration 10 μ l ligation mix, mix gently
After be centrifuged, 24 DEG C be incubated for 30 minutes.
10) it is incubated for 5 minutes for 95 DEG C, puts cold on ice set.
11) prepare REPLI-g SensiPhi amplification mix according to table 3.
Table 3:REPLI-g SensiPhi amplification mix
| Ingredient | Volume |
| REPLI-g sc Reaction Buffer | 29μl |
| REPLI-g SensiPhi DNA Polymerase | 1μl |
| Total volume | 30μl |
12) in 10) reaction solution of step each sample be added fresh configuration 30 μ l REPLI-g SensiPhi
Amplification mix is centrifuged after mixing gently, 30 DEG C of incubation 2h.
13) it is incubated for 5 minutes for 65 DEG C.
14) purifying cDNA is identical as the gDNA method for purifying general 70kb, is quantified with Qubit.
3, AR-V7 is detected
Utilize droplet type PCR (ddPCR) instrument (QX200TM Droplet DigitalTMPCR system, Bio-Rad,
1864001) AR-V7 is detected, detection primer is shown in Table 4.
Table 4:AR-V7ddPCR detection primer
1) 20 μ l ddPCR sonde method quantitative reaction systems are configured according to table 5.
Table 5:ddPCR reaction system
| Ingredient | Volume/amount |
| ddPCRTMSupermix for Probes | 10μl |
| Primer | System is added according to 4 reaction density of table |
| The cDNA product that step 2 step 14 obtains | 30ng |
| H2O | It supplies |
| Total volume | 20μl |
2) a new DG8cartridge is put into holder, pays attention to notch direction；20ul reaction solution is added to
One row of centre of DG8 cartridge.
3) 70 μ l droplets are respectively added in DG8cartridge most 8 holes of bottom next row and generate oily (DG Oil).
4) it is sealed with rubber mat.
5) the above holder is lightly steadily placed in droplet and generates instrument (QX200TMDroplet Generator) in,
Start to generate droplet, pays attention to LED status on instrument, generally about 2 minutes.
6) droplet (40ul) produced is transferred completely into 96 orifice plates.
7) there is the one side (anti-wet look) of red line mark to be placed on 96 orifice plates upward film and fix, with preheated
PX1 heat-sealing instrument carries out sealer, the operation program of recommendation to it are as follows: 180 DEG C, 10s, carries out secondary sealer without inverted orientation；
8) it is expanded using PCR instrument according to following reaction condition.
9) ddPCR Reader (QX200 is utilizedTMDroplet Reader) AR-V7 positive droplet data are read, 1 μ l is anti-
Liquid is answered to can detect 10 with positive droplet.
AR-V7 is detected using regular-PCR instrument, detection primer is shown in Table 6.
Table 6:
20 μ l ddPCR reaction systems are configured according to table 7.
Table 7:PCR reaction system
| Ingredient | Volume/amount |
| 2xKAPA Fast Multiplex Mix | 2μl |
| Primer | System is added according to 6 reaction density of table |
| The cDNA product that step 2 step 14 obtains | 30ng |
| H2O | It supplies |
| Total volume | 20μl |
It is expanded using PCR instrument according to following reaction condition.
Amplified production is detected using Agilent 2100Bioanalyzer system.
Comparative example 1
The same embodiment of operating method only only joined the Lysis Buffer of 4 μ l in RNA reverse transcription and amplification.
It was found that cell cracking is insufficient, cannot sufficiently suspend magnetic bead, and cDNA obtained is not used to AR-V7 detection.
Comparative example 2
The same embodiment of operating method only joined the Lysis of 2 μ l PBS and 4 μ l in RNA reverse transcription and amplification
Buffer。
It was found that cell cracking is insufficient, cannot sufficiently suspend magnetic bead, and cDNA obtained is not used to AR-V7 detection.
Comparative example 3
The same embodiment of operating method only joined the Lysis of 13 μ l PBS and 4 μ l in RNA reverse transcription and amplification
Buffer。
It was found that cell cracking is insufficient, cDNA obtained is not used to AR-V7 detection.
Comparative example 4
The same embodiment of operating method, only the reaction density of AR-V7 ddPCR detection primer is adjusted to 900nM, such as 8 institute of table
Show.
Table 8:
It was found that changing reaction density, ddPCR positive number of droplets reduces half than embodiment.
Comparative example 5
The same embodiment of operating method only adjusts AR-V7ddPCR detection primer and probe sequence, and sequence adjusted is such as
Shown in table 9.
