CN114214412B - PIK3CA gene and BRAF gene mutation multiplex detection primer probe and kit thereof - Google Patents

PIK3CA gene and BRAF gene mutation multiplex detection primer probe and kit thereof Download PDF

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CN114214412B
CN114214412B CN202111617387.1A CN202111617387A CN114214412B CN 114214412 B CN114214412 B CN 114214412B CN 202111617387 A CN202111617387 A CN 202111617387A CN 114214412 B CN114214412 B CN 114214412B
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洪专
潘文健
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Prius New Shanghai Biomedical Technology Co ltd
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Abstract

The invention relates to a multiplex detection primer probe for PIK3CA gene and BRAF gene mutation and a kit thereof, which comprise an upstream primer F-T790M, a downstream primer R-T790M, a wild type probe WP-T790 and a mutant probe MP-T790M for detecting the mutation of the PIK3CA gene and the BRAF gene 20 # exon T790M, an upstream primer F-L858R, a downstream primer R-L858R, a wild type probe WP-L858 and a mutant probe MP-L858R for detecting the deletion mutation of the PIK3CA gene and the BRAF gene 19 # exon fragment, and an upstream primer F-E19del, a downstream primer R-E19del, a wild type probe WP-E19 and a mutant probe MP-E19del for detecting the PIK3CA gene and the BRAF gene 19 # exon fragment. The PIK3CA gene and BRAF gene mutation multiplex detection primer probe and the kit thereof have the advantages of high speed, high efficiency, simple preparation of standard products, high specificity and sensitivity of a detection system, high utilization rate of free DNA and high accuracy.

Description

PIK3CA gene and BRAF gene mutation multiplex detection primer probe and kit thereof
Technical Field
The invention relates to a PIK3CA gene and BRAF gene mutation detection product, and belongs to the technical field of biology.
Background
The EGFR gene has two major downstream signaling pathways, the first of which is the Ras/Raf/MEK/ERK-MAPK pathway. The PIK3CA gene belongs to a lipid kinase coding gene, and belongs to a high-frequency somatic mutation gene in various tumors such as colorectal cancer, ovarian cancer, breast cancer, gastric cancer, liver cancer, lung cancer and the like. The frequency of gene-activated somatic cell changes encoding effectors in the Mitogen Activated Protein Kinase (MAPK) signaling pathway is high (70%), including members of the point mutated phosphoinositide 3-kinase (PI 3K) pathway such as PTEN, PIK3CA and AKT1. It has been pointed out that PIK3CA gene mutation is one of the common gene mutations of breast cancer besides HER2 gene amplification and p53 gene mutation, and the mutation occurs in a spiral region and a kinase region in approximately 80%, and corresponds to exon 9 (E542K, E545D, E K mutation site) and exon 20 (H1047R, H1047L) of PIK3CA gene respectively. Patients carrying PIK3CA gene mutations cannot benefit from drugs targeting EGFR, HER2 gene proteins.
Another important EGFR signaling pathway is the PI3K/Akt/mTOR pathway. BRAF mutation mediates tumor proliferation and survival by activating RAF-MEK-ERK pathway, promotes continuous proliferation of cells, inhibits apoptosis, and further leads to continuous development of tumor cells. About 43-66% of diagnosed melanomas carry BRAF mutations, the most common of which is V600E (80%), other mutations found less frequently; heterogeneity of BRAF mutations in V600K (12%), M (4%), R (5%) and D (5%) patients has been between primary tumors and metastases as well as between different metastases. The BRAF gene is located downstream of the cascade signaling pathway, and BRAF gene mutation is related to the efficacy of epidermal growth factor receptor inhibitors. Targeting mab such as cetuximab and panitumumab can be used for treating patients with negative BRAF gene, and is ineffective for patients with mutation in BRAF.
