CN109266744A - Multiple PCR primer, kit and the method for targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology - Google Patents
Multiple PCR primer, kit and the method for targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology Download PDFInfo
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Abstract
The invention belongs to genetic test field, in particular to multiple PCR primer, kit and the method for a kind of targeting sequencing detection lung cancer gene based on UMI (Unique molecular identifer) unimolecule label noise reduction technology.The common detection method for driving gene of lung cancer is captured and expanded with a pcr amplification reaction the invention particularly discloses the multiple PCR primer of the specific primer comprising the common driving gene of targeted capture lung cancer shown in SEQ ID NO:1 to SEQ ID NO:48, the kit containing above-mentioned primer and with above-mentioned primer or kit.Multiple PCR primer, kit and detection method with UMI molecular label of the invention, the common driving gene of the multiple lung cancer of multiple samples can be detected simultaneously, the UMI unimolecule label noise reduction technology of use can effectively eliminate bring false positive during PCR, detection sensitivity, accuracy rate are improved, while reducing costs, simplifying operating procedure.
Description
Technical field
The invention belongs to genetic test fields, in particular to a kind of to be based on UMI (Unique molecular
Identifer) multiple PCR primer, kit and the method for the targeting sequencing detection lung cancer gene of unimolecule label noise reduction technology.
Background technique
Lung cancer is the most common cancer in the whole world in many decades.Lung cancer is divided into non-small cell lung by the World Health Organization (WHO)
Cancer and Small Cell Lung Cancer, non-small cell lung cancer account for 80% or more of whole cases of lung cancer.As Obama releases " accurate medicine meter
Draw ", China is also carrying forward vigorously target gene detection, with target therapeutic agent joint development.The main lung cancer driving of FDA approval
Gene includes EGFR, KRAS, NRAS, BRAF, PIK3CA, ALK, ROS1 etc., and relevant targeting medicine includes the EGFR for EGFR
Tyrosine kinase inhibitor (TKI) and anti-egfr antibodies class；Buddhist nun, ALK inhibitor are replaced for gram azoles of ALK；For BRAF's
BRAF inhibitor, mek inhibitor etc..In addition, the genes such as HER2, MAP2K1, MET, RET, TP53 are also lung cancer common mutations base
The variation situation of cause, listed gene has Clinical significance of MG to conditions of patients monitoring, prognosis evaluation, medication guide etc..
New-generation sequencing technology (Next Generation Sequencing, NGS) is that polygenes multidigit point detects jointly,
The prefered method that sample size is few, at low cost, accuracy is high, susceptibility is high, specificity is good.It is past during the decade, two generations sequencing
Instrument manufacturer Illumina occupies most of sequenator market.However, Illumina two generation sequenator libraries enrichment and
The cluster generating process of sequenator is completed by PCR, be will appear genome to a certain extent and is covered non-uniform sequencing noise；This
Outside, it relies on being sequenced in synthesis for polymerase and is difficult to ensure base accuracy, the sequencing noise of base mistake, very great Cheng will be generated
Downstream data analysis is influenced on degree.Thus noise reduction technology is particularly important.
Summary of the invention
In consideration of it, a kind of based on UMI (Unique molecular identifer) it is necessary to provide regarding to the issue above
Multiple PCR primer, kit and the method for the targeting sequencing detection lung cancer gene of unimolecule label noise reduction technology.The present invention can
The multiple lung cancer driving genes of multiple samples are detected simultaneously, and the UMI unimolecule label noise reduction technology used can effectively eliminate PCR
Bring false positive in the process improves detection sensitivity, accuracy rate, while reducing costs, simplifying operating procedure.
The present invention is achieved by the following technical solutions:
A kind of multiple PCR primer of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology, it is described
Multiple PCR primer includes 24 pairs of specific primers, and every PCR primer structure includes UMI sequence and specific primer sequence
Column；The specific primer sequence such as SEQ ID NO:1 to SEQ ID NO:48.
Further, every PCR primer structure further includes bridging sequence；The bridging sequence are as follows:
CAGACGTGTGCTCTTCCGATCT。
Further, the specific primer sequence is the primer sequence Jing Guo special modification.
Further, the method for modifying of the specific primer sequence is thio-modification, phosphorylation modification, LNA and arm
One or more of modification etc..
