CN114214412A - 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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CN114214412A
CN114214412A CN202111617387.1A CN202111617387A CN114214412A CN 114214412 A CN114214412 A CN 114214412A CN 202111617387 A CN202111617387 A CN 202111617387A CN 114214412 A CN114214412 A CN 114214412A
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洪专
潘文健
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Abstract

The invention relates to a PIK3CA gene and BRAF gene mutation multiple detection primer probe and a kit thereof, which comprises an upstream primer F-T790M, a downstream primer R-T790M, a wild-type probe WP-T790 and a mutant-type probe MP 790M for detecting PIK3CA gene and BRAF gene exon 20T 790M mutation, an upstream primer F-L858R, a downstream primer R-L858R, a wild-type probe WP-L858 and a mutant-type probe MP-L858R for detecting PIK3CA gene and BRAF gene exon 21L 858R mutation, and an upstream primer F-E19del, a downstream primer R-E19del, a wild-type probe WP-E19 and a mutant-type probe MP-E19del for detecting PIK3CA gene and BRAF gene 19 exon fragment deletion mutation. The PIK3CA gene and BRAF gene mutation multiple detection primer probe and the kit thereof are quick and efficient, the preparation of the standard substance is simple, the specificity and the sensitivity of the detection system are high, the utilization rate of free DNA is high, and the accuracy is high.

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 mainly two downstream signal transduction 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 gene generating high-frequency somatic mutation in colorectal cancer, ovarian cancer, breast cancer, gastric cancer, liver cancer, lung cancer and other tumors. Genes encoding effector factors in the mitogen-activated protein kinase (MAPK) signaling pathway stimulate cellular changes in living cells at a high frequency (70%), including point mutation of members of the phosphoinositide 3-kinase (PI3K) pathway, such as PTEN, PIK3CA, and AKT 1. It is pointed out that the mutation of PIK3CA gene is one of common breast cancer gene mutations except HER2 gene amplification and p53 gene mutation, and about 80% of the mutations occur in the helical region and the kinase region, which correspond to the No.9 exon (E542K, E545D, E545K mutation site) and the No. 20 exon (H1047R, H1047L) of PIK3CA gene respectively. Patients carrying the mutation of the PIK3CA gene fail to benefit from drugs targeting EGFR and HER2 gene proteins.
Another important EGFR signaling pathway is the PI3K/Akt/mTOR pathway. The BRAF mutation mediates the proliferation and survival of the tumor by activating a RAF-MEK-ERK pathway, promotes the continuous proliferation of cells, inhibits the apoptosis and further leads to the continuous development of the tumor cells. About 43-66% of diagnosed melanomas carry BRAF mutations, with V600E (80%) being the most common, and other variations being found less frequently; the heterogeneity of BRAF mutations in V600K (12%), M (4%), R (5%) and D (5%) patients has been between primary tumors and metastases and between different metastases. The BRAF gene is positioned at the downstream of a cascade signal transduction pathway, and the mutation of the BRAF gene is related to the drug effect of an epidermal growth factor receptor inhibitor. Targeting mabs such as cetuximab and panitumumab may be used to treat patients who are negative for the BRAF gene, and are ineffective for patients with mutations in BRAF.
Before the targeted gene therapy of EGFR, HER2 and the like is carried out on cancer patients, the accurate detection of the mutation condition of the genes of BRAF, PIK3CA and the like of the cancer patients is particularly important. The traditional detection method for the two genes is mainly based on Polymerase Chain Reaction (PCR), and comprises first-generation sequencing, qPCR and the like. However, the conventional method has low sensitivity and strict requirements on the type of the sample, and is only suitable for surgical tissue samples, so that the mutation condition of the gene cannot be continuously observed in real time. Now, digital PCR (dpcr) is changing the field of traditional PCR and redefining the detection downline for mutation detection. For many detection methods, the sensitivity of dPCR is obviously higher than that of the traditional PCR analysis, so that designing a multiple ddPCR system and efficiently and accurately detecting the mutation states of the two genes are very important.