Before the cancer patients are subjected to drug treatment targeting EGFR, HER2 and other genes, the accurate detection of the mutation status of BRAF, PIK3CA and other genes of tumor patients is particularly important. Traditional methods for detecting these two genes are mostly based on Polymerase Chain Reaction (PCR), and there are one-generation sequencing, qPCR, and the like. However, the conventional method has low sensitivity, has strict requirements on sample types, and is only suitable for surgical tissue samples, so that mutation conditions of genes cannot be observed continuously in real time. Digital PCR (dPCR) is now changing the domain of traditional PCR and redefines the detection of mutation detection. For many detection methods, the sensitivity of dPCR is significantly higher than that of conventional PCR analysis, and therefore it is important to design a multiplex ddPCR system and to detect the mutation states of these two genes efficiently and accurately.
Disclosure of Invention
The invention aims to solve the technical problem of providing a PIK3CA gene and BRAF gene mutation multiplex detection primer probe which is rapid and efficient, has high specificity and sensitivity of a detection system, high utilization rate of free DNA and high accuracy, and a kit adopting the primer probe.
The invention provides a technical scheme for solving the technical problems, which is as follows: a multiplex detection primer probe for PIK3CA gene and BRAF gene mutation comprises an upstream primer F-E545, a downstream primer R-E545, a wild type probe WP-E545 and a mutant probe MP-E545K for detecting PIK3CA gene E545K mutation, an upstream primer F-H1047, a downstream primer R-H1047, a wild type probe WP-H1047 and a mutant probe MP-H1047R for detecting PIK3CA gene H1047R mutation, and an upstream primer F-V600, a downstream primer R-V600, a wild type probe WP-V600 and a mutant probe MP-V600E for detecting BRAF gene V600E mutation;
the nucleotide sequence of the upstream primer F-E545 is shown as SEQ ID No.1, the nucleotide sequence of the downstream primer R-E545 is shown as SEQ ID No.2, the nucleotide sequence of the wild probe WP-E545 is shown as SEQ ID No.3, and the nucleotide sequence of the mutant probe MP-E545K is shown as SEQ ID No. 4;
the nucleotide sequence of the upstream primer F-H1047 is shown as SEQ ID No.5, the nucleotide sequence of the downstream primer R-H1047 is shown as SEQ ID No.6, the nucleotide sequence of the wild type probe WP-H1047 is shown as SEQ ID No.7, and the nucleotide sequence of the mutant probe MP-H1047R is shown as SEQ ID No. 8;
the nucleotide sequence of the upstream primer F-V600 is shown as SEQ ID No.9, the nucleotide sequence of the downstream primer R-V600 is shown as SEQ ID No.10, the nucleotide sequence of the wild probe WP-V600 is shown as SEQ ID No.11, and the nucleotide sequence of the mutant probe MP-V600E is shown as SEQ ID No. 12.
The concentration of the mutant probe MP-E545K was 50nM, the concentration of the mutant probe MP-H1047R was 30nM, and the concentration of the mutant probe MP-V600E was 10nM.
All primers were used at a concentration of 50nM. All wild-type probes were used at a concentration of 20nM.
All wild type probes and mutant type probes are modified MGB probes, the 5' ends of all wild type probes are modified by VIC fluorescent groups, the 5' ends of all mutant type probes are modified by FAM fluorescent groups, and the 3' ends of all mutant type probes are modified by NFQ groups.
The invention provides another technical scheme for solving the technical problems as follows: a PIK3CA gene and BRAF gene mutation multiplex detection kit adopting the primer probes.
The multiplex detection kit for PIK3CA gene and BRAF gene mutation also comprises plasmids with PIK3CA gene E545K mutation, H1047R mutation and BRAF gene V600E mutation.
The invention has the positive effects that:
(1) The PIK3CA gene and BRAF gene mutation multiplex detection product adopts standard substances which are prepared by using normal cfDNA after enzyme digestion and mutant plasmids inserted with PIK3CA gene E545K, H1047R and BRAF gene V600E mutant fragments after enzyme digestion according to the proportion of copy numbers, and different mutation frequency standard substances play different roles. The cfDNA and the plasmid adopted by the standard substance can reduce and detect the characteristics of the sample to the greatest extent, thereby providing a lot of bases for optimizing the system and playing a decisive role in optimizing the system.
