CN107653320A - EML4 ALK fusion gene noninvasive detection kits - Google Patents
EML4 ALK fusion gene noninvasive detection kits Download PDFInfo
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Abstract
The invention discloses EML4 ALK fusion gene noninvasive detection kits.The invention provides primer set, including EFTUD2 F1, EFTUD2 R1, EML4 (13) ALK F2, EML4 (20) ALK F3, EML4 (6) ALK F4, EML4 (6/13/20) ALK R, detection probe ALK EML4 (6/13/20) TAR and internal reference probe EFTUD2 REF；The present invention surveys EML4 ALK fusion genes using noninvasive sampling blood plasma excretion physical examination, and optimal RNA extracts reagent formulas are groped, and as template, digital pcr detection is carried out with the optimal primer and probe groped, its detection sensitivity is high, quick, convenient and absolute quantitation, and guidance is provided for the medicine use for EML4 ALK fusion genes.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of EML4-ALK fusions noninvasive detection kit.
Background technology
Lung cancer is one of global incidence and fatal rate highest disease, and wherein non-small cell lung cancer accounts for the 80- of lung cancer
85%.At present, 10 kinds of driving genes are there are about to be found in adenocarcinoma of lung.Clinical practice is molecular targeted for these driving genes
Medicine, the treatment and the extension of life cycle for making lung cancer make remarkable progress, thus find more new therapy targets into
For the focus of current lung cancer research.In addition to gene mutation known to EGFR, KRAS etc., EML4-ALK fusions are also proved
For the therapy target of non-small cell lung cancer.Incidences of the EML4-ALK in Patients with Non-small-cell Lung is about 4%~5%, and
And the expression rate in without the adenocarcinoma patients of EGF-R ELISA EGFR mutation or K-Ras mutation is about 42.80%.
The EML4-ALK amalgamation modes having found have kind more than ten, wherein with V1 (EML4 13 exons are broken), V2 (No. 20 of EML4
Extron) and V3 variants (EML4 6 exons fracture) it is most commonly seen.Precise Identification goes out EML4-ALK fusions
NSCLC, and it is to be badly in need of to give corresponding treatment.
EML4-ALK can detect from tumor tissues, CTCs or the mRNA that is extracted from whole blood, but these methods
Have a substantial limitation, for example, biopsy assess the molecular evolution of tumour and heterogeneity be have it is invasive, and not always
Feasible, it is very high to separate CTCs cost and complexity, and mRNA is extracted from whole blood has the shortcomings that muting sensitivity.Therefore need to open
It is noninvasive to send out a kind of, easy and high sensitivity detection method.Blood plasma excretion body mRNA detection fusion gene mutations are a kind of fine
Method, can be used for the detection progression of disease of Noninvasive.Excretion body is extracellular vesica of the diameter in 30-150nm, can be led to
Cross all types cell to be secreted, and information is transported to other cells of body by inclusion, its inclusion includes：
Albumen, lipid and nucleic acid.Excretion body acts in common physiological processes and all being played in advancing of disease.
Being usually used in detecting the technology that ALK gene merges in lung cancer at present has a variety of, including immunohistochemical method
(IHC), fluorescence in situ hybridization technique (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR)；This three kinds of detection methods are made
Sample is tissue samples, is invasive sampling.It is comparatively laborious because carrying out detection fusion gene using DNA, it need to know interior
Specific breakpoint containing son can just detect, and the breakpoint of introne is very more and is mostly unknown.
Digital pcr is the nucleic acid molecules absolute quantitation technology for drawing attention and developing rapidly in recent years, due to biology
The needs and the mixing together of multidisciplinary technology, PCR for learning research have marched toward the third generation --- droplet type digital pcr (Droplet
Digital PCR, ddPCR) epoch.DdPCR technologies are that a kind of flux is higher, lower-cost absolute quantitation technology, can conduct
The primary dcreening operation technology of genetic test.The testing result coincidence rate of the technology and conventional method is high, and method uniformity is good, compared to current
The NGS of main flow, digital pcr protrude in following aspect advantage：Operation is quick, easy, without the bioinformatic analysis of complexity, inspection
Survey high sensitivity.The technology is counted based on single-molecule PCR method, mainly using the popular research field of present analysis chemistry
Micro-fluidic or droplet method, the nucleic acid solution after Macrodilution is dispersed in the microreactor or droplet of chip, each
The nucleic acid-templated number of reactor is less than or equal to 1.So pass through after PCR cycle, the fluorescence signal of droplet is divided one by one
Analysis statistics.According to relative scale and the volume of reactor, it is possible to extrapolate the nucleic acid concentration of original solution, eliminate the reliance on ct values
Or reference gene is that can determine that as little as single absolute number for copying target molecule to be checked.DdPCR is one of quantitative detection sensitive
Method, only need minimal amount of sample i.e. detectable, and high sensitivity and required sample size are few, meet clinical precious sample significantly
Such as puncture the demand of the trace detection of target sequence in sample, Pleural effusions, peripheral blood.