Disclosure of Invention
The invention aims to provide a PIK3CA gene and BRAF gene mutation multiple detection primer probe which is rapid and efficient, high in specificity and sensitivity of a detection system, high in utilization rate of free DNA and high in accuracy, and a kit adopting the primer probe.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a PIK3CA gene and BRAF gene mutation multiplex detection primer probe comprises an upstream primer F-E545, a downstream primer R-E545, a wild type probe WP-E545 and a mutant type 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 type 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 type 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-type probe WP-E545 is shown as SEQ ID No.3, and the nucleotide sequence of the mutant-type 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-type 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-type probe WP-V600 is shown as SEQ ID No.11, and the nucleotide sequence of the mutant-type 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 10 nM.
All primers were used at a concentration of 50 nM. All wild-type probes were used at a concentration of 20 nM.
All wild type probes and mutant 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 probes are modified by FAM fluorescent groups, and the 3 ' ends of all mutant probes are modified by NFQ groups.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: a PIK3CA gene and BRAF gene mutation multiplex detection kit adopting the primer probe.
The PIK3CA gene and BRAF gene mutation multiplex detection kit 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 multiple detection product adopts the standard substance prepared by using enzyme-digested normal cfDNA and enzyme-digested mutant plasmids inserted into PIK3CA gene E545K, H1047R and BRAF gene V600E mutant fragments according to copy ratio, and the standard substances with different mutation frequencies play different roles. The standard sample can reduce and detect the characteristics of the sample to the maximum extent by adopting cfDNA and plasmids, and provides a lot of bases for the optimization of a system, thereby playing a decisive role in the optimization of the system.
(2) The PIK3CA gene and BRAF gene mutation multiple detection product determines the end point fluorescence signal values generated by each mutation probe under different concentrations through the digital PCR detection result of the medium mutation frequency standard substance, so that the result of data statistics is more accurate. The PIK3CA gene E545K, H1047R and BRAF gene V600E mutation detection system of the invention determines the background threshold value of each mutation site of the detection system through the digital PCR detection result of a 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 product can determine the sensitivity of a detection system through the digital PCR detection result of a low mutation frequency standard product.
(4) The optimization of the PIK3CA gene and BRAF gene mutation multiple detection product of the invention optimizes the concentration of each probe by the traditional real-time fluorescence PCR detection result of a high mutation frequency standard product, and selects proper probe concentration according to the difference of the fluorescence intensity of the probes with different mutation concentrations after reaction.
(5) The primer probe of the PIK3CA gene and BRAF gene mutation multiple detection product is designed by self, the probe is an MGB probe, the sequence of the probe is shorter, and the specificity is good. The primer probe is optimized and selected through multiple combinations, the amplification efficiency is high, and the sensitivity is high.
(6) The PIK3CA gene and BRAF gene mutation multiple detection product is rapid, efficient, low in cost and high in accuracy, and can rapidly and accurately monitor various mutations of EGFR gene of a tumor patient 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 ctDNA detection, new gene mutation of the patient can be timely monitored, and a basis is provided for making and adjusting a clinical treatment scheme.
Drawings
FIG. 1 is a graph showing the results of reaction data for detecting a full wild-type template by the kit of the embodiment;
FIG. 2 is a graph showing the result of reaction data for detecting a template having a mutant wild ratio of 0.5 using the kit of the embodiment;
FIG. 3 is a graph showing the result of reaction data for detecting a template having a mutant wild ratio of 0.0001 using the kit of the embodiment;
FIG. 4 is a graph showing the intensity distribution of the fluorescence of the mutation signal FAM of the mutant probes at different concentrations.
Detailed Description
The following description of the present invention is provided by way of specific embodiments, in which: 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 disclosed herein. In the examples which follow, reagents used were, by default, analytically pure, and were all commercially available, except where specifically stated. The invention does not make a specific experimental method, and can be basically completed according to a conventional experimental method such as a basic biochemical molecular experimental method published in molecular cloning experimental manual published by scientific publishing house 2002 published by J. SammBruk, etc., or obtained according to an experimental method specifically indicated by a reagent supplier. Except where expressly indicated by a few, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
The main reagents are as follows:
the content of the patent relates to all reagent consumables used in experiments are purchased from a regular reagent consumable manufacturer, and the used main reagents are (a few general conventional reagents are not listed) as follows: 25 × Droplet stabilizer (RainDance), TaqMan GenotypingMaster Mix (2 ×) (Applied Biosystems), EcoR I endonuclease (TAKARA, 1040A), 10 × T Buffer (TAKARA, 1116A), 0.1% BSA (TAKARA, 1116A), plasmid miniprep Kit (D1100, Beijing Soileo technologies, Ltd.), QIAamp Circulating Nucleic Acid Kit (50) (55114, Qiagen), and QubitTMdsDNA HS Assay Kit (Q32854), and the like.
