CN109161594A - A kind of method and its kit detecting BRAF gene mutation - Google Patents
A kind of method and its kit detecting BRAF gene mutation Download PDFInfo
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Abstract
The present invention provides a kind of methods and its kit for detecting BRAF gene mutation, specifically the present invention provides a kind of method and its kit based on digital pcr technology detection BRAF gene mutation, which includes the enzyme for preparing digital pcr reaction solution and the primer combination of probe suitable for digital pcr.Compared with traditional PCR technique, the present invention overcomes the low disadvantage of traditional PCR technique detection limit for height, accuracy using digital pcr, and primed probe used in the present invention can detect multiple site mutations of BRAF simultaneously, substantially increase versatility.Using detection method and kit of the invention, sample requirements are few, and sensitivity and specificity are high, and detection efficiency greatly improves.
Description
Technical field
The invention belongs to biotechnologys and genetic test field, and in particular to a kind of detection BRAF gene V600 Positive mutants
Primer, probe and its kit.
Background technique
Positioned at BRAF gene encoding serine/Serineprotein kinase of chromosome 7q34, belonged to ARAF, CRAF
RAF family.The mutation of BRAF gene can occur in primary and metastatic tumo(u)r.Studies have shown that the tumour of 7-8% occurs
BRAF gene point mutation, including about 40-68% malignant mela noma, 5-20% thyroid papillary carcinoma, 5-15% colon cancer with
And 3% lung cancer etc..The BRAF gene mutation type having now been found that is predominantly located at the 15th of CR3 kinase domain up to more than 40
Exon and the 11st exon, wherein the most common mutant form of BRAF (about 95%) is the 1799th nucleosides of the 15th exon
Valine (T) sports glutamic acid (A) on acid, i.e. V600E, and what other be reported has V600D, V600K.BRAF is RAS-
The important transduced element of RAF-MEK-ERK MAPK signal path, BRAF mutation can directly result in inhibition of mitogen-activated extracellular letter
Number regulatory protein kinases (mitogen-activated extracellularsignal-regulated kinase, MEK) is living
Change and continue to activate ERK, cause MAPK access by sustained activation, and then cause cell cycle and circulation out of control, promotes tumour
Occur.
The problems such as can causing tumor drug resistance and patient's poor prognosis due to BRAF gene mutation, BRAF gene mutation is recognized
For an individual index for being tumor patient prognosis evaluation.Therefore, BRAF is carried out to tumor patient especially late tumor patient
The detection of gene mutation site is very necessary, this helps to judge therapeutic effect, it helps clinician, which chooses, to be corresponded to
Target therapeutic agent to formulating tumor individual therapy scheme.
For malignant mela noma, thyroid cancer, colorectal cancer, the BRAF gene occurred in the patients such as non-small cell lung cancer
Mutation, existing clinical common detection means specifically include that PCR sequencing PCR and real time fluorescent quantitative method etc..Both detection methods are equal
It needs to carry out detection in Gene Mutation after extracting DNA in tumor tissues, this carries out real-time monitoring band for patient over the course for the treatment of
Come difficult.
Therefore, sensitivity is stronger, accuracy is higher there is an urgent need in the art to developing, and the gene mutation that sample requirements are few
Detection method.
Summary of the invention
The purpose of the present invention is to provide a kind of methods and its kit for detecting BRAF gene mutation.
In the first aspect of the present invention, a kind of general drawing for digital pcr detection BRAF gene V600 mutation is provided
Object pair, the universal primer is to including general upstream primer F and general reverse primer R；Wherein, the general upstream primer F
Nucleotide sequence is as shown in SEQ ID NO.:1, and the nucleotide sequence of the general reverse primer R is as shown in SEQ ID NO.:2.
The second aspect of the present invention provides a kind of probe groups for digital pcr detection BRAF gene V600 mutation, institute
Stating probe groups includes one or more probes selected from the group below: probe T1, probe T2, probe T3 and probe T4；Wherein, described
Probe T1 nucleotide sequence is as shown in SEQ ID NO.:3, and the probe T2 nucleotide sequence is as shown in SEQ ID NO.:4, institute
Probe T3 nucleotide sequence is stated as shown in SEQ IDNO.:5, the probe T4 nucleotide sequence is as shown in SEQ ID NO.:6.