Further, the Tm value of the specific primer is 60-68 DEG C, preferably 62 DEG C.
Further, UMI (the Unique molecular identifer) sequence of the PCR primer is 4-16 random
Base.
Further, it includes the high sample of the DNA abundance such as tissue, FFPE, cell that the PCR primer, which is applicable in sample type,
It further include the DNA sample of the cell-free sample extraction such as plasma serum, bronchoalveolar lavage fluid and urine.
A kind of kit of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology, the reagent
Box includes the PCR primer of specific primer shown in the above-mentioned NO:1 to SEQ ID of ID containing SEQ NO:48.
A method of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology, operating procedure packet
It includes:
S3: the PCR primer of the specific primer shown in the above-mentioned NO:1 to SEQ ID of ID containing SEQ NO:48 carries out first
Step targeting amplification, it is therefore an objective to primer pair target fragment be allowed to carry out targeted capture.
S5: it after step (1) targeted capture product purification, carries out second step universal primer and expands enriched library, it is therefore an objective to
It will be used for the molecular label (Barcode) of sample differentiation on different sample bands and add sequencing primer, obtain sequencing library.
Further, the first step targeting amplification program is (operation duration 1 hour):
Further, second step library enrichment procedures are (operation duration 1.5 hours):
The invention has the advantages that:
PCR amplification mistake causes when the use of PCR primer with UMI sequence of the invention avoids low DNA applied sample amount
False positive, by subsequent UMI data analysis can remove PCR amplification mistake generation repetitive sequence, improve detection it is sensitive
Degree, is particularly suitable for tumour liquid biopsy field, and the detection sensitivity of somatic mutation can reach 0.05%, is much higher than current city
Surface technology (1%).
It not only includes the high sample of the DNA abundance such as tissue, FFPE, cell that PCR primer of the invention, which is applicable in sample type, also
DNA including the cell-free sample extraction such as plasma serum, bronchoalveolar lavage fluid and urine.DNA applied sample amount can be far below down to 1ng
The 100ng-1ug of market NGS library constructing method.
Detection method of the invention is simple, and library is built in the completion of two-step pcr amplification, and total time-consuming is 3 hours, is far below market skill
Art 8 hours.Multiplex PCR prize law of the present invention and common banking process (hybrid capture) on the market are different, when
Between, in terms of reagent cost and ease of handling have significant sexual clorminance.It can be on duty to operator's simple training.
Detailed description of the invention
Fig. 1 is specific primer specificity verification result electrophoretogram of the present invention, wherein M:Marker；N: negative control；P:
Positive control；1-3: plasma sample；4-6: bronchoalveolar lavage fluid；7-9: paraffin organization sample.
Fig. 2 is primer detection sensitivity verification result electrophoretogram of the present invention, wherein M:Marker；N: negative control；P: sun
Property control；1-3: plasma sample (5ng, 10ng and 20ng)；4-6: bronchoalveolar lavage fluid (5ng, 10ng and 20ng)；7-9: paraffin group
Knit sample (5ng, 10ng and 20ng).
Fig. 3 is primer detection repeatability verification result electrophoretogram of the present invention, wherein M:Marker；N: negative control；P: sun
Property control；1-3 is respectively A operator in a laboratory testing blood plasma, bronchoalveolar lavage fluid, paraffin organization sample results；4-6 difference
It is B operator in b laboratory testing blood plasma, bronchoalveolar lavage fluid, paraffin organization sample results.
Fig. 4 is the PCR primer structural schematic diagram of the first step of the present invention targeting amplification, wherein 51: bridging sequence；52:UMI
Sequence；53: specific primer sequences.
Fig. 5 is second step library enriching primer structural schematic diagram, wherein 61: sequence measuring joints sequence；62:index sequence；
63: bridging sequence.
Specific embodiment
The problem of solved in order to better illustrate the present invention, used technical solution and effect achieved, are now tied
It closes specific embodiment and related data is further described.It should be noted that the content of present invention is including but not limited to following implementation
Example and combinations thereof embodiment.
Particular technique or condition are not specified in the embodiment of the present invention, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available etc.
The conventional products that approach obtains.