The advantage and disadvantage such as table 1 below of above-mentioned four kinds of methods：
Table 1
So if want it is extremely difficult come all types of detection fusion gene by DNA, and because using probe it is more, meeting
Cause cost higher；If there is a kind of noninvasive method to obtain enough RNA, many costs can be reduced, and can detect
Relatively comprehensively, this focus for just turning into research.
The content of the invention
A purpose of the invention is to provide the primer set for digital pcr detection EML4-ALK fusion mutation.
Primer set provided by the invention, including internal reference upstream primer EFTUD2-F1, internal reference downstream primer EFTUD2-R1,
Expand forward primer EML4 (13)-ALK-F2 of EML4-ALK fusions, expand the forward primer of EML4-ALK fusions
EML4 (20)-ALK-F3, forward primer EML4 (the 6)-ALK-F4 for expanding EML4-ALK fusions, amplification EML4-ALK fusions
Reverse primer EML4 (6/13/20)-ALK-R, detection probe ALK-EML4 (6/13/20)-TAR and the internal reference probe of gene
EFTUD2-REF；
The nucleotides sequence of the internal reference upstream primer EFTUD2-F1 is classified as sequence 1；
The nucleotides sequence of the internal reference downstream primer EFTUD2-R1 is classified as sequence 2；
Forward primer EML4 (13)-ALK-F2 of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 3；
Forward primer EML4 (20)-ALK-F3 of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 4；
Forward primer EML4 (6)-ALK-F4 of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 5；
Reverse primer EML4 (6/13/20)-ALK-R of amplification EML4-ALK fusions nucleotides sequence is classified as sequence
Row 6；
The nucleotides sequence of detection probe ALK-EML4 (the 6/13/20)-TAR is classified as sequence 7；
The nucleotides sequence of the internal reference probe EFTUD2-REF is classified as sequence 8.
In above-mentioned primer set,
Detection probe ALK-EML4 (the 6/13/20)-TAR are different with the internal reference probe EFTUD2-REF end marks
Fluorophor；In embodiment, detection probe ALK-EML4 (6/13/20)-TAR 5' ends mark VIC, internal reference probe
EFTUD2-REF 5' ends flag F AM.
Or, the internal reference upstream primer EFTUD2-F1, internal reference downstream primer EFTUD2-R1, amplification EML4-ALK fusion bases
Forward primer EML4 (13)-ALK-F2 of cause, forward primer EML4 (the 20)-ALK-F3 for expanding EML4-ALK fusions, expand
Increase forward primer EML4 (6)-ALK-F4 of EML4-ALK fusions, expand the reverse primer EML4 of EML4-ALK fusions
(6/13/20)-ALK-R, detection probe ALK-EML4 (6/13/20)-TAR and probe EFTUD2-REF proportioning are 1:1:1：1:
1:1:0.25:0.25。
Another object of the present invention is to provide the reagent for digital pcr detection EML4-ALK fusion mutation.
Reagent provided by the invention, including above-mentioned primer set, reverse transcriptase, dithiothreitol (DTT) and contain reverse transcription primer
Buffer solution (BIO-RAD companies reagent Bio-rad SuperMix (containing reverse transcription primer)).