(2) The PIK3CA gene and BRAF gene mutation multiple detection products of the invention determine the endpoint fluorescence signal values generated by each mutation probe under different concentrations according to the digital PCR detection results of the medium mutation frequency standard substance, so that the data statistics results are more accurate. According to the PIK3CA gene E545K, H1047R and BRAF gene V600E mutation detection system, the background threshold value of each mutation site of the detection system is determined through the digital PCR detection result of the wild type template, and when the mutation copy number of a sample is detected, the mutation copy number is equal to the detection result minus the background threshold value, so that the result is more accurate.
(3) The PIK3CA gene and BRAF gene mutation multiple detection products can determine the sensitivity of a detection system through the digital PCR detection result of the low mutation frequency standard.
(4) The PIK3CA gene and BRAF gene mutation multiplex detection products are optimized by optimizing the concentration of each probe according to the traditional real-time fluorescence PCR detection result of the high mutation frequency standard, and proper probe concentration is selected according to the difference of fluorescence intensity after the reaction of probes with different concentrations of each mutation.
(5) The primer probe of the PIK3CA gene and BRAF gene mutation multiplex detection product is self-designed, the probe is an MGB probe, the probe sequence is shorter, and the specificity is good. The primer probe is optimized and selected through multiple combinations, so that the amplification efficiency and the sensitivity are high.
(6) The PIK3CA gene and BRAF gene mutation multiplex detection product is quick, efficient, low in cost and high in accuracy, and can quickly and accurately monitor various mutations of EGFR genes of tumor patients according to trace PIK3CA gene E545K, H1047R and BRAF gene V600E mutation ctDNA in a sample, so that a digital PCR platform can be used for detecting ctDNA, and can timely monitor the occurrence of new gene mutations of the patients, thereby providing a basis for the establishment and adjustment of clinical treatment schemes.
Drawings
FIG. 1 is a graph showing the results of the reaction data of the kit of the example for detecting an all wild-type template;
FIG. 2 is a graph showing the results of the reaction data of the template with a mutant wild ratio of 0.5 detected by the kit of the example;
FIG. 3 is a graph showing the results of the reaction data of the kit of the example for detecting a template with a mutant wild ratio of 0.0001;
FIG. 4 is a graph showing the intensity distribution of mutant signal FAM fluorescence of mutant probes at different concentrations.
Detailed Description
The following description of the present invention is made by way of specific examples, in which it is noted that: the following examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention, as any third party may make insubstantial modifications and adaptations of the methods and compositions described herein. In the examples that follow, reagents used were analytically pure by default and were commercially available unless specifically stated to be present. The invention does not make an explicit experimental method, and can be basically finished according to a conventional experimental method such as a basic biochemical molecular experimental method published in books of molecular cloning experiment guidelines published in 2002 by the scientific press of J.Sam Brooks et al, or according to an experimental method explicitly indicated by a reagent provider. Except where a few definitions are explicitly noted, all specialized and scientific terms referred to herein are consistent with the meanings commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention.
The main reagent comprises:
this patent disclosure relates to the purchase of reagent consumables from the manufacturer of regular reagent consumables for all experiments, with major reagents (few general routine reagents are not listed): 25X Droplet stabilizer (RaindanceC), taqMan GenotypingMaster Mix (2X) (Applied Biosystems), ecoRI endonuclease (TAKARA, 1040A), 10 XT Buffer (TAKARA, 1116A), 0.1% BSA (TAKARA, 1116A), plasmid miniprep kit (D1100, beijing Soxhaust apparatus technology Co., ltd.), QIAamp Circulating Nucleic Acid Kit (50) (55114, qiagen), qubit TM dsDNA HS Assay Kit (Q32854), etc.
The main instrument is as follows:
a magnetic frame, a vortex oscillator,3.0 fluorescent quantitative instrument, high-speed centrifuge, miniAmp PCR instrument (manufacturer: applied Biosystems), biosafety cabinet, water bath, rainDropSource (RainDance Technologies), pipette, rainDropSense (RainDance Technologies), etc.