A kind of multiplex PCR specific primer of the targeting sequencing lung cancer gene based on UMI unimolecule label of embodiment 1, examination
Agent box and method
1, the design of primer
It is described that University of California Santa Cruz (UCSC) is selected to lung cancer driving gene order
Database selects Primer3 primer-design software to carry out tumour and drives hotspot mutation design of primers, the mutation of scope of design
Hot spot derives from Catalogue of Somatic Mutations in Cancer (COMSIC) database, mainly and lung cancer
Targeting medicament curative effect related target.
The primer that the present invention carries out lung cancer driving gene (EGFR, KRAS, NRAS, BRAF, PIK3CA, ALK, ROS1) is set
Totally 24 pairs of meter, each pair of primer amplified target area size is 60-150bp, and amplification sub-piece is short, is suitable for short-movie section sample
The targeted capture of this DNA especially plasma DNA and FFPE degradation of dna.
Referring to fig. 4, special primer structure includes bridging sequence, UMI sequence and specific primer sequences.Bridge on special primer
It is identical or complementary as with the bridging sequence of Index sequence measuring joints to join sequence, the enrichment in library when reacting for second step PCR
(primer construction is referring to Fig. 5).UMI (Unique molecular identifer) sequence is 4-16 randomized bases, is used for area
The mutating alkali yl for dividing original DNA mutating alkali yl and amplified library mistake to generate.The random primer of the 4-16 base quantity of UMI with
The matching degree of template is high, specificity is good, and quantity leads to primer amplification low efficiency too much, and quantity is very few to lead to primer randomness not
By force, homogeneity is poor；Putting in order is random alignment.When occur UMI sequence it is identical when, can by other feature differentiations, such as
Specific primer sequence etc., but the identical probability for UMI occur is very small, it might even be possible to ignore.Specific primer sequences are applications
Primer3 primer-design software is directed to the primer sequence of selected shot design, can specifically capture target area.
The key point of UMI correction is: when special primer captures target fragment, the product for carrying most original mutation is just taken
Different molecular label.Unimolecule label technique principle is substantially are as follows: UMI label is first added to the amplified production of targeted capture
Then DNA molecular carries out being enriched with using universal primer library, at this time, universal primer is free from UMI sequence, DNA molecular meeting
It is copied into the molecule equally marked, then carries out the sequencing of machine high depth again.In sequencing data, pass through unimolecule label
Amplicon is subjected to clustering, the mistake of mistake and the interpretation of sequencing procedure base that archaeal dna polymerase duplication generates is in high depth
It is random scattered distribution in sequencing data, these faulty sequences being randomly generated will be filtered after counting consensus sequence
It removes, only remains band there are many original mutation sequence of UMI label, the precise sequence after so just being corrected can be used for
" absolute quantitation " is carried out to the frequency of mutation in entire sample.The efficiency of single UMI, the mutation error correction of UMI itself and difference
The optimization of the performances such as the uniformity of UMI is crucial.
Special primer introduces special sex modification when designing, and the primer specificity after modification is stronger, is not easy to be degraded by nuclease,
And there is relatively uniform Tm value, Uniting is 62 DEG C or so (± 2 DEG C)；The primer is shown very well in multiplex amplification
Specificity, stability and homogeneity, while be not present non-specific amplification.Specifically, the primer specific method of modifying is sulphur
One or more of generation modification, phosphorylation modification, LNA and arm modification.Primer sequence after the thio-modification is few core
The derivative that oxygen atom on thuja acid chain with double bond is replaced by sulphur atom can resist the degradation of nuclease, enhance primer stability
Property；Each deoxyribonucleotide takes phosphoric acid pole by 5 ' or 3 ' in primer oligonucleotide sequence after the phosphorylation modification
End can resist the degradation of nuclease, enhance primer stability；LNA (Locked Nucleic Acid) modification is with a type
Oligonucleotide derivative, structure be β-D-RIBOSE 2'-O, 4 '-C act on forming rigid structure by shrink, instead of
The thaw temperature (Tm value) of primer can be improved in deoxyribonucleotide, increases the specificity of primer and template matching；Described
Primer after arm (Spacer) modification can provide necessary interval for primer to reduce the interaction between oligonucleotides, drop
The generation probability of low DNA hairpin structure.
Sequencing primer of the invention also ensures its amplification efficiency while ensuring PCR amplification specificity.
2, detection lung cancer drives kit gene
The kit of detection lung cancer driving genetic method of the invention, comprising:
Reverse transcription reagents: being cDNA by the RNA reverse transcription of sample.