In mentioned reagent,
Internal reference upstream primer EFTUD2-F1, internal reference downstream primer EFTUD2-R1, amplification EML4- in the primer set
Forward primer EML4 (13)-ALK-F2 of ALK fusion gene, the forward primer EML4 (20) of amplification EML4-ALK fusions-
ALK-F3, forward primer EML4 (the 6)-ALK-F4 for expanding EML4-ALK fusions, expand the anti-of EML4-ALK fusions
To primer EML4 (6/13/20)-ALK-R, detection probe ALK-EML4 (6/13/20)-TAR and probe EFTUD2-REF described
Concentration in reagent is respectively 1uM, 1uM, 1uM, 1uM, 1uM, 1uM, 0.25uM and 0.25uM.
Kit containing above-mentioned primer set or mentioned reagent is also the scope of protection of the invention.
Mentioned reagent box also includes reagent and extraction excretion body RNA reagent needed for enrichment excretion body；
Reagent (ExoQuick-TM/TC (SBI)) needed for the enrichment excretion body specifically comes from ExoQuickTC
Exosomes Precipitation Solution kits；
Reagent (the exoRNeasy Serum/Plasma Midi Kit (QIAGEN, except going of the extraction excretion body RNA
Secrete body enriching section)) specifically come from exoRNeasy Serum/Plasma Midi Kit kits.
Above-mentioned primer set or the kit of mentioned reagent are in digital pcr detection sample to be tested excretion body is prepared
Application in EML4-ALK fusions catastrophe or the product of the frequency of mutation is also the scope of protection of the invention；
Or, above-mentioned primer set or the kit of the mentioned reagent EML4- in digital pcr detects sample to be tested excretion body
Application in ALK fusion gene mutation situation or the frequency of mutation is also the scope of protection of the invention.
3rd purpose of the invention is to provide EML4-ALK fusions mutation feelings in a kind of digital pcr detection sample to be tested
The method of condition.
Method provided by the invention, comprises the following steps：
1) sample to be tested excretion body is enriched with the excretion body enrichment reagents in above-mentioned kit, then with outside the extraction
Secrete body RNA reagent extraction sample to be tested excretion body RNA；
2) using the RNA as template, digital pcr detection is carried out with above-mentioned primer set, realizes that digital pcr detection is to be measured
EML4-ALK fusions catastrophe in sample.
In the above method, also comprise the following steps between enrichment and extraction：Add in the excretion body that above-mentioned enrichment is obtained
Enter PBS to incubate (37 DEG C of incubation 3min).
In above-mentioned, the sample to be tested is Blood plasma in vitro or urine.
The criterion of EML4-ALK fusion catastrophes is as follows in above-mentioned digital pcr detection sample to be tested：
Positive sample has VIC fluorescence signals and FAM fluorescence signals to produce, and wherein VIC fluorescence signals are got over strong positive sample and dashed forward
Frequency is higher；Negative sample only has the generation of FAM fluorescence signals.
Mentioned reagent box also includes reference substance and quality-control product.
A. reference substance is the RNA extractions of normal person, surveys after concentration and to be diluted to 10ng/ul stand-by；
B. quality-control product is ordered and tested：Quality-control product sequence first by finding corresponding sequence in ncbi database,
And carried out being spliced into corresponding mutation type according to fused type, it is then issued to commercial company and is synthesized；After quality-control product receives
A certain amount of ultra-pure water is added, after being quantified using Q-bit, -20 degree preserve；It is dense that used time further constant gradient is diluted to working solution
Degree.
The technology path of the present invention is as shown in Figure 3.
The experiment proves that surveying EML4-ALK fusions using noninvasive sampling blood plasma excretion physical examination, and grope
Optimal RNA extracts reagents combination, and as template, digital pcr detection is carried out with the optimal primer and probe groped, its
Detection sensitivity is high, quick, convenient and absolute quantitation, and guidance is provided for the medicine use for EML4-ALK fusions.
Brief description of the drawings
Fig. 1 is DDPCR detects schematic diagrams.
Fig. 2 is that DDPCR detects two-dimensional representation.
Fig. 3 is the technology path of the present invention.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1, for digital pcr detection EML4-ALK fusions primer design and screening
1st, the design of primed probe
It is used to detect its primer according to tri- kinds of mutation (table 2) design synthesis of EML4-ALK fusions variant1/2/3
And probe.