In another preferred example, the probe groups include: probe T1, probe T2, probe T3 and probe T4.
In another preferred example, the 5 ' ends of the probe T1 include fluorescent reporter group VIC；And/or the probe T1
3 ' end include quenching group MGB.
In another preferred example, the 5 ' ends of the probe T2, the probe T3 or the probe T4 include fluorescence report
Group FAM；And/or 3 ' the ends of the probe T2, the probe T3 or the probe T4 include quenching group MGB.
The third aspect of the present invention provides a kind of kit for digital pcr detection BRAF gene V600 mutation, institute
Stating kit includes primer pair described in first aspect present invention.
In another preferred example, the kit further includes probe groups described in second aspect of the present invention.
In another preferred example, the kit includes drawing visit mixed liquor 1, draw to visit mixed liquor 2 and draw and visit mixed liquor 3；Its
In,
It is described draw visit mixed liquor 1 include: the general upstream primer F, the general reverse primer R, the probe T1 and
The probe T2；
It is described draw visit mixed liquor 2 include: the general upstream primer F, the general reverse primer R, the probe T1 and
The probe T3；
It is described draw visit mixed liquor 3 include: the general upstream primer F, the general reverse primer R, the probe T1 and
The probe T4.
In another preferred example, the kit further includes positive control and/or negative control.
In another preferred example, the kit further includes digital pcr reaction enzymes；And/or digital pcr buffer.
In another preferred example, the general upstream primer F and general reverse primer R in the reaction system final
Concentration is 500~2000nM；More preferably 1000nM.
In another preferred example, the ultimate density of the probe T1 in the reaction system is 100~150nM；Preferably
125nM。
In another preferred example, the probe T2, the probe T3, and/or the probe T4 be in the reaction system most
Final concentration of 100~500nM；Preferably 250nM.
The fourth aspect of the present invention provides a kind of method of digital pcr detection BRAF gene V600 mutation, the method
Comprising steps of
(1) DNA sample of object to be detected is provided；
(2) digital pcr reaction system is prepared:
The digital pcr reaction system include step (1) provide the DNA sample, described in first aspect present invention
Probe groups described in primer pair and second aspect of the present invention；
(3) the digital pcr reaction system that step (2) obtain is prepared as digital pcr reaction droplet, and carries out PCR
Amplification；
(4) droplet detects:
After PCR amplification, testing result is analyzed, and target molecules are calculated based on Poisson distribution Statistics
Copy number.
In another preferred example, the method is non-diagnostic purpose detection method.
In another preferred example, in the step (2), the general upstream primer F and the general reverse primer R are in institute
Stating the ultimate density in digital pcr reaction system is 500~2000nM；More preferably 1000nM.
In another preferred example, ultimate density of the probe T1 in the digital pcr reaction system be 100~
150nM；Preferably 125nM.
In another preferred example, the probe T2, the probe T3, and/or the probe T4 are reacted in the digital pcr
Ultimate density in system is 100~500nM；Preferably 250nM.
The fifth aspect of the present invention provides universal primer pair described in first aspect present invention, and/or the present invention second
The purposes of probe groups described in aspect is used to prepare digital pcr detection kit, and the digital pcr detection kit is for examining
Survey BRAF gene V600 mutation.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Figure 1A to Fig. 1 D shows digital pcr sensitivity technique result；
Fig. 2A to Fig. 2 F shows kit detection tumor patient blood plasma sample results of the present invention；
Fig. 3 A to Fig. 3 C shows kit detection tumor patient tissue samples result of the present invention；
Fig. 4 shows the testing result using control primer pair 1；
Fig. 5 shows the testing result using control primer pair 2；
Fig. 6 shows the testing result using control probe group 1；
Fig. 7 shows the testing result using control probe group 2.
Specific embodiment
The present inventor is obtained a kind of based on digital pcr technology detection BRAF gene mutation by extensive and in-depth research
Method and its kit, the kit include that enzyme for preparing digital pcr reaction solution and the primer suitable for digital pcr are visited
Needle combination.Compared with traditional PCR technique, digital pcr overcomes the low disadvantage of traditional PCR technique detection limit for height, accuracy, this hair
Primed probe used in bright can detect multiple site mutations of BRAF simultaneously, substantially increase versatility.Using of the invention
Detection method and kit, sample requirements are few, and sensitivity and specificity are high, and detection efficiency greatly improves.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this
Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and
And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated
" about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method
And material, place enumerates preferred method and material herein.