Specific primer: for expanding multiple target areas in sample to be tested nucleic acid, amplification range at least coverage goal
The hot spot mutation of gene.
Universal primer: for being carried out again to the amplified production of special primer amplification target area in library construction process
The sequencing library of different samples to be tested is marked in secondary amplification, and then distinguishes different samples.
PCR reaction solution: including enzymatic mixture, nuclease-free water；The enzymatic mixture includes that high-fidelity DNA polymerase (is used
Amount be 1.25U/ react), PCR Buffer (dosage is 2 ×), dNTP mixture (dosage is 200 μM/kind/reaction).
Positive DNA quality-control product: it is mixed with the positive lung cancer clinical sample of mutation with the DNA that lung cancer positive cell strain is extracted
Object.
Negative DNA quality-control product: it is extracted without the lung cancer clinical sample and the strain of lung cancer negative cells of detection site mutation
DNA mixture.
Positive RNA sample: the RNA that lung cancer clinical sample and the strain of lung cancer positive cell with ALK, ROS1 fusion extract is mixed
Close object.
Negative RNA quality-control product: it is extracted without the lung cancer clinical sample and the strain of lung cancer negative cells of ALK, ROS1 fusion
RNA mixture.
3, the method for the targeting sequencing lung cancer gene based on UMI unimolecule label
The following steps are included:
S1: using nanoscale, superparamagnetic carboxyl magnetic bead under different salinity and hydrophobic environment with the reversible suction of nucleic acid molecules
Attached principle, Specific adsorption nucleic acid molecules obtain the sample of nucleic acid of high-purity, use by subsequently washing and elution step
Qubit or NanoDrop carries out concentration and the measurement of purity uses 10mM Tris according to the sample of nucleic acid concentration results of measurement
Sample of nucleic acid is diluted to 1-50ng/ μ L (DNA), 20-200ng/ μ L (RNA), preferred 10-20ng/ μ L DNA), 50-
100ng/ μ L (RNA) builds the initial concentration in library as amplification.
S2: RNA product is subjected to reverse transcription with reverse transcription reagents of the invention and synthesizes cDNA.
Reverse transcription reaction system are as follows: 2 μ L of reverse transcription reagents；RNA template/quality-control product 400ng；Nuclease-free water mends volume
Enough to 10 μ L.
Reverse transcription reaction program are as follows:
25 DEG C, reverse transcription 60min；
65 DEG C, inactivate 10min；
S3: the PCR primer or examination of the specific primer shown in the NO:1 to SEQ ID of ID containing SEQ NO:48 of the invention
Agent box carries out first time PCR amplification to the target gene target area of the cDNA in sample to be tested DNA in S1 and S2, collects targeting
Capture product.
Pcr amplification reaction system are as follows: 2 μ L of special primer mixture；DNA profiling/quality-control product 50ng；10 μ L of cDNA product；
12.5 μ L of enzymatic mixture；Volume is complemented to 25 μ L by nuclease-free water.
Pcr amplification reaction program are as follows:
S4: the targeted capture product of each sample is purified respectively using purifying magnetic bead.It removes remaining in PCR product
Primer and dNTP etc. recycle targeted capture product；
S5: second of PCR expansion is carried out to the targeted capture product purified in S4 with universal primer or kit of the invention
Increase, enriched library product.
Pcr amplification reaction system are as follows: 2 μ L of universal primer；12.5 μ L of enzymatic mixture；Volume is complemented to 25 by nuclease-free water
μL。
When sample to be tested has it is multiple when, need to be expanded respectively using the universal primer of different Index, to distinguish difference
Sample.
Second step library enrichment procedures (operation duration 1.5 hours):
S6: the library production of each sample is purified respectively using purifying magnetic bead.Remove remaining primer in PCR product
And dNTP etc., recycle library enriched product.
S7: concentration, purity testing and 1% agarose gel electrophoresis test sample quality, the mark of the sample qualification are carried out
Standard is nucleic acid concentration > 2ng/ μ L, OD260/OD280=1.7-2.0,1% agarose gel electrophoresis test sample band are complete, equal
One, without obvious hangover.By all qualified nucleic acid samples (being no more than 96), concentration is diluted to 2nmol/L respectively, and mixes in equal volume
Sample, machine sequencing in unification.