Table 9:
| Title | Sequence (5 ' -3 ') | Reaction density |
| AR-V7-F1 | CGGAAATGTTATGAAGCAGGGATGA | 500nM |
| AR-V7-R1 | CCAGGTTTCTCCAGACTATCCAC | 500nM |
| Probe 1 | [6FAM]TCTGGGAGAAAAATTCCG[BHQ1] | 250nM |
It was found that changing primer and probe sequence, ddPCR positive number of droplets reduces 1/4 than embodiment.
Comparative example 6
The same embodiment of operating method only adjusts the annealing temperature of the amplified reaction of AR-V7ddPCR detection, adjusted
Reaction condition is as described below:
It was found that changing annealing temperature, ddPCR positive number of droplets reduces half than embodiment.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
SEQUENCE LISTING
<110>Beijing lotus and Co., Ltd, medical test institute
<120>method and its application of the full transcript amplified production of cell are constructed
<130> PIDC3176275
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial
<220>
<223>ddPCR primer
<400> 1
cagcagaaat gattgcacta ttgat 25
<210> 2
<211> 25
<212> DNA
<213> Artificial
<220>
<223>ddPCR primer
<400> 2
ctggtcattt tgagatgctt gcaat 25
<210> 3
<211> 27
<212> DNA
<213> Artificial
<220>
<223>probe of ddPCR amplification
<400> 3
aattccgaag gaaaaattgt ccatctt 27
<210> 4
<211> 23
<212> DNA
<213> Artificial
<220>
<223>primer of PCR amplification
<400> 4
ccaggtttct ccagactatc cac 23
Claims (10)
1. a kind of method for constructing the full transcript amplified production of cell characterized by comprising
Cell sample is subjected to magnetic capture processing, to obtain cell to be processed；And
The cell to be processed is subjected to unicellular full transcript amplification, is expanded to obtain the full transcript of the cell to be processed
Increase production object, wherein further comprise carrying out the cell to be processed at cracking before carrying out unicellular full transcript amplification
Reason, cracking processing are by PBS being added into the cell to be processed and lysate carries out, based on 1~100 wait locate
Cell is managed, the dosage of the PBS is 7 microlitres, and the dosage of the lysate is 4 microlitres, and the lysate includes 100mM Tris-
HCl、500mM KCl、25mM MgCl2, 10%NP40,0.1M DTT, the RNase inhibitor of 40U/ μ l, 0.5 μM of UP1 primer,
2.5mM dNTP and nuclease-free water.
2. the method according to claim 1, wherein the cell sample derives from peripheral blood；
Optionally, the peripheral blood is blood of cancer patients；
Optionally, the cell to be processed is circulating tumor cell；
Optionally, the cracking processing includes inhale to the cell to be processed with pipettor playing resuspension processing at least 5 times.
3. according to the method described in claim 2, it is characterized in that, magnetic capture processing is to carry out in the following way
:
1) 5mL peripheral blood first being carried out with 100 μ L magnetic beads to mix, the magnetic bead advances with PBS and carries out the first resuspension processing,
First mixing is that 30min is carried out under conditions of 5rpm；
2) first mix products are stood into 3min on magnetic frame, to remove the first supernatant；
3) precipitating obtained by step 2) is subjected to the second resuspension processing, the second resuspension processing is carried out in 5ml PBS；
4) processing product is resuspended by second and stands 1min on magnetic frame, to remove the second supernatant；
5) step 3) and 4) twice is repeated；
6) precipitating obtained by step 5) is subjected to third resuspension processing, the third resuspension processing is carried out in 1ml PBS；
7) processing product is resuspended in third and stands 1 minute on magnetic frame, to obtain the circulating tumor cell；
Wherein, the magnetic bead is coated with the antibody for identifying the circulating tumor cell；
Optionally, the antibody includes at least one of EpCAM, Her2.
4. according to the method described in claim 2, it is characterized in that, the unicellular full transcript amplification is in the following way
It obtains:
1) circulating tumor cell is carried out to cracking 5 minutes under conditions of 24 DEG C, is then cracked 3 minutes under conditions of 95 DEG C,
It is cooled to 4 DEG C；
2) pyrolysis product that step 1) obtains is stood on magnetic frame 1 minute, draws third supernatant；
3) the obtained third supernatant of step 2) is mixed with 2 μ l gDNA Wipeout Buffer, the mixing is 42
It is carried out 10 minutes under conditions of DEG C；
4) the obtained product of step 3) is mixed with 6 μ l Quantiscript RT mix, the mixing is at 42 DEG C
Under the conditions of carry out 60 minutes；
5) step 4) products therefrom is incubated for 3 minutes under conditions of 95 DEG C, puts cold on ice set；
6) it is mixed in step 5) products therefrom with the ligation mix of 10 μ l, the mixing is that 30 points are carried out under the conditions of 24 DEG C
Clock；
7) step 6) products therefrom is stood 5 minutes under the conditions of 95 DEG C, puts cold on ice set；
8) step 7) products therefrom is mixed with the REPLI-g SensiPhi amplification mix of 30 μ l, it is described
Mixing is to carry out 2h under the conditions of 30 DEG C；
9) step 8) products therefrom is incubated for 5 minutes under conditions of 65 DEG C；
10) step 9) products therefrom is subjected to purification process, expanded to obtain the full transcript of the circulating tumor cell genome
Increase production object.