Digital pcr
Digital pcr, that is, Digital PCR (dPCR), it is a kind of nucleic acid molecules absolute quantitation technology, by by one
Sample is divided into tens to tens of thousands of parts, is assigned to different reaction members, each unit contains at least one the target molecule of copy
(DNA profiling) carries out PCR amplification to target molecule respectively in each reaction member, to each reaction member after amplification
Fluorescence signal carry out statistical analysis.
Compared with digital pcr, real time fluorescent quantitative method is had the following deficiencies: 1. real time fluorescent quantitative methods be carry out it is qualitative or
Person's relative quantitative assay can not carry out absolute quantification analysis, therefore be unable to get accurately result；2. real time fluorescent quantitative method without
Method directly obtains objective results, artificially carries out interpretation to result, leads to the subjective of result；3. relative to digital pcr
0.1% sensitivity, the conventional method of only 1% sensitivity seem that sensitivity is low, and being unable to satisfy specific clinical detection needs
It asks.
And the appearance of liquid Biopsy carries out real-time monitoring for patient over the course for the treatment of and brings convenience.With it is traditional
Tissue biopsy is compared, and liquid biopsy has rapid, convenient, the damaging many merits such as small.Clinician can be monitored with it
Tumor recurrence is predicted in reaction of the tumour to treatment.Liquid biopsy can also help doctor when patient does not occur any symptom
Wait the tumour of discovery initial stage.CtDNA is called Circulating tumor DNA, is that apoptosis or downright bad tumour cell are secreted into blood and released
The DNA fragmentation put.The genomic information obtained from the ctDNA in blood is even it can be pointed out that the position that in-vivo tumour rises.
But since the ctDNA in tumour early stage, blood samples of patients is also seldom, and ctDNA segment is relatively small, at present to swollen
The clinical testing procedure of tumor related gene is not particularly suited for the detection to ctDNA.Digital pcr use with " endpoint signal have or
Nothing " quantifies nucleic acid molecules as quantitative approach, compared with other PCR, accuracy, accuracy and the sensitivity of result
More preferably.
Compared with other detection techniques, digital pcr is had the advantage that
1. highly sensitive
The interference that wild type is detected sequence can be excluded, to improve the sensitivity of rare mutation detection.
2. pinpoint accuracy
Digital pcr, can be accurately by the quantity and ratio of calculating positive reaction member in a reaction members up to ten thousand
Detect the aim sequence difference varied less.
3. height endurability
Digital pcr technology can make background sequence and PCR response inhabitation object uniform during reaction system is distributed
It is assigned to each reaction member, and in most of reaction member and does not contain aim sequence, low-abundance aim sequence is opposite
It is enriched in certain reaction members, reaction is done to reduce background sequence and mortifier in these reaction members significantly
It disturbs.
4. absolute quantitation
Digital pcr, which is used, quantifies nucleic acid molecules using " endpoint signal with or without " as quantitative approach, without according to
Rely and can be carried out accurate absolute quantitation detection in Ct value and standard curve.
But requirement of the digital pcr to primer and probe is all very high, a little change of primer and probe sequence just will affect
The specificity and sensitivity of detection.
That the technical problem to be solved in the present invention is to provide a kind of sample requirements is few, sensitivity and specificity are high, detection effect
The BRAF gene V600 Positive mutants detection primer probe and its kit that rate greatly improves.
A kind of technical solution that the present invention proposes to solve above-mentioned technical problem is: a kind of BRAF gene V600 Positive mutants
Detection primer probe, including general upstream primer F and general reverse primer R, it is wild for detecting the 600th codon of BRAF gene
Type TaqMan probe T1, the TaqMan mutant probe T2 in the 600th site codon V600E, for detecting the 600th codon
The TaqMan mutant probe T3 in the site V600D and TaqMan mutant probe for detecting the 600th site codon V600K
T4；
The nucleotide sequence of the general upstream primer F as shown in SEQ ID NO.:1, the general reverse primer R's
Nucleotide sequence is as shown in SEQ ID NO.:2；
The nucleotide sequence of the TaqMan probe T1 for detecting the 600th codon wild type of BRAF gene is such as
Shown in SEQ ID NO.:3；
The nucleotide sequence such as SEQ for being used to detect the 600th site codon V600E TaqMan mutant probe T2
Shown in ID NO.:4；
The nucleotide sequence such as SEQ of the TaqMan mutant probe T3 for detecting the 600th site codon V600D
Shown in ID NO.:5；
The nucleotide sequence such as SEQ of the TaqMan mutant probe T4 for detecting the 600th site codon V600K
Shown in ID NO.:6.