Check experiment:
Positive control reaction system includes: 2 μ L of special primer mixture；12.5 μ L of enzymatic mixture；Positive DNA quality-control product
50ng；The 10 μ L of cDNA product of positive RNA quality-control product；Volume is complemented to 25 μ L by nuclease-free water.Positive control is for checking
Whether whether normal and sample is abnormal for the reverse transcription and amplification ability of reaction system.
Negative control reaction system includes: 2 μ L of special primer mixture；12.5 μ L of enzymatic mixture；Negative DNA quality-control product
50ng；The 10 μ L of cDNA product of negative RNA quality-control product；Volume is complemented to 25 μ L by nuclease-free water.Negative control is for checking
Reaction system is polluted with the presence or absence of nucleic acid substances.
Response procedures of the response procedures of positive control and negative control with step sample.
2 UMI sequence validation verification of embodiment
The special primer of UMI sequence of the design with 0,4,10,16 base, other sequences (bridging sequence of special primer
Column and specific primer sequence) it is identical, according to detection method in embodiment 1 respectively to 0.05%, 0.1%, 0.5%,
1%, the 5% mutation positive is detected with negative standards' product, after the completion of detection, is analyzed sequencing result, as a result such as following table
It is shown.
The mutant proportion that 1 standard items ratio of table is 0.05% analyzes result
The mutant proportion that 2 standard items ratio of table is 0.1% analyzes result
| EGFR T790M | EGFR E19del | KRAS G12D | BRAF V600E | ALK-EML4 fusion | |
| Standard items ratio | 0.1% | 0.1% | 0.1% | --- | 5% |
| UMI(0) | 0.08% | --- | 0.27% | 0.25% | Detection |
| UMI(4) | 0.11% | 0.15% | 0.12% | --- | Detection |
| UMI(10) | 0.12% | 0.10% | 0.18% | --- | Detection |
| UMI(16) | 0.09% | 0.11% | 0.09% | --- | Detection |
The mutant proportion that 3 standard items ratio of table is 0.5% analyzes result
| EGFR T790M | EGFR E19del | KRAS G12D | BRAF V600E | ALK-EML4 fusion | |
| Standard items ratio | --- | 0.5% | 0.5% | 0.5% | 5% |
| UMI(0) | 0.14% | --- | 0.17% | 0.25% | Detection |
| UMI(4) | --- | 0.54% | 0.52% | 0.57% | Detection |
| UMI(10) | --- | 0.60% | 0.58% | 0.78% | Detection |
| UMI(16) | --- | 0.65% | 0.59% | 0.67% | Detection |
The mutant proportion that 4 standard items ratio of table is 1% analyzes result
| EGFR T790M | EGFR E19del | KRAS G12D | BRAF V600E | ALK-EML4 fusion | |
| Standard items ratio | 1% | 1% | 1% | 1% | --- |
| UMI(0) | 0.94% | 0.23% | 1.98% | 1.57% | It is not detected |
| UMI(4) | 1.24% | 0.94% | 1.12% | 1.23% | It is not detected |
| UMI(10) | 1.30% | 0.96% | 1.14% | 1.12% | It is not detected |
| UMI(16) | 1.15% | 0.98% | 1.17% | 0.97% | It is not detected |
The mutant proportion that 5 standard items ratio of table is 5% analyzes result
| EGFR T790M | EGFR E19del | KRAS G12D | BRAF V600E | ALK-EML4 fusion | |
| Standard items ratio | 5% | 5% | 5% | 5% | 5% |
| UMI(0) | 4.64% | 2.23% | 7.24% | 4.35% | Detection |
| UMI(4) | 4.97% | 4.87% | 5.14% | 5.35% | Detection |
| UMI(10) | 5.30% | 4.79% | 5.57% | 4.81% | Detection |
| UMI(16) | 5.12% | 4.64% | 5.15% | 5.27% | Detection |
Wherein table 1 is not provided with this mutation into the expression standard items that 5 Plays product ratio of table is " --- ", such as corresponding
Detection group in detected this be mutated percentage (i.e. there is mutant proportion in the corresponding table of UMI (0)-UMI (16))
It is then false positive.
Testing result is shown, when special primer is without UMI sequence, 1% mutant proportion pattern detection false positive below and vacation
Negative probability rises significantly, and detects the mutant proportion mutant proportion original with sample and differ bigger than normal；And special primer includes
When UMI sequence, the mutant proportion and sample original mutation ratio of detection coincide substantially, and are obviously improved in accuracy.