Table 2 is the type of fusion
In ncbi database, each gene nucleotide series are found, setting for primer and probe is carried out using software oligo7
Meter, on the basis of meeting cardinal principle as far as possible, it is desirable to which upper and lower primer extension product is less than 200bp, and probe annealing temperature ratio draws
Thing is higher by 5 DEG C, it is contemplated that each gene designs each 5-6 sets primer, and 1 set of probe, designs internal control primer in addition and internal reference probe is (specific
It is shown in Table 3)：
Table 3 is the primer sequence and probe sequence of design：
This research and design primer of internal reference, probe and the primer of target area, probe, reference gene are one section of conservative sequences
Row will not morph substantially, and the purpose for selecting internal reference is to judge the presence of wild type.No matter negative or positive sample (only
If it were not for the pure positive) can all there is the amplification of wild type, the purpose of this research and design wild type, it is to judge final sample
The frequency of mutation.
2nd, the screening of primer
Respectively with the positive plasmid containing fusion EML4 (13)-ALK (20) partial sequence, contain fusion EML4
(20) positive plasmid of-ALK (20) partial sequence and the positive plasmid containing fusion EML4 (6)-ALK (20) partial sequence
For template, using respectively set primer enters performing PCR amplification respectively corresponding to each fusion in above-mentioned table 3, PCR amplification productions are obtained
Thing.Agarose gel electrophoresis detects above-mentioned pcr amplification product, the results showed that：PCR expands obtained band and expected consistent and bar
With single, illustrate that primer specificity is good.
Positive plasmid containing fusion EML4 (13)-ALK (20) partial sequence is the fusion shown in by table 4
EML4 (13)-ALK (20) partial sequence is cloned on pUC57 carriers (Jin Sirui standard vectors), obtained plasmid.
Positive plasmid containing fusion EML4 (20)-ALK (20) partial sequence is the fusion shown in by table 4
EML4 (20)-ALK (20) partial sequence is cloned on pUC57 carriers (Jin Sirui standard vectors), obtained plasmid.
Positive plasmid containing fusion EML4 (6)-ALK (20) partial sequence is the fusion shown in by table 4
EML4 (6)-ALK (20) partial sequence is cloned on pUC57 carriers (Jin Sirui standard vectors), obtained plasmid.
Table 4 is the fusion partial sequence needed for clone's positive plasmid
Again corresponding to each fusion shown in table 3 each set primer respectively in connection with internal control primer, internal reference probe, 1 set
Probe to positive sample (positive plasmid containing EML4 (13)-ALK (20) fusion partial sequence, containing EML4 (20)-
The positive plasmid of ALK (20) fusion partial sequence, the positive matter containing EML4 (6)-ALK (20) fusion partial sequence
The mass mixings such as grain obtain positive sample) carry out digital pcr amplification.
As a result show：EML4 (13)-ALK-F2, EML4 (20)-ALK-F3, EML4 (6)-ALK-F4 and EML4 (6/13/
20) this group of primer PCR of-ALK-R expands obtained band and expected consistent and band is single, illustrates that primer specificity is good；Analysis
It is qualified that negative, positive, water three fruiting area point is evident as, and illustrates to detect the special of ALK-EML4 (6/13/20)-TAR probes
Property is good.
Therefore, the optimal primer and probe of tri- kinds of mutation of digital pcr detection EML4-ALK fusions variant1/2/3
Sequence is as shown in table 5 and table 6：
Table 5 is that digital pcr detects the optimal primer sequence of EML4-ALK fusions
Table 6 is that digital pcr detects EML4-ALK fusion probe sequences
| Probe | 5'->3' |
| EFTUD2-REF (5' ends flag F AM) | GGACATCCTTTGGCTTTTGA (sequence 7) |
| ALK-EML4 (6/13/20)-TAR (5' ends mark VIC) | CTTGCAGCTCCTGGTGCTTCC (sequence 8) |
The foundation of embodiment 2, digital pcr detection EML4-ALK fusion methods
First, excretion body RNA is extracted
Sample to be tested is that EML4-ALK fusions mutational cell line (is purchased from bio tech ltd of Nanjing section one hundred；Carefully
The EML4-ALK frequencies of mutation of born of the same parents system itself are to have been demonstrated by the sequencing of two generations, its frequency of mutation in excretion body 45%),
The reagent screening that excretion body is enriched with and excretion body RNA extractions are used is as follows：
1st, excretion body is enriched with
Use ExoQuickTC Exosomes Precipitation Solution, EXOTC10A-1, system
Bioscience (SBI) kit carries out excretion body enrichment：
1) 10ML cell culture fluids centrifugation (3000g, 15min, 4 DEG C), takes supernatant (being precipitated as cell or cell fragment)；
2) supernatant is shifted to sterile test tube, is added 2ML ExoQuick-TC, is overturned test tube and mix.