The main apparatus is as follows:
a magnetic frame, a vortex oscillator,
Figure BDA0003436764730000041
3.0 fluorescence quantifier, high speed centrifuge, MiniAmp PCR instrument (manufacturer: Applied Biosystems), biosafety cabinet, water bath, RainDropSource (RainDance technologies), pipette gun, RainDropSense (RainDance technologies), and the like.
Examples
The PIK3CA gene and BRAF gene mutation multiplex detection kit comprises an upstream primer F-E545, a downstream primer R-E545, a wild-type probe WP-E545 and a mutant-type 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-type probe MP-H1047R for detecting PIK3CA gene H1047R mutation, an upstream primer F-V600, a downstream primer R-V600, a wild-type probe WP-V600 and a mutant-type probe MP-V600E for detecting BRAF gene V600E mutation, and plasmids with PIK3CA gene E K mutation, H1047R mutation and BRAF gene V600E mutation.
The design of the primers, probes and plasmids in this example was designed by the laboratory technicians of the Precissin (Shanghai) Biomedical science and technology GmbH, the synthesis unit was completed by Nanjing Kingsler Biotechnology GmbH, and the nucleotide sequences of the primers and probes are shown in Table 1.
TABLE 1 primer Probe characterization Table
Figure BDA0003436764730000051
The primer is a conventional primer, the probe is a modified MGB probe, the 5 'end fluorescence modifying 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 5 'end fluorescence modifying groups of all mutant probes are FAM fluorescence (MP-E545K, MP-H1047R and MP-V600E), and the 3' end is modified by NFQ groups.
The wild type amplification template used in the embodiment adopts free DNA (cfDNA) of healthy people of non-tumor patients, and the mutant type amplification template adopts artificially constructed plasmid in a laboratory, and the plasmid is subjected to endonuclease digestion treatment to form a linear structure.
Preparing a template with theoretical mutation frequency from a wild template and a mutant template according to different copy number ratios, wherein the preparation method comprises the following steps:
1. and (4) extracting a sample.
Plasma extraction was performed using the QIAamp Circulating Nucleic Acid Kit (50) (55114, Qiagen) Kit, and the specific procedures can be referred to the product instructions of the Kit.
The extraction of the plasmid adopts a plasmid small quantity extraction kit (D1100, Beijing Sorleibao science and technology Co., Ltd.), is carried out according to the product instruction, and the specific operation steps can refer to the official instruction of the kit.
Concentration determination of cfDNA and mutant plasmids.
The obtained cfDNA and plasmid are extracted to be
Figure BDA0003436764730000061
3.0 fluorescence quantifier, using a QubitTMThe dsDNA HS Assay Kit (Q32854) was used for quantification, and the specific experimental procedures were described in the Kit with reference to the instructions contained therein.
3. And (5) preparing a standard substance.
1) And (3) carrying out enzyme digestion linearization on the plasmid template and recovering.
Plasma free DNA is a highly fragmented nucleic acid duplex, and fragmentation is not required.
The quantified mutant plasmids were digested as shown in Table 2 below.
TABLE 2 digestion reaction System for various mutant plasmids
Composition (I) Dosage of
10×SEBuffer 10μl
BSA 10μl
EcoRⅠ 1.0μl
Plasmid DNA 79.0μl
The method for recovering the mutation after enzyme digestion by a magnetic bead method comprises the following steps:
a. and (3) transferring all the enzyme-digested products to a 1.5ml EP tube, adding AmPure XP Reagent magnetic beads into the EP tube, repeatedly blowing, uniformly mixing, standing at room temperature, and timing for 5 min.
b. Transferring the EP to a magnetic frame, standing for 10min, separating magnetic beads from the solution, sucking the liquid by a liquid transfer device after the magnetic beads are completely adsorbed on the tube wall, and discarding the solution.
c. 200 μ l of freshly prepared 80% aqueous ethanol solution was added to the cells, and the beads were washed.
