CN109811041A - A kind of specific primer detecting the site PIK3CA gene H1047R is to, probe and kit - Google Patents
A kind of specific primer detecting the site PIK3CA gene H1047R is to, probe and kit Download PDFInfo
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Abstract
The present invention provides a kind of specific primers for detecting the site PIK3CA gene H1047R to, probe and kit, and the nucleotide sequence of specific primer pair of the invention is as shown in SEQ ID NO.1-2, and the nucleotide sequence of probe is as shown in SEQ ID NO.3-4.Primer of the present invention is designed based on the site PIK3CA gene H1047R, using above-mentioned specific primer to and probe, the site PIK3CA gene H1047R in blood free nucleic acid to be measured is detected using the method for digital pcr, specific good and high sensitivity, testing result accuracy is high, the frequency of mutation based on site can be directly given simultaneously in detection mutation, there is important application value.
Description
Technical field
The present invention relates to technical field of gene detection, specifically, being related to a kind of detection proto-oncogene PIK3CA gene
The specific primer in the site H1047R is to, probe and contains its kit.
Background technique
PIK3CA gene encodes p110 α albumen, it is a subunit of phosphatidyl-inositol 3-kinase (PI3K).p110α
Albumen is catalytic subunit, because it executes the effect of PI3K, and another subunit (being generated by different genes) adjusts the activity of enzyme.
As other kinases, PI3K adds an oxygen and the cluster (phosphate) of phosphorus atoms passes through Phosphorylation events.PI3K phosphorylation
A little signaling molecules trigger a series of reactions for transmitting chemical signal in the cell.PI3K signal transduction is living for many cells
Property (including cell is grown and division (proliferation), the migration of cell, the generation of novel protein, the transhipment of intra cellular material and cell
Survival) it is critically important.Research shows that PI3K signal transduction may participate in adjusting several hormones, and may be in the maturation of fat cell
It plays a role.
It is thin with the presence of the body of patient's PIK3CA gene of 1%-3% in the patient of all non-small cell lung cancers (NSCLC)
Cytoplasmic process becomes.PIK3CA mutation can also occur simultaneously with the mutation of EGFR.In the patient of NSCLC, PIK3CA gene H1047R
Mutation account for PIK3CA mutation 12.9%.H1047R mutation causes the position 1047 in PIK3CA gene that an ammonia has occurred
The replacement of base acid, becomes arginine from histidine.This mutation occurs in highly conserved kinase region.The PIK3CA egg of variation
White matter improves catalytic activity, leads to the enhancing of downstream signal and the conversion of internal tumorigenesis.Preclinical data are shown, will be swashed
PIK3CA variation living is introduced into the lung cancer cell line of EGFR variation the drug resistance given for EGFR TKI therapy.In addition, small one
The change of PIK3CA is had found in the lung cancer partially to make a variation (less than 5%) for the EGFR of the acquired resistance of EGFR TKI therapy
It is different.
Digital pcr (Digital-PCR) is a kind of novel PCR detection method, and working principle is DNA or cDNA
Sample segment is many independent, parallel PCR reaction systems, these reactions of part contain target molecules (positive), and other
Not comprising (feminine gender).Individual molecule can be amplified 1,000,000 times or more, can be improved the performance of existing TaqMan measurement, from
And realize higher sensitivity, accuracy, avoid the appearance of false negative result.Standard PCR reaction system after droplet occurs,
Be assigned in about 20000 droplets, each droplet include or not comprising one or more copy target molecules (DNA profiling),
It realizes " unimolecule template PCR amplifications ", after amplification, the droplet containing nucleic acid templates can provide fluorescence signal, not have
The droplet of template is just generated without fluorescence signal.Finally according to Poisson distribution principle and the ratio of positive droplet, software is analyzed
The concentration or copy number for providing target molecule to be checked can be calculated.Digital pcr can directly calculate the copy number of target sequence, therefore nothing
It need to can be carried out accurate absolute quantitation detection dependent on control sample and standard curve；Further, since digital pcr is carrying out
As a result only judge when interpretation with/without two kinds of amplification states, therefore also do not need the intersection point of detection fluorescence signal and given threshold line,
It is completely independent of the identification of Ct value, therefore the reaction of digital pcr is influenced to substantially reduce by amplification efficiency, PCR is reacted and is pressed down
The tolerance of object processed greatly improves；The process of digital pcr experiment Plays reaction system distribution can be reduced largely
With the background sequence concentration of the competitive effect of target sequence, the concentration or copy number for providing target molecule to be checked are calculated.