3) 4 DEG C of refrigerated overnights (at least 12h) (test tube needs to stand during refrigeration, does not rotate).
4) (1500g, 30min, 4 DEG C or room temperature) is centrifuged, after centrifugation, excretion body is ecru/white globules of ttom of pipe.
5) supernatant is removed, (1500g, 5min) is centrifuged again, removes all liquid as far as possible, be careful not to encounter precipitation, obtain
To enrichment excretion body.
2nd, excretion body RNA extraction
Respectively cell NCI- is extracted with following QIAGEN companies exoRNeasy Serum/Plasma Midi Kit kits
The RNA of H2228 (sale company is bio tech ltd of Nanjing section one hundred) culture supernatant excretion body, is comprised the following steps that：
1) PBS that 100ul is added in being precipitated to above-mentioned excretion body (is purchased from), 37 DEG C of incubation 3min；
It is very hard to tolerate because excretion body precipitation is directly directly dissolved using QIAzol Lysis, and QIAzol Lysis are easy
Volatilization, if piping and druming is too long, influence extraction efficiency, and even if piping and druming mixture be for a long time also difficult to it is completely molten
Solution；But PBS is added, 37 DEG C of warm bath is carried out, then shakes, be then very beneficial for dissolving.
2) the QIAzol Lysis of 5 times of volumes are added into above-mentioned pipe, mediates and mixes；
3) it is incubated at room temperature 5min；
4) pillar is loaded into collecting pipe, the above-mentioned solution of 600ul is transferred in pillar；
5) chloroform (such as 200ul sample, adding 200ul chloroform) of initial sample same volume is added,
Acutely concussion；
6) it is incubated at room temperature 2-3min；
7) 4 DEG C of centrifugation 5min of 12000g；
8) supernatant is shifted, is careful not to be drawn onto intermediate layer, adds the absolute ethyl alcohol of 1.5 times of volumes, piping and druming mixes；
9) 700ul samples are drawn into adsorption column, room temperature 8000g, 15s is centrifuged, abandons waste liquid；
10) remaining sample is adsorbed onto in same pillar with same method；
11) 700ul RWT buffer, 8000g centrifugation 15s are added, abandon waste liquid；
12) 500ul RPE buffer, 8000g centrifugation 15s are added, abandon waste liquid；
13) 500ul RPE buffer, 8000g centrifugation 2min are added, abandon waste liquid；
14) uncap centrifugation 5min at full speed, is easy to desciccator diaphragm, abandons waste liquid；
15) new 1.5ml centrifuge tubes are used instead, 14ul RNase-free water is added to film, centrifuges 1min at full speed；
16) pillar is abandoned, the RNA of extraction is put -80 DEG C immediately.
Obtain SBI methods enrichment excretion body+QIAGEN extraction excretion bodies RNA.
With ExoQuickTC Exosomes Precipitation Solution, the EXOTC10A-1 of SBI companies,
System bioscience (SBI) kit carries out the enrichment of excretion body and excretion body RNA extractions, obtains the enrichment extraction of SBI companies
Excretion body RNA.
The enrichment of excretion body and excretion are carried out with QIAGEN companies exoRNeasy Serum/Plasma Midi Kit kits
Body RNA is extracted, and obtains QIAGEN companies enrichment extraction excretion body RNA.
2nd, digital pcr detects
Respectively using the RNA of extraction as template, one-step method is carried out with the probe shown in the optimal primer and table 6 shown in table 5
DDPCR detections (the One-step RT-ddPCR Advanced Kit for Probes kits for using BIO-RAD companies),
It is specific as follows：
1st, reaction solution is prepared：
By 4 pairs of optimal primers, 2 probes, above-mentioned one obtained RNA, ddPCRsupermix (BIORAD), Reverse
Transcriptase, DTT are configured to 22ul reaction solutions, are added to droplet and occur on card.