Examples
The multiplex detection kit for PIK3CA gene and BRAF gene mutation in the embodiment comprises an upstream primer F-E545, a downstream primer R-E545, a wild probe WP-E545 and a mutant probe MP-E545K for detecting PIK3CA gene H1047R mutation, an upstream primer F-H1047, a downstream primer R-H1047, a wild probe WP-H1047 and a mutant probe MP-H1047R for detecting BRAF gene V600E mutation, an upstream primer F-V600, a downstream primer R-V600, a wild probe WP-V600 and a mutant probe MP-V600E for detecting PIK3CA gene E545K mutation, H1047R mutation and BRAF gene V600E mutation.
The design of primers, probes and plasmids involved in this example was designed by the skilled laboratory technician of Pressin (Shanghai) biomedical science and technology, and the synthesis unit was completed by Nanjing Jinsi biomedical science and technology, and the nucleotide sequences of the primers and probes are shown in Table 1.
TABLE 1 primer probe characterization table
The primer is a conventional primer, the probe is a modified MGB probe, the 5 '-end fluorescence modification groups of all wild probes are VIC fluorescence (WP-E545, WP-H1047 and WP-V600), and the 3' -end is modified by NFQ groups; the fluorescence modification group at the 5 'end of all mutant probes is FAM fluorescence (MP-E545K, MP-H1047R, MP-V600E), and the 3' end is modified by the NFQ group.
The wild type amplification template used in this example was non-tumor patient healthy human free DNA (cfDNA), and the mutant amplification template was a plasmid constructed artificially in the laboratory, which was digested with endonucleases to form a linear structure.
The wild template and the mutant template are prepared into templates with theoretical mutation frequency according to different copy number ratios, and the preparation method is as follows:
1. and (5) sample extraction.
Plasma extraction was performed using a QIAamp Circulating Nucleic Acid Kit (50) (55114, qiagen) kit, and specific procedures can be found in the product description of this kit.
The plasmid is extracted by using a plasmid small-scale extraction kit (D1100, beijing Soy Bao technology Co., ltd.) according to the product specification, and specific operation steps can be referred to the official specification of the kit.
Concentration determination of cfdna and mutant plasmid.
The cfDNA and plasmid obtained by extraction are used as3.0 fluorescence quantitative instrument, using Qubit TM dsDNA HS Assay Kit (Q32854) are quantified and specific experimental procedures are referred to the instructions contained in the kit.
3. And (5) preparation of a standard substance.
1) And (5) enzyme digestion and lineation of the plasmid template and recovery.
Plasma free DNA is highly fragmented nucleic acid duplex, without fragmentation.
The quantified mutant plasmids were subjected to cleavage as shown in Table 2 below.
TABLE 2 cleavage reaction System for various mutant plasmids
Composition of the components Dosage of
10×SEBuffer 10μl
BSA 10μl
EcoRⅠ 1.0μl
Plasmid DNA 79.0μl
The recovery method of the mutation after enzyme digestion by adopting a magnetic bead method is as follows:
a. transferring all the products after enzyme digestion into an EP tube with the volume of 1.5ml, adding AmPure XP Reagent magnetic beads into the EP tube, repeatedly blowing and mixing uniformly, standing at room temperature, and timing for 5min.
b. Transferring EP onto a magnetic rack, standing for 10min, separating magnetic beads from the solution, sucking liquid by a pipette after the magnetic beads are completely adsorbed on the pipe wall, and discarding the solution.
c. 200 μl of freshly prepared 80% aqueous ethanol was added and the beads were cleared.
d. The washed ethanol solution was discarded, the previous step was repeated, the ethanol solution was discarded, and a small amount of the remaining solution was sucked off with a 10. Mu.l pipette, and dried.
e. After the beads were dried, 80. Mu.l of deionized water was added to the EP tube, the beads were dissolved, and the mixture was blown and mixed, placed on a magnetic stand, and allowed to stand for about 10 minutes until the beads were completely separated from the liquid.
f. Carefully pipette 79. Mu.l of the clarified DNA solution into a new EP tube.
g. Purifying to obtain enzyme-digested product, taking 1 μl for use3.0, quantification was performed.