The verifying of 3 primer specificity of embodiment
Using S1 method in embodiment 1 from plasma sample (number: 1-3), bronchoalveolar lavage fluid sample (number: 4-6), paraffin
Nucleic acid is extracted in tissue sample (number: 7-9), after carrying out concentration, purity testing, qualified samples is taken to be detected.Utilize implementation
Method detects above-mentioned 9 qualified samples in example 1, and library production is carried out 1% agarose gel electrophoresis detection.9 samples
This is detected using lung cancer driving gene specific primer amplification and detection method.Check experiment with embodiment 1, detection method with
Sample testing method is identical.Testing result is shown in Fig. 1.
Testing result indicates that negative control is in addition to residual primers, without any non-specific band, illustrates that reaction system is no dirt
Dye；Positive control band is bright, and size is correct, illustrates that the amplification ability of reaction system is normal.The final amplified production master of 9 samples
300bp is concentrated on, without other non-specific bands, is consistent with desired design, while primer dimer content is few, and primer
Dimer size and amplification target fragment difference are obvious.It can thus be seen that PCR primer and detection method of the invention can be broken through
Conventional method generates the limitation of a large amount of primer dimers, enhances primer specificity.
4 primer detection sensitivity of embodiment verifying
Using S1 method in embodiment 1 from the plasma sample of quality inspection qualification (number: 1), bronchoalveolar lavage fluid sample (number:
4), paraffin organization sample (number: 7) extracts sample nucleic and carries out primer detection sensitivity verifying.Each sample distinguishes loading
5ng, 10ng and 20ng.Above-mentioned 9 qualified diluted samples are carried out using method in embodiment 1 to build library, 9 samples use this hair
Bright lung cancer driving gene specific primer amplification and detection method are detected.Check experiment is the same as embodiment 1, detection method and sample
This detection method is identical.Testing result is shown in Fig. 2.
Testing result indicates that negative control is in addition to residual primers, without any non-specific band, illustrates that reaction system is no dirt
Dye；Positive control band is bright, and size is correct, illustrates that the amplification ability of reaction system is normal.The final amplified production master of 9 samples
300bp is concentrated on, without other non-specific bands, is consistent with desired design, primer dimer content is few, and primer dimerization
Body size and amplification target fragment difference are obvious, easily differentiate.Meanwhile even if template content down to 5ng, still being capable of Successful amplification
Target stripe out improves it can thus be seen that PCR primer and detection method of the invention can break through the limitation of conventional method
Detection sensitivity.
The verifying of 5 primer detection repeatability of embodiment
Using method in embodiment 1 from the slurry samples of quality inspection qualification (number: 1), bronchoalveolar lavage fluid sample (number: 4), stone
Wax tissue sample (number: 7) extracts sample nucleic and carries out the verifying of primer detection repeatability.By 2 different operators at 2 not
Same lab platform expands above-mentioned 3 samples according to method in embodiment 1 respectively, applied sample amount 100ng, 3 samples
This 6 amplification and detection method progress for driving gene specific primer to be obtained by different operation person in different experiments room using lung cancer
Detection.Check experiment is as in the first embodiment, detection method is identical as sample testing method.Testing result is shown in Fig. 3.