Summary of the invention
The object of the present invention is to provide one kind sites detection PIK3CA gene H1047R at low cost, accuracy high sensitivity
Method.
Present invention firstly provides a pair of for detecting the specific primer pair in the site PIK3CA gene H1047R, described special
The nucleotide sequence of property primer pair is as shown in SEQ ID NO.1-2.
Forward direction primer: (SEQ ID NO.1) CAAGAGGCTTTGGAGTATTTCATG
Reverse primer: (SEQ ID NO.2) TTCTCAGTTACTTTTCAGTTCAATGC
The present invention provides with above-mentioned specific primer to the probe combinations being used cooperatively, the nucleotide of the probe combinations
Sequence is as shown in SEQ ID NO.3-4.
Further, 5 ' label VIC, 3 ' label MGB-NFQ, shown in SEQ ID NO.4 of probe shown in SEQ ID NO.3
5 ' flag F AM, 3 ' label MGB-NFQ of probe.
The probe combinations that the present invention designs, particular sequence are
VIC:5'-VIC-ATGCACATCATGGTGG-MGB-NFQ-3'；
FAM:5'-FAM-AATGATGCACGTCATG-MGB-NFQ-3'.
Kit containing probe shown in specific primer pair shown in SEQ ID NO.1-2 and/or SEQ ID NO.3-4 or
Detection reagent belongs to the protection scope of the application.
The present invention provides above-mentioned specific primer to and probe combinations or containing its kit detection PIK3CA base
Because of the application in the site H1047R.
The present invention provides above-mentioned specific primer to and probe combinations preparation detect the site PIK3CA gene H1047R
Application in kit.
The present invention contain specific primer shown in SEQ ID NO.1-2 to and SEQ ID NO.3-4 shown in probe reagent
Box, working procedure are as follows:
(1) genomic DNA of sample to be tested is extracted；
(2) using the genomic DNA of extraction as template, using specific primer shown in SEQ ID NO.1-2 to and nucleosides
Acid sequence 2 probes as shown in SEQ ID NO.3-4 carry out digital pcr amplified reaction；
(3) result is determined according to the copy number that fluorescence signal provides.
The wherein program of step (2) digital pcr amplified reaction are as follows: 95 DEG C of initial denaturation 10min；94 DEG C of denaturation 20s, 56 DEG C are moved back
Fire and extension 45s, totally 40 recycle；98 DEG C of solidification droplet 10min.
The present invention provides a kind of method for detecting the site PIK3CA gene H1047R in free nucleic acid in blood, including as follows
Step: (1) genomic DNA of sample to be tested is extracted；
(2) using the genomic DNA of extraction as template, using specific primer shown in SEQ ID NO.1-2 to and nucleosides
Acid sequence 2 probes as shown in SEQ ID NO.3-4 carry out digital pcr amplified reaction；
(3) result is determined according to the copy number that fluorescence signal provides.
In the above method, the program of digital pcr amplified reaction is carried out in step (2) are as follows: 95 DEG C of initial denaturation 10min；94℃
It is denaturalized 20s, 56 DEG C of annealing and extension 45s, totally 40 circulations；98 DEG C of solidification droplet 10min.
The present invention provides the methods and inspection for detecting the site PIK3CA gene H1047R in blood circulation free nucleic acid
Test agent box.The present invention uses droplet digital pcr technology, designs a pair of of specificity for PIK3CA gene H1047R site mutation
Primer pair and two probes used in conjunction with.
It is applicable not only to provided by the present invention for the method for detecting PIK3CA gene H1047R to tumor tissues, paraffin packet
It buries tissue-derived nucleic acid to be detected, be more applicable for PIK3CA gene H1047R mutation in blood circulation free nucleic acid
The detection in site；And it is accurate to combine the detection architecture and droplet type digital pcr system that can carry out to the frequency of mutation of sample of nucleic acid
Detection.Specific primer and probe provided by the invention can accurately detect blood free nucleic acid, same in detection mutation
When can directly give the frequency of mutation of gene loci, and combine digital pcr system to the detection of mutation up to five one thousandths,
High sensitivity.
Detailed description of the invention
Fig. 1 is (B01/02:61.4 DEG C of the selection result figure in digital pcr reaction process of the present invention for annealing temperature；
C01/02:60.3℃；D01/02:58.4℃；E01/02:55.9℃；F01/02:54.0 DEG C), it is final to determine 56 DEG C of annealing temperature
For optimum annealing temperature.