2) Configuring standard substance for experiment
(1) Theoretically, human DNA was calculated at about 300ng per 1ng single gene copy number, and plasma free DNA (cfDNA) was serially diluted to about 2.0X10 5 copies/μl。
(2) Plasmid copy number calculation after cleavage, 9.1X10 8 Plasmid concentration (ng/. Mu.l)/(plasmid length (2800 bp); the resulting plasmid was serially diluted to 2.0X10 6 copies/μl、2.0×10 5 copies/μl、2.0×10 2 copies/μl、20copies/μl。
(3) Standard substances required by the configuration experiment: 10ul of 2.0X10 are taken 5 COPIES/. Mu.lcfDNA and equivalent volume of 2.0X10 5 copies/μl mutant plasmid solution is mixed uniformly, and deionized water of the same volume is added to obtain 1.0X10 5 The copies/. Mu.l template was mutated at a frequency of 0.5 and 10ul of 2.0X10 5 COPIES/. Mu.lcfDNA and 1ul volume of 2.0X10 3 Mixing the mutant plasmid solution of copies/. Mu.l, and adding 9ul deionized water to obtain 1.0X10 5 The mutation frequency of the copies/. Mu.l template was 0.001 template; 10ul of 2.0X10 are taken 5 COPIES/. Mu.lcfDNA and 1ul volume of 2.0X10 2 Mixing the mutant plasmid solution of copies/. Mu.l, and adding 9ul deionized water to obtain 1.0X10 5 The mutation frequency of the copies/. Mu.l template was 0.0001 template.
In this example, the optimum working concentration of each mutant probe was measured by using a MiniAmp PCR apparatus (manufacturer: applied Biosystems) to distinguish each mutant site at the end of the experimental reaction.
The present example uses a real-time fluorescent quantitative (qPCR) reaction for optimization of probe concentration.
According to the purpose of development, working concentrations of three groups of primers (50 nM) and three wild type probes (20 nM) are determined to be constant, the three groups of primers and the three-site wild type probes are uniformly mixed in advance to form MIX1, and the working concentrations of the mutant probes are respectively regulated so as to maximally separate the mutant probes from each other:
the final concentrations of the mutant probes at each mutation site were determined to be 10nM, 20nM, 30nM, 40nM, 50nM, and 60nM, respectively. The mutant template and the wild template are used for preparing an amplification template according to the copy number ratio of 1:1, and a qPCR reaction system is prepared according to a qPCR reaction system component table shown in Table 3.
TABLE 3 component Table of qPCR reaction System
Wherein x represents the volume of the mutant probe.
qPCR reactions were performed according to the qPCR reaction procedure shown in table 4. Reactions were performed at 28 cycles on a MiniAmp PCR apparatus (manufacturer: applied Biosystems).
TABLE 4 reaction procedure for qPCR
After the qPCR reaction is finished, under the condition that the concentration of the mutation probe is 1:1, the mutation fluorescent signal value is recorded under the condition that the concentration of the mutation template and the wild template is 1:1, and according to the summary statistics of the fluorescent signal values, the most suitable concentration of each probe in a multiple system is comprehensively and comprehensively selected, so that the mutual interference of each reaction droplet in the digital PCR (ddPCR) reaction is avoided.
TABLE 5 qPCR reaction results
The intensity distribution diagram of the mutation signal FAM fluorescence shown in fig. 4 is made according to the end point fluorescence signal value of each probe, and the concentration value of each mutation probe in the multiple ddPCR system can be reasonably selected according to the position of the fluorescence value of each mutation signal in the diagram.