Testing result indicates that negative control is in addition to residual primers, without any non-specific band, illustrates that reaction system is no dirt
Dye；Positive control band is bright, and size is correct, illustrates that the amplification ability of reaction system is normal.The totally 6 final amplifications of 3 samples
Product is concentrated mainly on 300bp, without other non-specific bands, is consistent with desired design, while primer dimer content is few,
And primer dimer size and amplification target fragment difference are obvious.Meanwhile different operators and different experiment porch detect
As a result no significant difference.It can thus be seen that PCR amplification primer of the invention and detection method are reproducible.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Guangzhou Qi Hui Biotechnology Co., Ltd
<120>multiple PCR primer, the kit of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology
And method
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<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gcctgtggct gtcagtattt g 21
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gactggttct cactcaccgg 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
attggggtga gcctgcaatc 20
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tgttcaaact gatgggaccc a 21
<210> 8
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atttcttcat gaagacctca cagt 24
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gctcccaacc aagctctctt 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
tgtgccaggg accttacctt 20
<210> 11
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tggatcccag aaggtgagaa ag 22
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cccacacagc aaagcagaaa 20
<210> 13
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cacactgacg tgcctctcc 19
<210> 14
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ggtggaggtg aggcagatg 19
<210> 15
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tacgtgatgg ccagcgtg 18
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gacatagtcc aggaggcagc 20
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
cctcacagca gggtcttctc 20
<210> 18
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
tgatcttgac atgctgcggt 20
<210> 19
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gtgaaaacac cgcagcatgt 20
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
gccacctcct tactttgcct 20
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ttgtccccag gaagcatacg 20
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
agggcataag ctgtgtcacc 20
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
ctggtccctc attgcactgt 20
<210> 24
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
actgtgtttc tcccttctca gg 22
<210> 25
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
tgttggatca tattcgtcca caa 23
<210> 26
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
aggcctgctg aaaatgactg a 21
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
tgcagaagaa gctggaggag 20
<210> 28
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
tgatcttctc aaagtcgtca tcct 24
<210> 29
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ttctgggcac tgggtcaaag 20
<210> 30
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
actcttgcat cgtagcgaac t 21
<210> 31
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gttcgctacg atgcaagagt 20
<210> 32
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tggaaaagta gctcggtagt ct 22
<210> 33
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
tacacagagg aagccttcgc 20
<210> 34
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
aaacaagtgg ttatagatgg tgaaac 26
<210> 35
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
agtggttctg gattagctgg a 21
<210> 36
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
acaggttctt gctggtgtga 20
<210> 37
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tgacaaagaa cagctcaaag ca 22
<210> 38
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
agcacttacc tgtgactcca 20
<210> 39
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gcaagaggct ttggagtatt tca 23
<210> 40
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
tgcatgctgt ttaattgtgt gga 23
<210> 41
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
gtctccctcc tgtttgcaca 20
<210> 42
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
ggtcttttgg aattctgatt tggga 25
<210> 43
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
atcccaactg cctaccgttg 20
<210> 44
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
gcagcttgga gtttgtctgc 20
<210> 45
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
tgtccactgc tgttccttca 20
<210> 46
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
aatcttcctg ccttccctcg 20
<210> 47
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
cagcctgggc atccttgag 19
<210> 48
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
cctcctctgt tgctgcagat 20
Claims (10)
1. a kind of multiple PCR primer of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology, feature
It is, every PCR primer structure includes UMI sequence and specific primer sequence；The specific primer sequence such as SEQ
Shown in ID NO:1 to SEQ ID NO:48.
2. multiple PCR primer according to claim 1, which is characterized in that the UMI sequence of the PCR primer is 4-16
Randomized bases.
3. multiple PCR primer according to claim 1, which is characterized in that every PCR primer structure further includes bridging
Sequence.
4. multiple PCR primer according to claim 1, which is characterized in that the specific primer sequence is by modification
Primer sequence；The method of modifying is one or more of thio-modification, phosphorylation modification, LNA and arm modification.
5. multiple PCR primer according to claim 1, which is characterized in that the Tm value of the specific primer is 60-68
℃。
6. multiple PCR primer according to claim 1, which is characterized in that it includes group that the PCR primer, which is applicable in sample type,
It knits, FFPE, cell, plasma serum, bronchoalveolar lavage fluid or urine.
7. a kind of kit of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology, which is characterized in that
The kit includes multiple PCR primer described in claim 1 to 6 any one.
8. a kind of method of the targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology, which is characterized in that behaviour
Include: as step
S3: the multiple PCR primer described in claim 1 to 6 any one or kit as claimed in claim 7 are to sample
Carry out first step targeting amplification, targeted capture target fragment；
S5: carrying out second step universal primer for step (1) targeted capture product and expand enriched library, makes in product band for sample
The molecular label of differentiation simultaneously adds sequencing primer, obtains sequencing library.