The ultimate density of above-mentioned general upstream primer F and general reverse primer R in the reaction system is 1000nM, described
Probe T1 ultimate density in the reaction system be 125nM, the ultimate density of T2, T3, T4 in the reaction system is
250nM。
5 ' the ends of above-mentioned TaqMan probe T1 include fluorescent reporter group VIC, and 3 ' ends include quenching group MGB；
5 ' the ends of described TaqMan probe T2, T3, the T4 include fluorescent reporter group FAM, and 3 ' ends include quenching group MGB.
A kind of technical solution that the present invention proposes to solve above-mentioned technical problem is: a kind of using above-mentioned primed probe
BRAF gene V600 Positive mutants testing product.
A kind of BRAF gene V600 Positive mutants detection kit: including for preparing digital pcr reaction solution enzyme,
Buffer and primed probe mixed liquor；
The primed probe mixed liquor includes general upstream primer F and general reverse primer R, for detecting BRAF gene
600 codon wild type TaqMan probe T1, the TaqMan mutant probe T2 in the 600th site codon V600E, for detecting the
The TaqMan mutant probe T3 in 600 sites codon V600D and TaqMan for detecting the 600th site codon V600K
Mutant probe T4；
The nucleotide sequence of the general upstream primer F is as shown in SEQ ID NO.:1, the nucleosides of the downstream primer R
Acid sequence is as shown in SEQ ID NO.:2；
The nucleotide sequence of the TaqMan probe T1 for detecting the 600th codon wild type of BRAF gene is such as
Shown in SEQ ID NO.:3；
The nucleotide sequence such as SEQ for being used to detect the 600th site codon V600E TaqMan mutant probe T2
Shown in ID NO.:4；
The nucleotide sequence such as SEQ of the TaqMan mutant probe T3 for detecting the 600th site codon V600D
Shown in ID NO.:5；
The nucleotide sequence such as SEQ of the TaqMan mutant probe T4 for detecting the 600th site codon V600K
Shown in ID NO.:6.
The ultimate density of the general upstream primer F and general reverse primer R in the reaction system is 1000nM, described
Probe T1 ultimate density in the reaction system be 125nM, the ultimate density of T2, T3, T4 in the reaction system is
250nM。
5 ' ends of the kit of above-mentioned detection BRAF gene V600 Positive mutants, the TaqMan probe T1 include glimmering
Light reporter group VIC, 3 ' ends include quenching group MGB；5 ' the ends of described TaqMan probe T2, T3, the T4 include fluorescence
Reporter group FAM, 3 ' ends include quenching group MGB.
Above-mentioned BRAF gene V600 Positive mutants detection kit includes 7.5 μ L in reaction system when reaction
Mastermix, 0.75 μ L Solution, 1 μ L primed probe mixed liquor, 5 μ L sample DNA templates or reference substance, add sterile water to mend
Football Association's volume is to 16 μ L.
It is connected with MGB (Minor Groove Binder) modification group on probe, the Tm value of probe can be improved 10 DEG C
Left and right.Therefore in order to obtain same Tm value, MGB probe can be designed shorter than general T aqMan probe, both reduce conjunction
At cost, but also the success rate of probe design greatly improves.The quenching group of MGB probe uses non-fluorescence quenching group
(Non-Fluorescent Quencher), itself does not generate fluorescence, can substantially reduce the intensity of background signal.
Main advantages of the present invention are:
1, the present invention provides the primer pairs and probe sequence that use digital pcr, compared with traditional PCR technique, the present invention
The low disadvantage of traditional PCR technique detection limit for height, accuracy is overcome using digital pcr.
2, primed probe used in the present invention can detect multiple site mutations of BRAF simultaneously, substantially increase general
Property.