Shown in reaction solution table 7 specific as follows：
Table 7 is that one-step method DDPCR detects reaction solution
In above-mentioned reaction solution EML4 (6/13/20)-ALK-F be by EML4 (13)-ALK-F2, EML4 (20)-ALK-F3 and
EML4 (6)-ALK-F4 equimolars are mixed to get.
In above-mentioned reaction solution, final concentration of 1uM of each primer in reaction solution, end of each probe in reaction solution be dense
Spend for 0.25uM.
2nd, droplet is prepared：Droplet generation card is put into drop generators, by each 22 microlitres of reaction solutions in 2.5 minutes
It is divided into 20000 drops；
3rd, PCR is expanded：Droplet is transferred on 96 orifice plates, in being expanded in PCR instrument；
4th, droplet is detected：96 orifice plates are put into droplet analyzer, sequentially draw each sample droplet and with carrier fluid stream by
One passes through double-colored detector.
It is containing 3 kinds of at least one mutation to have the droplet of VIC fluorescence signals and FAM fluorescence signals, for the positive, is as mutated
Type droplet；The wherein VIC fluorescence signals more strong positive sample frequency of mutation is higher；Only the droplet of FAM fluorescence signals is not contain 3
Any mutation of kind, for feminine gender, as wild type droplet.
5th, analyze data：
The ratio of positive number of droplets in each sample of software records.
6th, result is shown：Software automatically analyzes data, and shows testing result in many ways.
SBI methods enrichment excretion body+QIAGEN extracts excretion body RNA results as depicted in figs. 1 and 2,
In Fig. 1, A09：Single use SBI methods enrichment extraction excretion body RNA testing results；C09：Single use QIAGEN methods
Testing result after enrichment extraction excretion body RNA extractions；E09：SBI methods enrichment excretion body+QIAGEN extraction excretion body RNA detection knots
Fruit；F09：Positive sample (by the positive plasmid containing EML4 (13)-ALK (20) fusion partial sequence, contains EML4
(20) positive plasmid of-ALK (20) fusion partial sequence, contain EML4 (6)-ALK (20) fusion partial sequence
The mass mixings such as positive plasmid obtain positive sample)；G09：Negative control (YH cells (Yan Di and Huang Di, two legendary rulers of remote antiquity's No.1 " cell line) extraction
RNA)；H09：Water.
1) upper figure represents the FAM passage expanding effects of five samples, and figure below is VIC passage expanding effects；
2)Event Number：Overall reaction number；
3)Ch1Amplitude：Refer to FAM fluorescence channel expanding effects；
4)Ch1Pos:9890Neg:58527:It is 9890 to have fluorescence signal overall reaction number in FAM fluorescence channels, and no signal is total
Stoichiometric number is 58527；
5) each point meaning in figure：Each point represents droplet amplification situation in figure；
Ch2 passages in Fig. 1, the passage are mainly the fluorescence channel of saltant type, from the F09 of ch2 passages (positive control,
As have mutation) and the droplet amplification situation of G09 (negative sample, as without mutation) the two samples can be seen that in baseline
On, positive sample has obvious amplification, and negative sample is without amplification, so wild type and saltant type can be distinguished substantially.
In Fig. 2, ordinate is passage 1 (FAM fluorescence detection channels)；Abscissa is passage 2 (VIC fluorescence detection channels)；
It is wild type droplet that square, which encloses the droplet come, and it is saltant type droplet that ellipse, which encloses the droplet come,；Ring gets up micro-
Drip not include DNA droplet.
Optimal primer and probe specificity is can be seen that from the result in figure preferably, and wild type and saltant type can be obvious
Distinguish.
Using same method detection SBI method enrichment extraction excretion body RNA and QIAGEN method enrichment extraction excretion bodies RNA.
Count SBI methods enrichment extraction excretion body RNA, QIAGEN method enrichment extraction excretion body RNA and SBI method enrichment excretion body
+ QIAGEN extraction excretion bodies RNA the frequency of mutation.
The percentage of the frequency of mutation=saltant type number of droplets/(saltant type number of droplets+wild type number of droplets)；
Frequency of mutation result is as shown in table 8.