9. detection method according to claim 8, which is characterized in that the first step targets amplification program are as follows:
10. detection method according to claim 8, which is characterized in that second step library enrichment procedures are as follows:
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110491448A (en) * | 2019-07-15 | 2019-11-22 | 广州奇辉生物科技有限公司 | A kind of method, system, platform and storage medium handling PCR primer |
| CN112708664A (en) * | 2019-10-25 | 2021-04-27 | 益善生物技术股份有限公司 | Multi-gene mutation sequencing library construction method and kit for lung cancer driving gene |
| CN112852918A (en) * | 2021-01-20 | 2021-05-28 | 深圳百人科技有限公司 | Two-step PCR technique |
| CN113122615A (en) * | 2021-05-24 | 2021-07-16 | 广州赛哲生物科技股份有限公司 | Single-molecule label primer applied to multiple PCR amplification of absolute quantitative high-throughput sequencing and application thereof |
| CN113215663A (en) * | 2021-06-02 | 2021-08-06 | 中国科学院合肥物质科学研究院 | Construction method and primers of gastric cancer targeted therapy genome library based on high-throughput sequencing |
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| CN113621693A (en) * | 2021-08-09 | 2021-11-09 | 阅尔基因技术（苏州）有限公司 | Method and kit for detecting human HER2 gene copy number variation |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305625A (en) * | 2013-07-08 | 2013-09-18 | 广东省人民医院 | Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application |
| CN105102635A (en) * | 2013-03-15 | 2015-11-25 | 生命技术公司 | Classification and actionability indices for lung cancer |
| CN107988362A (en) * | 2017-10-26 | 2018-05-04 | 广东省人民医院(广东省医学科学院) | A kind of related 33 gene targets capture sequencing kit of lung cancer and its application |
| CN108486230A (en) * | 2018-05-18 | 2018-09-04 | 中国人民解放军陆军军医大学第附属医院 | Kit and preparation method thereof for Non-invasive detection MITF gene mutations |
-
2018
- 2018-09-14 CN CN201811073924.9A patent/CN109266744A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105102635A (en) * | 2013-03-15 | 2015-11-25 | 生命技术公司 | Classification and actionability indices for lung cancer |
| CN103305625A (en) * | 2013-07-08 | 2013-09-18 | 广东省人民医院 | Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application |
| CN107988362A (en) * | 2017-10-26 | 2018-05-04 | 广东省人民医院(广东省医学科学院) | A kind of related 33 gene targets capture sequencing kit of lung cancer and its application |
| CN108486230A (en) * | 2018-05-18 | 2018-09-04 | 中国人民解放军陆军军医大学第附属医院 | Kit and preparation method thereof for Non-invasive detection MITF gene mutations |
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| CN110491448A (en) * | 2019-07-15 | 2019-11-22 | 广州奇辉生物科技有限公司 | A kind of method, system, platform and storage medium handling PCR primer |
| CN112708664A (en) * | 2019-10-25 | 2021-04-27 | 益善生物技术股份有限公司 | Multi-gene mutation sequencing library construction method and kit for lung cancer driving gene |
| CN112852918A (en) * | 2021-01-20 | 2021-05-28 | 深圳百人科技有限公司 | Two-step PCR technique |
| CN113122615A (en) * | 2021-05-24 | 2021-07-16 | 广州赛哲生物科技股份有限公司 | Single-molecule label primer applied to multiple PCR amplification of absolute quantitative high-throughput sequencing and application thereof |
| CN113215663A (en) * | 2021-06-02 | 2021-08-06 | 中国科学院合肥物质科学研究院 | Construction method and primers of gastric cancer targeted therapy genome library based on high-throughput sequencing |
| CN113215663B (en) * | 2021-06-02 | 2023-04-25 | 中国科学院合肥物质科学研究院 | Construction method of gastric cancer targeted therapy genome library based on high-throughput sequencing and primers |
| CN113278717A (en) * | 2021-06-10 | 2021-08-20 | 广州赛哲生物科技股份有限公司 | Primer pool, kit and method for detecting bloodstream infection by targeted sequencing method |
| CN113278717B (en) * | 2021-06-10 | 2024-04-19 | 湖南赛哲智造科技有限公司 | Primer pool, kit and method for detecting blood flow infection by targeted sequencing method |
| CN113621693A (en) * | 2021-08-09 | 2021-11-09 | 阅尔基因技术（苏州）有限公司 | Method and kit for detecting human HER2 gene copy number variation |
| CN114277114A (en) * | 2021-12-30 | 2022-04-05 | 深圳海普洛斯医学检验实验室 | Method for adding unique identifier in amplicon sequencing and application |
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Application publication date: 20190125 |