3, using detection method and kit of the invention, sample requirements are few, and sensitivity and specificity are high, detection efficiency
It greatly improves.
4, the probe of BRAF gene V600 Positive mutants detection kit of the invention carries out MGB modification, improves and is directed to
The specificity of the respective probe in wild type site and mutational site, achievees the purpose that probe and target sequence specific binding, with ARMS
Method (amplification refractory mutation system, be mutated amplification system) compares, can by wild type and
Saltant type BRAF gene detected simultaneously.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002)
Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
The composition of 1 kit of embodiment
The BRAF gene V600 Positive mutants detection kit of the present embodiment, including digital pcr Mastermix,
Solution, primed probe mixed liquor, positive control and negative control.Each ingredient of kit is as shown in table 1.
1 Kit components table of table
Reagent constituents are described as follows in above-mentioned table 1:
1, digital pcr enzyme is 2 × Mastermix (purchased from JN Medsys company, article No. are as follows: 12013).
2, Solution be 20 × buffer, (be purchased from JN Medsys company, article No. are as follows: 10011)
3, draw that visit mixed liquor 1 include general upstream primer F, general reverse primer R, the 600th codon of BRAF gene is wild
The TaqMan probe T1 of type and the 600th site codon V600E TaqMan mutant probe T2.
4, draw that visit mixed liquor 2 include general upstream primer F, general reverse primer R, the 600th codon of BRAF gene is wild
The TaqMan probe T1 of type and the 600th site codon V600D TaqMan mutant probe T3.
5, draw that visit mixed liquor 3 include general upstream primer F, general reverse primer R, the 600th codon of BRAF gene is wild
The TaqMan probe T1 of type and the 600th site codon V600K TaqMan mutant probe T4.
5 ' the ends of TaqMan probe T1 include fluorescent reporter group VIC, and 3 ' ends include quenching group MGB；Described
5 ' the ends of TaqMan probe T2, T3, T4 include fluorescent reporter group FAM, and 3 ' ends include quenching group MGB.
6, positive control includes the biological sheet degree of BRAF V600E, V600D, V600K site mutation, (by raw work biology
Engineering (Shanghai) limited liability company provides).
7, negative control is the Hela cellular nucleic acid extracted by our company.
The design of 2 primed probe of embodiment and sensitivity technique
1, primer probe sequence of the invention is as shown in table 2.
2 primer probe sequence table of table
2, primed probe is synthesized by Da'an Gene Company, Zhongshan University, and wherein primer is with 1 × TE according to saying
Bright book is diluted to mother liquor by dry powder, then mother liquor is diluted to working solution with 1 × TE；Probe with MGB dilution to specifications by
Dry powder is diluted to mother liquor, then by mother liquor with MGB diluted at working solution.
3, the ultimate density of general upstream primer F and general reverse primer R in the reaction system is 1000nM, described
The ultimate density of TaqMan probe T1 in the reaction system is 125nM, and the ultimate density of T2, T3, T4 in the reaction system is
250nM。
Using positive control to above-mentioned primer and probe combine carry out sensitivity technique, sensitivity technique the result shows that, make
Digital pcr is carried out with primer combination of probe of the invention, 0.1% sensitivity can be obtained, it is sensitive more than ARMS method 1%
Degree.For V600E sensitivity technique result as shown in Figure 1A to Fig. 1 D, what testing result showed digital pcr of the present invention detects mutation
Rate is 0.1%.
The kit provided by the invention of embodiment 3 detects plasma specimen
1, the extraction of blood plasma ctDNA
Collect 5mL tumor patient (determining that saltant type is respectively V600E, V600D and V600K through gene sequencing) and Healthy People
The blood sample of (doing negative control use), high speed centrifugation separate supernatant, obtain blood plasma.The peace limited public affairs of gene share are reached with Zhongshan University
The plasma DNA extracts kit (article No.: cat.#DA0670 or cat.#DA0680) provided is provided, is said according to kit operation
Dissociative DNA in bright book extracting patients blood plasma.After obtaining plasma sample dissociative DNA, it is divided using 2000 ultramicron of NanoDrop
Photometer or same quasi-instrument measure plasma sample dissociative DNA concentration and purity, can be loaded or be placed in -20 DEG C immediately and save backup.