5. a kind of method of predetermined factor in detection cell characterized by comprising
The full transcript amplified production of cell is constructed according to the described in any item methods of Claims 1 to 4；And
The PCR amplification for predetermined factor is carried out based on the full transcript amplified production, is made a reservation for detect in the cell
The factor.
6. according to the method described in claim 5, it is characterized in that, the cell is prostate cancer circulating tumor cell；
Optionally, the predetermined factor is AR-V7.
7. according to the method described in claim 6, ddPCR primer has it is characterized in that, the PCR amplification is ddPCR amplification
Nucleotide sequence shown in NO:1~2 SEQ ID；
Preferably, the probe of ddPCR amplification has nucleotide sequence shown in SEQ ID NO:3；
Preferably, the reaction condition of ddPCR amplification is
8. according to the method described in claim 6, it is characterized in that, the primer of the PCR amplification has SEQ ID NO:1 or 4
Shown in nucleotide sequence；
Preferably, the reaction condition of PCR amplification is
9. the system of predetermined factor in a kind of detection cell characterized by comprising
The full transcript amplified production device of cell is constructed, the full transcript amplified production device of building cell is for constructing cell
Full transcript amplified production, and
PCR amplification device, the PCR amplification device are connected with the full transcript amplified production device of building cell, are used for base
The PCR amplification for predetermined factor is carried out in the full transcript amplified production, to detect predetermined factor in cell；
Wherein, the full transcript amplified production device of building cell further comprises:
Magnetic capture unit, the magnetic capture unit are used to capture the cell to be processed in cell sample；
Unit is cracked, the cracking unit is connected with the magnetic capture unit, for cracking the cell to be processed
Processing, cracking processing be by PBS being added into the cell to be processed and lysate carries out, be based on 1~100 to
Cell is handled, the dosage of the PBS is 7 microlitres, and the dosage of the lysate is 4 microlitres, and the lysate includes 100mM
Tris-HCl、500mM KCl、25mM MgCl2, 10%NP40,0.1M DTT, the RNase inhibitor of 40U/ μ l, 0.5 μM of UP1
Primer, 2.5mM dNTP and nuclease-free water；
Unicellular full transcript amplification unit, the unicellular full transcript amplification unit are connected with the cracking unit, are used for
Unicellular full transcript amplification is carried out to cracking processing product, to obtain the full transcript amplified production of cell to be processed.
10. the kit of AR-V7 in a kind of detection prostate cancer circulating tumor cell characterized by comprising reagent, it is described
Reagent includes nucleic acid, and the nucleic acid has nucleotide sequence shown in NO:1~3 SEQ ID,
Optionally, the reagent further comprises the nucleic acid with nucleotide sequence shown in SEQ ID NO:4.
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| CN109321569A (en) * | 2018-10-29 | 2019-02-12 | 凯杰（苏州）转化医学研究有限公司 | A kind of primer combination of probe object and its application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140213485A1 (en) * | 2013-01-28 | 2014-07-31 | Yale University | Methods For Preparing cDNA From Low Quantities of Cells |
| US20160264973A1 (en) * | 2013-10-21 | 2016-09-15 | The General Hospital Corporation | Methods relating to circulating tumor cell clusters and the treatment of cancer |
| US20160312298A1 (en) * | 2013-12-20 | 2016-10-27 | The General Hospital Corporation | Methods and assays relating to circulating tumor cells |
-
2017
- 2017-12-29 CN CN201711475632.3A patent/CN109988825B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140213485A1 (en) * | 2013-01-28 | 2014-07-31 | Yale University | Methods For Preparing cDNA From Low Quantities of Cells |
| US20160264973A1 (en) * | 2013-10-21 | 2016-09-15 | The General Hospital Corporation | Methods relating to circulating tumor cell clusters and the treatment of cancer |
| US20160312298A1 (en) * | 2013-12-20 | 2016-10-27 | The General Hospital Corporation | Methods and assays relating to circulating tumor cells |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109321569A (en) * | 2018-10-29 | 2019-02-12 | 凯杰（苏州）转化医学研究有限公司 | A kind of primer combination of probe object and its application |
| CN109321569B (en) * | 2018-10-29 | 2022-04-12 | 迈杰转化医学研究(苏州)有限公司 | Primer probe composition and application thereof |
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