Table 8 is DDPCR testing results
SBI or QIAGEN methods extraction excretion body RNA is used alone as can be seen from Table 8 and carries out digital pcr detection, its
The detection frequency of mutation is very low or does not have, and both results use, and the frequency of mutation of detection is up to 42%, with cell line in itself
The EML4-ALK frequencies of mutation it is very close, illustrate both combine extraction excretion body RNA ratios be more suitable for digital pcr detection
Fusion is mutated.
Therefore, SBI methods enrichment excretion body+QIAGEN extraction excretion body RNA combine for optimal excretion body RNA extracts reagents.
3rd, the preparation of the kit of EML4-ALK fusions is detected for digital pcr
Embodiment 1 is screened to obtained optimal primer and optimal probe, ExoQuickTC Exosomes
Excretion body enrichment related reagent and exoRNeasy Serum/Plasma Midi in Precipitation Solution kits
Remaining reagent of excretion body enrichment is removed in Kit kits, prepares the reagent for digital pcr detection EML4-ALK fusions
Box.
The component of this kit is as shown in table 9：
Table 9 is the component of kit
Embodiment 3, digital pcr detection EML4-ALK fusion methods
First, blood plasma excretion body RNA is extracted
The SBI methods enrichment excretion body+QIAGEN extraction excretion body RNA extraction lung cancer EML4-ALK filtered out with embodiment 2
The excretion body of fusion mutated patient (have determined through sequencing and be mutated containing a kind of fusions of EML4 (13)-ALK (20))
RNA。
2nd, digital pcr detects
Digital pcr inspection is carried out to blood plasma excretion body RNA with optimal primer and optimal probe according to two method of embodiment 2
Survey.
It is containing 3 kinds of at least one mutation to have the droplet of VIC fluorescence signals and FAM fluorescence signals, for the positive, is as mutated
Type droplet；The wherein VIC fluorescence signals more strong positive sample frequency of mutation is higher；Only the droplet of FAM fluorescence signals is not contain 3
Any mutation of kind, for feminine gender, as wild type droplet.
As a result believe in lung cancer EML4-ALK fusions mutated patient's testing result containing VIC fluorescence signals and FAM fluorescence
Number droplet, for the positive, i.e., containing EML4-ALK fusions be mutated.
Sequence table
<110>Shenzhen Hua Da life science institute
<120>EML4-ALK fusion noninvasive detection kits
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
ggtcttgcca gacaccaaag 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
tgagaggaca cacgcaaaac 20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
actactgtag agcccacacc t 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
gtctaactcg ggagactatg a 21
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
gttaccaaaa ctgcagacaa g 21
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
tctgcagctc catctgcat 19
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
ggacatcctt tggcttttga 20
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
cttgcagctc ctggtgcttc c 21
Claims (10)
1. for the primer set of digital pcr detection EML4-ALK fusion mutation, including internal reference upstream primer EFTUD2-
F1, internal reference downstream primer EFTUD2-R1, forward primer EML4 (the 13)-ALK-F2 for expanding EML4-ALK fusions, amplification
Forward primer EML4 (20)-ALK-F3 of EML4-ALK fusions, the forward primer EML4 for expanding EML4-ALK fusions
(6)-ALK-F4, reverse primer EML4 (6/13/20)-ALK-R, the detection probe ALK-EML4 of amplification EML4-ALK fusions
(6/13/20)-TAR and internal reference probe EFTUD2-REF；
The nucleotides sequence of the internal reference upstream primer EFTUD2-F1 is classified as sequence 1；
The nucleotides sequence of the internal reference downstream primer EFTUD2-R1 is classified as sequence 2；
Forward primer EML4 (13)-ALK-F2 of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 3；
Forward primer EML4 (20)-ALK-F3 of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 4；
Forward primer EML4 (6)-ALK-F4 of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 5；
Reverse primer EML4 (6/13/20)-ALK-R of amplification EML4-ALK fusions nucleotides sequence is classified as sequence 6；
The nucleotides sequence of detection probe ALK-EML4 (the 6/13/20)-TAR is classified as sequence 7；
The nucleotides sequence of the internal reference probe EFTUD2-REF is classified as sequence 8.