2, prepared by digital pcr reaction solution
The digital pcr specific enzyme (2 × mastermix) of 7.5 μ L in kit, 0.75 μ L Solution, 1 μ L is taken to draw
The template of 5 μ L is added in physical prospecting needle mixed liquor, and sterilizing ultrapure water is added and mends to 16 μ L, digital pcr reaction solution is made, prepares quantitative
Reaction system, oscillation are centrifuged off bubble after mixing.Template refers to that tumor patient blood plasma sample ctDNA, positive control, feminine gender are right
It is compareed according to NTC (No Template Control), wherein positive control is the cellular nucleic acid of V600 Positive mutants, negative right
It compares according to for human normal plasma sample ctDNA, NTC as sterilizing ultrapure water.
3, PCR reacts droplet preparation
The dPCR reaction solution for taking 15 μ L to prepare, by ClarityTMAutomatic sample adding instrument is automatic by it, is equably subdivided into 10
The reaction member of 000 nanoliter level；Using ClarityTMSealing instrument is added sealing fluid and is sealed to reaction member.
4, PCR amplification
PCR amplification is carried out in dedicated PCR instrument, response procedures are as follows: 95 DEG C of the condition in initial denaturation stage, 10min；1
Circulation；94 DEG C of 30s, 56 DEG C of 30s, 40 circulations；98 DEG C, 10min, 1 circulations；4 DEG C, heat preservation.
5, droplet detects
PCR after reaction, uses ClarityTMSystem software analyzes testing result, and is counted based on Poisson distribution
Principle calculates the copy number of target molecules in each sample, and mutation includes the mutation of V600E, V600D and V600K, as a result as schemed
Shown in 2A to Fig. 2 F, it is shown that the present embodiment detects the dPCR result in the mutational site V600E in plasma specimen.Fig. 2A is this implementation
The dPCR result of the example detection mutational site V600E positive control, Fig. 2 B are the dPCR knot for detecting the mutational site V600E negative control
Fruit, Fig. 2 C are that the dPCR result in the mutational site V600E, Fig. 2 D are the detection mutational site V600E NTC control in detection plasma specimen
DPCR result, Fig. 2 E be that detect the dPCR result in the mutational site V600D, Fig. 2 F in plasma specimen be in detection plasma specimen
The dPCR result in the mutational site V600K.
The kit provided by the invention of embodiment 4 detects paraffin embedding tumor tissues sample
1, the extraction of tumor tissues DNA
Tumor tissues (determining that saltant type is respectively V600E, V600D and V600K through gene sequencing) nucleic acid of paraffin embedding
Extract the nucleic acid extraction and the purified reagent (number of putting on record: Guangdong fringe tool that Da'an Gene Company, Zhongshan University's offer is provided
It is No. 20170666 standby), concrete operation step is please guided according to kit specification and is carried out.Use 2000 ultramicron of Nano-Drop
Spectrophotometer carries out definite value to the nucleic acid after extraction with quasi-instrument.Inner mark solution in this kit participates in extraction process,
So must mating extraction reagent use.The nucleic acid DNA of extraction can be loaded or be placed in -20 DEG C immediately and save backup.
2, prepared by digital pcr reaction solution
The digital pcr specific enzyme (2 × mastermix) of 7.5 μ L in kit, 0.75 μ L Solution, 1 μ L is taken to draw
The template of 5 μ L is added in physical prospecting needle mixed liquor, and sterilizing ultrapure water is added and mends to 16 μ L, digital pcr reaction solution is made, prepares quantitative
Reaction system, oscillation are centrifuged off bubble after mixing.Template refers to that tumor patient blood plasma sample ctDNA, positive control, feminine gender are right
It is compareed according to NTC (No Template Control), wherein positive control is the cellular nucleic acid of V600 Positive mutants, negative right
It compares according to for human normal plasma sample ctDNA, NTC as sterilizing ultrapure water.
3, PCR reacts droplet preparation
The dPCR reaction solution for taking 15 μ L to prepare, by ClarityTMAutomatic sample adding instrument is automatic by it, is equably subdivided into 10
The reaction member of 000 nanoliter level；Using ClarityTMSealing instrument is added sealing fluid and is sealed to reaction member.