As can be seen from FIG. 4, when the working concentrations of the three sets of primers (50 nM) and the three wild-type probes (20 nM) were constant, the fluorescence value increased continuously with the increasing working concentration of the mutant probe, and then reached the plateau phase, and at this time, the fluorescence value did not increase with the increasing concentration, and the fluorescence concentration with the largest difference in the end point signals of the mutant sites was selected as ddPCR according to the difference in the fluorescence signals of the probes at different concentrations, and then the subsequent detection experiment and the development of the kit were performed. From the above principle, the most suitable probe concentrations for each mutant probe were selected as follows: the PIK3CA gene E545K mutation probe MP-E545K is 50nM; the mutation MP-H1047R of the PIK3CA gene H1047R is 30nM; the concentration of the BRAF gene V600E mutation detection probe MP-V600E is 10nM. When the concentrations of the three probes are combined, the mutation signal values have the smallest interaction, so that false positive can be greatly reduced and the detection sensitivity can be improved.
The multiplex detection kit for mutation of PIK3CA gene and BRAF gene of the embodiment carries out the next ddPCR reaction according to the experimental conclusion, the effectiveness of the system is verified, all primer probes are prepared into MIX2 according to the conclusion, and then the public reagent components and the reaction template are added for carrying out the subsequent experiments, wherein the ddPCR reaction system is shown in Table 6.
TABLE 6 component Table of ddPCR reaction System
The mutant templates and wild-type templates were used to prepare corresponding mutant templates according to the mutation frequencies shown in Table 7.
TABLE 7 ddPCR reaction mutant templates
The ddPCR reaction was performed and the ddPCR reaction procedure is shown in Table 8.
TABLE 8 ddPCR reaction program Table
After the reaction is finished, desktop software RainDropAnalyst II (V1.0.0) carried by RainDance company instruments is adopted for subsequent analysis, and the following steps are adopted:
1. double-click opens the RainDropAnalyst II software, and left-click 'Add sample' machine automatically reads fcs data file and imports the software, ready for subsequent analysis.
2. The first step selects sample data with mutation frequency of 0.5 for analysis, at this time, a negative point set, a wild probe VIC fluorescent point set and a mutant point set with FAM fluorescence can be roughly selected, and the "Apply spectral compensation" function button is used, the ordinate is set to the FAM fluorescence signal value, and the abscissa is set to the VIC signal value.
3. The software interface 'elastic Gate' function box can select and adjust the area range of aggregation points, the selected aggregation points are concentrated as much as possible, the positive mutation frequency of the aggregation points is as close as possible to the mutation frequency of 50% of the theoretical value, the selected area range of the standard is used as the standard selection range standard, and the analysis of other samples is applied.
In the data analysis using the reaction system of this example, it was necessary to carry out the same experiment system and experiment conditions with the standard having a mutation at each known site of 0.5. Firstly, analyzing data obtained by a standard experiment with the mutation frequency of 0.5, so as to determine the most suitable areas of the wild type aggregation point areas and the aggregation areas of all mutation sites, and providing the most suitable selection areas for sample analysis. Meanwhile, calculating the proportionality constant K of the wild mutation site of each mutation site from a standard with the mutation frequency of 0.5, wherein k=tt/Na, the constant K is used for calculating the number of wild type sites with different mutation frequency samples in the batch reaction, as shown in fig. 2, and the k= 1323679/5970176 =0.221 is shown as E545K; h1047r, k= 1409329/5970176 =0.236; V600E: k= 1012225/5970176 =0.169.
In the process of using the PIK3CA gene and BRAF gene mutation multiplex detection kit of the embodiment, the same system and method are adopted to carry out accompanying experiments by setting all-wild type standard products, and the all-wild type standard products are analyzed to obtain false positive background values (Tf) with the number of positive sites in a mutation area being the same batch of samples, as shown in figure 1, E545K:6, H1047R:7 and V600E:9. The true positive site value (Tt) =total number of sites (Tn) -false positive background value (Tf) as shown in fig. 3, E545K: 161-6=155, H1047R: 171-7=164, V600E: 126-9=117.