Fig. 2 is mutant plasmids loading concentrations in the embodiment of the present invention 3 from 0.00001ng/ul to 0.00000001ng/ul
When (B01/02:0.00001ng/ul, C01/02:0.000001ng/ul, D01/02:0.0000001ng/ul, E01/02:
0.00000001ng/ul), mutant probe in detecting is differed from 0-21.3 copy.
Fig. 3 is wild plasmid loading concentrations in the embodiment of the present invention 3 from 0.00001ng/ul to 0.00000001ng/ul
When (B01/02:0.00001ng/ul, C01/02:0.000001ng/ul, D01/02:0.0000001ng/ul, E01/02:
0.00000001ng/ul), the average copy number of WT probe in detecting two multiple holes in 20ul copy from 0 to 20.8 differs.
Fig. 4 (saltant type) is that mixed type plasmid in the embodiment of the present invention 3 (saltant type: wild type=1:1) loading concentrations are
Saltant type probe is detected as 125-169 etc. in 20ul system when 0.00001ng/ul, and overall repeatability is preferable, with two germplasm
Grain mixing ratio is consistent.
Fig. 5 is that mutation rate detects (saltant type) experimental result picture in the embodiment of the present invention 3, is (prominent in mixing plasmid ratio
Modification: wild type=1:1；1:10；1:100；1:1000；1:5000；1:10000) saltant type probe is examined in the system of 20ul
Measure the copy number results come, it can be seen that the reduction proportional to ratio of saltant type copy number.
Fig. 6 is that mutation rate detects (wild type) experimental result picture in the embodiment of the present invention 3, is (prominent in mixing plasmid ratio
Modification: wild type=1:1；1:10；1:100；1:1000；1:5000；1:10000) wild-type probe is examined in the system of 20ul
Measure the copy number results come, it can be seen that the copy number of wild type does not have too large deviation variation.
Fig. 7 is to use the standard items HD701 input quantity of Horizon company in 40ng and 4ng feelings in the embodiment of the present invention 3
Saltant type copy detection result figure under condition.
Fig. 8 is to use the standard items HD701 input quantity of Horizon company in 40ng and 4ng feelings in the embodiment of the present invention 3
Wild type copies under condition detect result figure.
Fig. 9 is to measure digital pcr saltant type experimental result picture using tumour patient blood sample in the embodiment of the present invention 3.
Figure 10 is to measure digital pcr wild type experimental result picture using tumour patient blood sample in the embodiment of the present invention 3.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1 be used to detect the specific primer in the site PIK3CA gene H1047R to and specific probe combination set
Meter
1, design of primers: searching the DNA sequence dna of people's PIK3CA gene from NCBI, utilizes primer6.0 software design
The upstream and downstream primer comprising mutational site (c.3140A > G) segment can be amplified out, therefrom choose 3 to (before respectively following
To primer 1 and reverse primer；Forward direction primer 2 and reverse primer；Forward direction primer 3 and reverse primer), the segment of 3 pairs of primer amplifications
Size is differed from 125bp to 127bp；Synthesize 3 pairs of primers；3 pairs of primers are tested using round pcr.
QPCR experiment is carried out to gained primer, verifies the amplification efficiency of different primers group, QPCR identical amplification as the result is shown
Under system and amplification condition, the CT value of QPCR occurs earliest when primer sets amplified production length is 126bp, shows the effect of amplification
Rate is optimal, therefore has selected amplification length in the pair of primers group (reverse primer and forward direction primer 1) of 126bp, nucleotides sequence
It is classified as:
Reverse primer: 5'-TTCTAGTTATCTTTCAGTTCAATGC-3'(SEQ ID NO.2)
Forward direction primer 1:5'-CAAGAGGCTTTGGAGTATTTCATG-3'(SEQ ID NO.1)
Forward direction primer 2: 5'-GCAAGAGGCTTTGGAGTATTTCA-3'(SEQ ID NO.5)
Forward direction primer 3:5'-GCAAGAGGCTTTGGAGTATTTCAT-3'(SEQ ID NO.6)
And concentration optimization is carried out to the primer that screening obtains, different concentration is respectively set in forward direction primer and reverse primer,
Final concentration is verified from 300nM to 900nM using QPCR, and primer working concentration is between 300nM-900nM as the result is shown
The Ct value of qPCR has no significant difference, therefore ensures sufficient primer amount using 900nM concentration.