4, PCR amplification
PCR amplification is carried out in dedicated PCR instrument, response procedures are as follows: 95 DEG C of the condition in initial denaturation stage, 10min；1
Circulation；94 DEG C of 30s, 56 DEG C of 30s, 40 circulations；98 DEG C, 10min, 1 circulations；4 DEG C, heat preservation.
5, droplet detects
PCR after reaction, analyzes testing result with ClarityTM system software, and is united based on Poisson distribution
Meter principle calculates the copy number of target molecules in each sample, and mutation includes the mutation of V600E, V600D and V600K.As a result such as
Shown in Fig. 3 A- Fig. 3 C, it is shown that the present embodiment detects the dPCR knot in the mutational site V600E in paraffin embedding tumor tissues sample
Fruit.Fig. 3 A detects the dPCR result in the mutational site V600E in paraffin embedding tumor tissues sample, Fig. 3 B detects paraffin embedding tumour
The dPCR result in the mutational site V600D, Fig. 3 C detect the mutational site V600K in paraffin embedding tumor tissues sample in tissue specimen
DPCR result.
Comparative example 1
The present inventor in the course of the research, has screened tens of pairs of digital pcr primers, wherein the overwhelming majority is unable to satisfy number
The demand of word PCR.Typical general primer sequence and detection effect data are as follows:
Compare primer pair 1:
Upstream primer 1:5 '-CATAATGCTTGCTCTGATAGG-3 ' (SEQ ID NO.:7)；
Downstream primer 1:5 '-AGCCTCAATTCTTACCATCC-3 ' (SEQ ID NO.:8).
Compare primer pair 2:
Upstream primer 2:5 '-CTCTTCATAATGCTTGCTCTG-3 (SEQ ID NO.:9)；
Downstream primer 2:5 '-TAGCCTCAATTCTTACCATCC-3 ' (SEQ ID NO.:10).
Specific detecting step, testing conditions, the same above embodiments of probe sequence, use the testing result of control primer pair 1
As shown in figure 4, testing result shows that the primer pair specificity is very poor.Using control primer pair 2 testing result as shown in figure 5,
Testing result shows that the primer pair specificity is very poor.Control primer pair 1 and control primer pair 2 are unable to satisfy wanting for clinical detection
It asks.
Comparative example 2
The present inventor is optimized with probe sequence repeatedly in the course of the research, to digital pcr, finds some in research
Although only a poor nucleotide can significantly affect detection specificity to probe, wherein the overwhelming majority is unable to satisfy the need of digital pcr
It asks.Typical average probe sequence and detection effect data are as follows:
Control probe group 1:
Wild probe T1:5 '-VIC-CTAGCTACAGTGAAATCTCG-MGB-3 ' (SEQ ID NO.:3)；
Mutant probe T2-2:5 '-FAM-CCTCAATTCTTACCATCCACAA-MGB-3 ' (SEQ ID NO.:11)；
Mutant probe T3:5 '-FAM-GCTACAGATAAATCTCGATGG-MGB-3 ' (SEQ ID NO.:5)；
Mutant probe T4:5 '-FAM-AGCTACAAAGAAATCTCGATGG-MGB-3 ' (SEQ ID NO.:6).
Control probe group 2:
Wild probe T1-2:5 '-VIC-CAACTGTTCAAACTGATGGGAC-MGB-3 ' (SEQ ID NO.:12)；
Mutant probe T2:5 '-FAM-GCTACAGAGAAATCTCGATGG-MGB-3 ' (SEQ ID NO.:4)；
Mutant probe T3:5 '-FAM-GCTACAGATAAATCTCGATGG-MGB-3 ' (SEQ ID NO.:5)；
Mutant probe T4:5 '-FAM-AGCTACAAAGAAATCTCGATGG-MGB-3 ' (SEQ ID NO.:6).
Specific detecting step, testing conditions, the same above embodiments of primer sequence, use wild probe in control probe group 1
The testing result in the T1 and mutant probe T2-2 detection mutational site V600E is as shown in fig. 6, testing result shows the spy of the probe groups
The opposite sex is poor, sensitivity is poor.