Using the reaction system and the reaction procedure of this example, when calculating the mutation frequency at each site, the mutation frequency (V) =tt/(tt+nt) ×100%, where Nt represents the number of wild-type mutation sites at that site, nt=k×na, N a is the total number of wild-type sites, and k is calculated from a standard experiment system with a mutation frequency of 0.5. As shown in FIG. 3, the sensitivity of the kit can reach one ten thousandth, the specificity is good, and the signal to noise ratio is high because E545K is 0.011%, H1047R is 0.011%, and V600E is 0.011%.
The PIK3CA gene and BRAF gene mutation multiplex detection kit provided by the invention can be used for solving the problems of insufficient mutation sensitivity, low accuracy, insufficient plasma free cfDNA and the like in plasma free cfDNA detection, and the detection method system can also be used for whole blood, cerebrospinal fluid, hydrothorax and ascites and tissue samples.
The invention provides a multiple reaction system for multipoint mutation of PIK3CA genes and BRAF genes based on a digital PCR system, which improves the detection sensitivity and accuracy of trace tumor DNA (ctDNA) PIK3CA gene E545K, H1047R and BRAF gene V600E mutation in plasma free DNA (cfDNA), and simultaneously detects multiple hot spot mutation of 3 EGFR genes by an independent ddPCR reaction system, improves the utilization efficiency of the plasma free DNA, reduces the detection cost and develops a kit by an optimization system in the later stage.
The wild type template is normal human cfDNA after enzyme digestion, and the mutant template is mutant plasmid inserted with PIK3CA gene E545K, H1047R and BRAF gene V600E mutant fragments after enzyme digestion. Preparing a mutant template and a wild template into a medium mutation frequency standard according to a copy number ratio of 1:1, firstly performing full PCR reaction on a common fluorescent quantitative qPCR instrument, wherein the reaction template is a template with mutation frequency of 0.5 and a pure wild template, the probe concentration is set to be 10nM, 20nM, 30nM, 40nM, 50nM and 60nM, the end point fluorescence values generated by the mutant probes are collected after the PCR reaction, and the difference of the fluorescence values corresponding to the end point fluorescence probes is used. And (3) marking the fluorescence value corresponding to the mutation probe under each concentration in a coordinate system, and selecting the reasonable configuration of the concentration of each probe of the multiplex dPCR. Because the reaction is carried out in the unified reaction tube, the fluorescence end point values generated by the wild type probes are consistent, so that the difference between the fluorescence signal values corresponding to the mutant type probes is maximized, namely, the reasonable probe collocation is selected. And carrying out a reaction by adopting a digital PCR (polymerase chain reaction) with a medium mutation frequency standard as a template, preparing a data statistical graph according to reaction data, and selecting a wild type fluorescent region and a mutant type fluorescent region, wherein the ratio of the copy number of a fluorescent signal in the mutant type fluorescent region to that of a fluorescent signal in the wild type fluorescent region is the same as the copy number ratio of the mutant template and the wild type template in the medium mutation frequency standard.
And (3) performing a reaction by using a wild template as a template through digital PCR, wherein the copy number of a fluorescent signal in a mutant fluorescent region is the background threshold of the detection system.
Preparing a mutant template and a wild template into a low mutation frequency standard according to a copy number ratio of 1:1 and 1:10000, adopting digital PCR (polymerase chain reaction) with the low mutation frequency standard as a template, and if the copy number of fluorescent signals appearing in a mutant fluorescent region is greater than 2.5 times of the background threshold value, enabling the sensitivity of the detection system to reach the value with the same copy number ratio of the low mutation frequency standard.
It is apparent that the above examples are merely illustrative of the present invention and are not limiting of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While remaining within the scope of the invention, obvious variations or modifications are incorporated by reference herein.