2, probe designs: for the obtained specific primer pair of screening, designing to obtain through primer design and cooperates with it
The specific probe used can be used together when detecting the PIK3CA gene mutational site H1047R.
Due to MGB group to DNA double chain ditch have high compatibility, PCR reaction in, MGB probe compared to
Average probe Tm value is much higher, to be usually higher by 10-20 DEG C.MGB probe has high Tm value, can be very good that GC is overcome to contain
Difficulty of the low template in probe design is measured, waits the Tm value between site mutations sequence widely different, improves the special of detection
Property.Meanwhile MGB probe includes a not fluorescent quenching group (NFQ), it can really eliminate what traditional quenching group generated
Background fluorescence improves signal-to-noise ratio, to promote detection sensitivity.
The nucleotide sequence of the probe are as follows:
VIC:5'-VIC-ATGCACATCATGGTGG-MGB-NFQ-3'；(SEQ ID NO.3)
FAM:5'-FAM-AATGATGCACGTCATG-MGB-NFQ-3'(SEQ ID NO.4)
And concentration optimization is carried out to the probe of design: using the primer (SEQ ID NO.1-2) of above-mentioned screening and optimized
Concentration, template concentrations 0.00001ng/ul.Using different concentration and probe concentrations, respectively 200nM-500nM, between being with 50nM
Every, the template of 7 concentration is detected respectively using QPCR, as the result is shown when concentration and probe concentration is 250nM, low concentration template
The earliest therefore most suitable final concentration of 250nM of probe of CT value that occurs of QPCR.
Embodiment 2 detects the foundation of the digital pcr detection method in the site PIK3CA gene H1047R
1, it prepares reaction system to be determined according to embodiment 1 in best primer and probe, determines reaction system 20ul, 2 × dd
To the wild with VIC fluorescent marker of 1.8 μ l, 10uM reverse primer of primer 1.8 μ l, 10uM before PCR premixed liquid 10 μ l, 10uM
Saltant type probe (SEQ ID NO.4) 0.5 μ l with FAM label of type probe (SEQ ID NO.3) 0.5 μ l, 2uM, water 3.4
μ l, 2 μ l of template, totally 20 μ l.
2, after completing step 1, reaction system is transferred to digital pcr droplet and is generated in the system well of card, and anti-
It is reacted using the digital pcr that 70 μ l are added in oily well and uses oil, digital pcr droplet is generated into card and is transferred to Droplet
In Generator drop generators, starts instrument, obtain micro system.
PCR reaction solution is prepared, is stored at room temperature after configuration 3 minutes.PCR reaction solution after standing is transferred to DG8 droplet hair
Sample cell in raw card adds droplet and special oil occurs.It is put into QX200 Droplet Generator and carries out droplet
Reaction.Generated droplet is shifted to 96 orifice plates.It is covered with PCR viscosity envelope paillon and carries out sealer using heat-sealing instrument.It is subsequently transferred to
PCR to carry out amplification reaction, reaction condition be 95 DEG C 10 minutes；94 DEG C 20 seconds, (B03:61.4, C03:60.3, D03:58.4,
E03:55.9, F03:54) DEG C 45s, 40 circulations；98 DEG C 10 minutes；12 DEG C continue.It is put into after the reaction was completed
QX200Droplet Reader carry out signal-obtaining, according to the result figure of QuantaSoft software select optimum annealing temperature for
56 DEG C, see Fig. 1.Data are analyzed according to the result figure of QuantaSoft software, wherein the frequency of mutation=saltant type copy
Number/total copy number (saltant type copy number+wild type copies number)
Embodiment 3 carries out specificity and sensitivity technique according to the method that embodiment 2 is established
1, specificity and sensitivity experiment
Building includes the mutant plasmid (mutant) in the mutational site PIK3CA gene H1047R and includes site open country respectively
The plasmid (WT) of raw type, and using the two plasmids as template, with droplet type digital pcr system to the specificity and sensitivity of probe
It is tested.Reaction system includes 2 × mix buffer, 10 μ l, template 2ul (WT 0.00002ng/20 μ l；WT
0.000002ng/20 μ l, WT 0.0000002ng/20 μ l, WT 0.00000002ng/20 μ l；Mutant plasmid 0.00002ng/20
μl；Mutant plasmid 0.000002ng/20 μ l, mutant plasmid 0.0000002ng/20 μ l；Mutant plasmid 0.0000002ng/20 μ l；
Mutant plasmid 0.00000002ng/20 μ l), 1.8 μ l of forward direction primer, 1.8 μ l, WT probe of reverse primer, 0.5 μ l, saltant type probe
0.5 μ l, 3.4 μ l of ultrapure water, totally 20 μ l reaction system.Reaction condition is 95 DEG C, 10 minutes；94 DEG C, 20 seconds, 56 DEG C, 45s (40
A circulation)；98 DEG C, 10 minutes；4℃.