2. primer set according to claim 1, it is characterised in that：
Glimmering different with the internal reference probe EFTUD2-REF end marks of detection probe ALK-EML4 (the 6/13/20)-TAR
Light group；
Or the internal reference upstream primer EFTUD2-F1, internal reference downstream primer EFTUD2-R1, amplification EML4-ALK fusions
Forward primer EML4 (13)-ALK-F2, forward primer EML4 (the 20)-ALK-F3 for expanding EML4-ALK fusions, amplification
Forward primer EML4 (6)-ALK-F4 of EML4-ALK fusions, the reverse primer EML4 for expanding EML4-ALK fusions
(6/13/20)-ALK-R, detection probe ALK-EML4 (6/13/20)-TAR and probe EFTUD2-REF proportioning are 1:1:1：1:
1:1:0.25:0.25。
3. the reagent being mutated for digital pcr detection EML4-ALK fusions, including the primer set of claim 1 or 2,
Reverse transcriptase, dithiothreitol (DTT) and contain reverse transcription primer buffer solution.
4. reagent according to claim 3, it is characterised in that：
Internal reference upstream primer EFTUD2-F1, internal reference downstream primer EFTUD2-R1, amplification EML4-ALK in the primer set
Forward primer EML4 (13)-ALK-F2 of fusion, forward primer EML4 (the 20)-ALK- for expanding EML4-ALK fusions
F3, forward primer EML4 (the 6)-ALK-F4 for expanding EML4-ALK fusions, the reverse of amplification EML4-ALK fusions are drawn
Thing EML4 (6/13/20)-ALK-R, detection probe ALK-EML4 (6/13/20)-TAR and probe EFTUD2-REF are in the reagent
In concentration be respectively 1uM, 1uM, 1uM, 1uM, 1uM, 1uM, 0.25uM and 0.25uM.
5. the kit containing the primer set described in claim 1 or 2 or the reagent of claim 3 or 4.
6. kit according to claim 5, it is characterised in that：The kit also includes reagent needed for enrichment excretion body
With extraction excretion body RNA reagent；
Reagent needed for the enrichment excretion body specifically comes from ExoQuickTC Exosomes Precipitation Solution
Kit；
The reagent of the extraction excretion body RNA specifically comes from exoRNeasy Serum/Plasma Midi Kit kits.
7. the kit of primer set or claim 3 or 4 reagent described in claim 1 or 2 is preparing digital pcr inspection
Survey the application in EML4-ALK fusions catastrophe or the product of the frequency of mutation in sample to be tested excretion body；
Or, primer set described in claim 1 or 2 or the kit of the reagent of claim 3 or 4 detect in digital pcr
Application in sample to be tested excretion body in EML4-ALK fusions catastrophe or the frequency of mutation.
8. the method for EML4-ALK fusion catastrophes, comprises the following steps in a kind of digital pcr detection sample to be tested：
1) the excretion body enrichment reagents enrichment sample to be tested excretion body in kit described in claim 6 is used, then described in
Extract excretion body RNA reagent extraction sample to be tested excretion body RNA；
2) using the RNA as template, digital pcr detection is carried out with the primer set described in claim 1 or 2, realizes digital pcr
Detect EML4-ALK fusion catastrophes in sample to be tested.
9. according to the method for claim 8, it is characterised in that：
Also comprise the following steps between enrichment and extraction：PBS is added in the excretion body that above-mentioned enrichment is obtained to incubate.
10. apply according to claim 7 or 8 or 9 methods describeds, it is characterised in that：
The sample to be tested is Blood plasma in vitro or urine.
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| CN110157806A (en) * | 2019-05-30 | 2019-08-23 | 深圳海普洛斯医学检验实验室 | A kind of kit detecting EML4-ALK and its detection method and application |
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| CN110157806A (en) * | 2019-05-30 | 2019-08-23 | 深圳海普洛斯医学检验实验室 | A kind of kit detecting EML4-ALK and its detection method and application |
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| CN113960313A (en) * | 2021-12-22 | 2022-01-21 | 上海思路迪医学检验所有限公司 | Exosome ALK fusion protein magnetic immunochemiluminescence detection kit |
| CN113960313B (en) * | 2021-12-22 | 2022-04-12 | 上海思路迪医学检验所有限公司 | Exosome ALK fusion protein magnetic immunochemiluminescence detection kit |
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