Sequence listing
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Claims (4)

1. A PIK3CA gene and BRAF gene mutation multiplex detection primer probe is characterized in that: the method comprises the steps of detecting an upstream primer F-E545, a downstream primer R-E545, a wild type probe WP-E545 and a mutant probe MP-E545K of a PIK3CA gene E545K mutation, detecting an upstream primer F-H1047, a downstream primer R-H1047, a wild type probe WP-H1047 and a mutant probe MP-H1047R of a PIK3CA gene H1047R mutation, and detecting an upstream primer F-V600, a downstream primer R-V600, a wild type probe WP-V600 and a mutant probe MP-V600E of a BRAF gene V600E mutation;
the nucleotide sequence of the upstream primer F-E545 is shown as SEQ ID No.1, the nucleotide sequence of the downstream primer R-E545 is shown as SEQ ID No.2, the nucleotide sequence of the wild probe WP-E545 is shown as SEQ ID No.3, and the nucleotide sequence of the mutant probe MP-E545K is shown as SEQ ID No. 4;
the nucleotide sequence of the upstream primer F-H1047 is shown as SEQ ID No.5, the nucleotide sequence of the downstream primer R-H1047 is shown as SEQ ID No.6, the nucleotide sequence of the wild type probe WP-H1047 is shown as SEQ ID No.7, and the nucleotide sequence of the mutant probe MP-H1047R is shown as SEQ ID No. 8;
the nucleotide sequence of the upstream primer F-V600 is shown as SEQ ID No.9, the nucleotide sequence of the downstream primer R-V600 is shown as SEQ ID No.10, the nucleotide sequence of the wild probe WP-V600 is shown as SEQ ID No.11, and the nucleotide sequence of the mutant probe MP-V600E is shown as SEQ ID No. 12;
the use concentration of the mutant probe MP-E545K is 50nM, the use concentration of the mutant probe MP-H1047R is 30nM, and the use concentration of the mutant probe MP-V600E is 10nM; all primers were used at a concentration of 50nM; all wild-type probes were used at a concentration of 20nM.
2. The PIK3CA gene and BRAF gene mutation multiplex detection primer probe of claim 1, wherein: all wild type probes and mutant type probes are modified MGB probes, the 5' ends of all wild type probes are modified by VIC fluorescent groups, the 5' ends of all mutant type probes are modified by FAM fluorescent groups, and the 3' ends of all mutant type probes are modified by NFQ groups.
3. A multiplex assay kit for PIK3CA gene and BRAF gene mutation using the primer probe of claim 1.
4. The multiplex assay kit for mutation of PIK3CA gene and BRAF gene according to claim 3, wherein: also comprises plasmids with PIK3CA gene E545K mutation, H1047R mutation and BRAF gene V600E mutation.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104818320A (en) * 2014-12-03 2015-08-05 厦门艾德生物医药科技有限公司 Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
CN106987640A (en) * 2017-04-28 2017-07-28 上海赛安生物医药科技有限公司 PIK3CA detection in Gene Mutation primed probe and its 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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104818320A (en) * 2014-12-03 2015-08-05 厦门艾德生物医药科技有限公司 Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
CN106987640A (en) * 2017-04-28 2017-07-28 上海赛安生物医药科技有限公司 PIK3CA detection in Gene Mutation primed probe and its 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

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
George Karlin-Neumann等.Very low abundance single-cell transcript quantification with 5-plex ddPCR assays.Digital PCR:Methods and Protocols, Methods in Molecular Biology.2018,第1768卷423-444. *
Irene Lurkin等.Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS,BRAF,NRAS and PIK3CA genes.PLOS ONE.2010,第5卷(第1期),e8802第1-6页. *
Kaitlyn Zenner等.Cell-free DNA as a diagnostic analyte for molecular diagnosis of vascular malformations.Genetics in Medicine.2020,全文. *
Vicky Rowlands等.Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA.SCIENTIFIC REPORTS.2019,第9卷摘要、第2-3页materials and methods部分、第5-9页、supplementary table 1. *
Xiaozhou Li等.Surface enhanced raman spectroscopy (SERS) for the multiplex detection of Braf, Kras, and Pik3ca mutations in plasma of colorectal cancer patients.Theranostics.2018,第8卷(第6期),第1678-1689页. *

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