Testing result is as shown in Figure 2 to 3.When wild plasmid (WT) final concentration of 0.00001ng, WT probe in detecting
116 to 170 copies (Fig. 2)；When the final concentration of 0.00001ng of mutant plasmids, saltant type probe detects 109 to 174 and copies
Shellfish；When the final concentration of 0.000001ng of mutant plasmids, mutant probe detects 39-41 and copies.Table 1 is in the present embodiment with packet
The mutant plasmid (mutant) in the mutational site the H1047R of gene containing PIK3CA and plasmid (WT) comprising the site wild type are mould
Plate carries out test result to the specificity of probe and sensitivity with droplet type digital pcr system.
Table 1
In conclusion wild-type probe will not identify the mutational site of H1047R, and saltant type probe will not identify it is non-
Mutational site；And wild type and saltant type probe be under the premise of plasmid final concentration is at 10 times of reductions, the copy number of detection
At 10 times of reduction, it was demonstrated that the probe has good specificity.
2, mutation rate detection experiment
Mutant plasmids and wild plasmid are mixed in the ratio of 1:1, concentration is respectively 10-5, 10-6, 10-7；Use droplet
Formula digital pcr system detects the mutation detector efficiency of the method for the present invention, as the result is shown saltant type and wild plasmid
Melting concn is 10-7When digital pcr detection saltant type and wild type ratio be 1:1, and the number of copy number also with concentration
Relationship in gradient, therefore method of the invention can reach the copy number of units to the detection of mutation (see Fig. 4-Fig. 5, table 1).
Separately mutant plasmids and wild plasmid are mixed according to the ratio of 1:1,1:10,1:100,1:5000,1:10000
It closes, is detected with mutation detector efficiency of the droplet type digital pcr system to the method for the present invention, as the result is shown droplet type number
PCR detector efficiency is probably at five one thousandths (Fig. 6, table 2)
Table 2
3, accuracy and clinical application test
The test experience of PIK3CA H1047R probe primer is carried out to the standard items HD701 of Horizon company, it is defeated respectively
Enter 40ng, 4ng, 0.4ng standard items carry out the digital pcr detection of 20 μ l systems, testing result such as Fig. 7-Fig. 8.Use the present invention
Method testing result be no matter 40ng input or 4ng input, the frequency of mutation (see the table below 3) all with Horizon company offer
Data 17.5% be consistent, and deviation is little.
Table 3
Intravenous blood collection is carried out to 5 tumor patients, and blood plasma is separated.Circulation trip is carried out with the blood plasma after separation
The extraction of freestone acid, with recycle the sample of dissociative DNA respectively with the primer and probe of the invention to the site PIK3CA H1047R into
Row detection, while the site PIK3CA H1047R is detected using the method that two generations were sequenced, testing result is as shown in Figure 9.Make
It is to there are wild type copies to detect (Figure 10) in 5 detection samples with method testing result of the invention, sample 1,2,3 has
There is (Fig. 9) in PIK3CA H1047R site mutation, the frequency of mutation is shown in Table 4；With two generations be sequenced method to same 5 samples into
Row detection, as a result show that, with the presence of same three samples H1047R mutation, the frequency of mutation is shown in Table 4.In conclusion side of the invention
Method can accurately detect sample to be existed with the presence or absence of PIK3CA gene H1047R site mutation, and can calculate the frequency of mutation, and
And there is opposite consistency with the frequency of mutation of two generation sequencing approaches detection.In addition by digital pcr side below 5/1000ths
The frequency of mutation that method detects, needs to further use a variety of method validations, and false positive probability is higher.
Table 4
The above results show the specific primer (SEQ in the detection site PIK3CA gene H1047R provided by embodiment 1
ID NO.1-2) and the detection effect of probe (SEQ ID NO.3-4) it is best, be suitable for clinical sample to tissue or peripheral blood
The detection in the middle site PIK3CA gene H1047R.