In control probe group 2, use wild probe T1-2, wild probe T1-2 and mutant probe T2 detection V600E prominent
The testing result of point is conjugated as shown in fig. 7, testing result shows that the specificity of the probe groups is poor, sensitivity is poor.Similarly,
Wild probe T1-2 and mutant probe T3 detection the mutational site V600D testing result show the probe groups specificity it is poor,
The poor requirement for being unable to satisfy digital pcr detection clinical application of sensitivity.And wild probe T1-2 and mutant probe T4 detection
The testing result in the mutational site V600K shows that the probe groups have good specificity and sensitivity.
And the present inventor has found in R&D process, in the primer and probe group screened, only few combination energy
The target gene in blood plasma is enough detected, preliminary analysis reason may be too low for the target gene content in blood plasma, most of primer and spy
Needle group sensitivity is poor, leads to not detect the target gene in blood plasma.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
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Claims (10)
1. a kind of universal primer pair for digital pcr detection BRAF gene V600 mutation, which is characterized in that the universal primer
To including general upstream primer F and general reverse primer R；Wherein, the nucleotide sequence such as SEQ ID of the general upstream primer F
Shown in NO.:1, the nucleotide sequence of the general reverse primer R is as shown in SEQ ID NO.:2.
2. a kind of probe groups for digital pcr detection BRAF gene V600 mutation, which is characterized in that the probe groups include choosing
From one or more probes of the following group: probe T1, probe T2, probe T3 and probe T4；Wherein, the probe T1 nucleotides sequence
Column are as shown in SEQ ID NO.:3, and the probe T2 nucleotide sequence is as shown in SEQ ID NO.:4, the probe T3 nucleotide
Sequence is as shown in SEQ ID NO.:5, and the probe T4 nucleotide sequence is as shown in SEQ ID NO.:6.
3. probe groups as claimed in claim 2, which is characterized in that the probe groups include: probe T1, probe T2, probe T3,
With probe T4.
4. probe groups as claimed in claim 2, which is characterized in that the 5 ' ends of the probe T1 include fluorescent reporter group
VIC；And/or 3 ' the ends of the probe T1 include quenching group MGB.
5. probe groups as claimed in claim 2, which is characterized in that the probe T2, the probe T3 or the probe T4's
5 ' ends include fluorescent reporter group FAM；And/or 3 ' the ends of the probe T2, the probe T3 or the probe T4 include
There is quenching group MGB.
6. a kind of kit for digital pcr detection BRAF gene V600 mutation, which is characterized in that the kit includes power
Benefit require 1 described in primer pair.
7. kit as claimed in claim 6, which is characterized in that the kit further includes probe as claimed in claim 2
Group.
8. kit as claimed in claim 6, which is characterized in that the kit includes drawing visit mixed liquor 1, draw spy mixed liquor
2 and draw visit mixed liquor 3；Wherein,
It is described to draw that visit mixed liquor 1 include: the general upstream primer F, the general reverse primer R, the probe T1 and described
Probe T2；
It is described to draw that visit mixed liquor 2 include: the general upstream primer F, the general reverse primer R, the probe T1 and described
Probe T3；
It is described to draw that visit mixed liquor 3 include: the general upstream primer F, the general reverse primer R, the probe T1 and described
Probe T4.
9. a kind of method for digital pcr detection BRAF gene V600 mutation, which is characterized in that the method includes the steps:
(1) DNA sample of object to be detected is provided；
(2) digital pcr reaction system is prepared:
The digital pcr reaction system includes the DNA sample, the primer described in first aspect present invention that step (1) provides
And second aspect of the present invention described in probe groups；
(3) the digital pcr reaction system that step (2) obtain is prepared as digital pcr reaction droplet, and carries out PCR amplification；
(4) droplet detects:
After PCR amplification, testing result is analyzed, and copying for target molecules is calculated based on Poisson distribution Statistics
Shellfish number.
10. the purposes of universal primer pair described in claim 1, and/or probe groups as claimed in claim 2, which is characterized in that
It is used to prepare digital pcr detection kit, the digital pcr detection kit is for detecting BRAF gene V600 mutation.
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Address after: No.19 Xiangshan Road, high tech Development Zone, Guangzhou, Guangdong 510665 Applicant after: Guangzhou Da'an gene Co.,Ltd. Address before: No.19 Xiangshan Road, high tech Development Zone, Guangzhou, Guangdong 510665 Applicant before: DAAN GENE CO., LTD. OF SUN YAT-SEN University |
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