Sequence table
<110>Guangzhou Man Rui biology information technology Co., Ltd
<120>a kind of specific primer for detecting the site PIK3CA gene H1047R is to, probe and kit
<130> KHP171115168.1
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caagaggctt tggagtattt catg 24
<210> 2
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttctcagtta cttttcagtt caatgc 26
<210> 3
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgcacatca tggtgg 16
<210> 4
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aatgatgcac gtcatg 16
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gcaagaggct ttggagtatt tca 23
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gcaagaggct ttggagtatt tcat 24
Claims (10)
1. a kind of for detecting the specific primer pair in the site PIK3CA gene H1047R, the nucleotide of the specific primer pair
Sequence is as shown in SEQ ID NO.1-2.
2. the probe combinations contain 2 spies with specific primer described in claim 1 to the probe combinations being used cooperatively
Needle, nucleotide sequence is respectively as shown in SEQ ID NO.3-4.
3. probe combinations as claimed in claim 2,5 ' label VIC, 3 ' label MGB-NFQ of probe shown in SEQ ID NO.3,
5 ' flag F AM, 3 ' label MGB-NFQ of probe shown in SEQ ID NO.4.
4. the kit containing probe combinations described in specific primer pair described in claim 1 and/or claim 2.
5. the detection reagent containing probe combinations described in specific primer pair described in claim 1 and/or claim 2.
6. specific primer described in claim 1 to and claim the 2-3 any probe combinations or claim 4 institute
Application of the detection reagent described in the kit or claim 5 stated in the detection site PIK3CA gene H1047R.
7. kit as claimed in claim 4, which is characterized in that its working procedure are as follows:
(1) genomic DNA of sample to be tested is extracted；
(2) using the genomic DNA of extraction as template, using specific primer described in claim 1 to and claim 2 described in
Probe combinations, carry out digital pcr amplified reaction；
(3) result is determined according to the copy number that fluorescence signal provides.
8. kit as claimed in claim 7, which is characterized in that the digital pcr amplified reaction of step (2) in its working procedure
Program are as follows: 95 DEG C of initial denaturation 10min；94 DEG C of denaturation 20s, 56 DEG C of annealing and extension 45s, totally 40 recycle；98 DEG C of solidifications are micro-
Drip 10min.
9. a kind of method for detecting the site PIK3CA gene H1047R in free nucleic acid in blood, which is characterized in that including as follows
Step:
(1) genomic DNA of blood sample to be measured is extracted；
(2) using the genomic DNA of extraction as template, using specific primer described in claim 1 to and claim 2 described in
Probe combinations, carry out digital pcr amplified reaction；
(3) result is determined according to the copy number that fluorescence signal provides.
10. according to the method described in claim 9, it is characterized in that, carrying out the program of digital pcr amplified reaction in step (2)
Are as follows: 95 DEG C of initial denaturation 10min；94 DEG C of denaturation 20s, 56 DEG C of annealing and extension 45s, totally 40 recycle；98 DEG C of solidification droplets
10min。
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| CN116219015A (en) * | 2023-02-06 | 2023-06-06 | 中日友好医院(中日友好临床医学研究所) | PCR-based detection kit and detection method for PIK3CA gene mutation in DNA |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101445832A (en) * | 2008-12-23 | 2009-06-03 | 广州益善生物技术有限公司 | PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof |
| CN109022573A (en) * | 2017-06-12 | 2018-12-18 | 深圳华大基因研究院 | The combination of breast cancer PIK3CA hot spot mutation detection probe primer sequence and kit |
| CN109295224A (en) * | 2018-10-12 | 2019-02-01 | 上海赛安生物医药科技股份有限公司 | The optimization method and testing product of PIK3CA gene H1047R mutation digital pcr detection architecture |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101445832A (en) * | 2008-12-23 | 2009-06-03 | 广州益善生物技术有限公司 | PIK3CA gene mutation detection probe, detection liquid phase chip and detection method thereof |
| CN109022573A (en) * | 2017-06-12 | 2018-12-18 | 深圳华大基因研究院 | The combination of breast cancer PIK3CA hot spot mutation detection probe primer sequence and kit |
| CN109295224A (en) * | 2018-10-12 | 2019-02-01 | 上海赛安生物医药科技股份有限公司 | The optimization method and testing product of PIK3CA gene H1047R mutation digital pcr detection architecture |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116219015A (en) * | 2023-02-06 | 2023-06-06 | 中日友好医院(中日友好临床医学研究所) | PCR-based detection kit and detection method for PIK3CA gene mutation in DNA |
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