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 aspirated by a 10. mu.l pipette and dried.
e. And (3) after the magnetic beads are dried, adding 80 mu l of deionized water into an EP tube, dissolving the magnetic beads, blowing and uniformly mixing, placing on a magnetic frame, and standing for about 10min until the magnetic beads and the liquid are completely separated.
f. Transfer 79. mu.l of the clarified DNA solution carefully pipetted into a new EP tube.
g. The purified enzyme-digested product was used in 1. mu.l
Figure BDA0003436764730000062
3.0 for quantification.
2) Preparing standard substances required by experiments
Theoretically, plasma-free DNA (cfDNA) is diluted stepwise to about 2.0X 10, calculated as about 300ng of copy number of a single gene per 1ng of human DNA5copies/μl。
② the copy number of the plasmid obtained after enzyme digestion is calculated, 9.1 multiplied by 108Xplasmid concentration (ng/. mu.l). plasmid length (2800 bp); the resulting plasmid was diluted stepwise to 2.0X 106copies/μl、2.0×105copies/μl、2.0×102copies/μl、20copies/μl。
Preparing a standard product required by the experiment: taking 10ul of 2.0 × 105copies/. mu.lcfDNA and equivalent volumes of 2.0X 105The copies/μ l mutant plasmid solution was mixed well and added to the same volumeDeionized water to give 1.0X 105copies/μ l template, mutation frequency 0.5, taking 10ul of 2.0 × 105copies/. mu.lcfDNA and 1ul volume of 2.0X 103copies/. mu.l mutant plasmid solution, mixed well, then added with 9ul deionized water to obtain 1.0X 105copies/μ l template, mutation frequency of 0.001 template; taking 10ul of 2.0 × 105copies/. mu.lcfDNA and 1ul volume of 2.0X 102copies/. mu.l mutant plasmid solution, mixed well, then added with 9ul deionized water to obtain 1.0X 105copies/. mu.l template, mutation frequency 0.0001 template.
In this example, the optimum working concentration of each mutation site mutation type probe was measured using a MiniAmp PCR instrument (manufactured by Applied Biosystems) to distinguish each mutation site at the end of the experimental reaction.
This example employs a real-time fluorescent quantitation (qPCR) reaction for optimization of probe concentration.
According to the development purpose, the working concentration of three groups of primers (50nM) and three wild probes (20nM) is constant, the three groups of primers and the wild probes at three sites are mixed uniformly to MIX1 in advance, and the working concentration of each mutant probe is respectively adjusted to make the differentiation of each mutant probe independent to each other to the maximum extent:
the final concentrations of the mutant probes for each mutation site were determined to be 10nM, 20nM, 30nM, 40nM, 50nM, and 60nM, respectively. An amplification template was prepared using the mutant template and the wild template in a copy number ratio of 1: 1, and a qPCR reaction system was prepared according to the qPCR reaction system component table shown in Table 3.
TABLE 3 qPCR reaction System Components Table
Figure BDA0003436764730000071
Wherein x represents the volume of the mutant probe.
The qPCR reactions were performed according to the qPCR reaction procedure shown in table 4. Each cycle was performed on a MiniAmp PCR instrument (manufacturer: Applied Biosystems) 28.
TABLE 4 reaction procedure for qPCR
Figure BDA0003436764730000081
And after the qPCR reaction is finished, recording the mutant fluorescence signal values under the concentration of each mutant probe under the condition that the mutant template and the wild template are in a ratio of 1: 1, summarizing and counting according to the fluorescence signal values, comprehensively and comprehensively selecting the most suitable concentration of each probe in the multiplex system, and avoiding the mutual interference of reaction microdroplets in digital PCR (ddPCR).
TABLE 5 qPCR reaction results
Figure BDA0003436764730000082
The intensity distribution of the FAM fluorescence of the mutation signal is shown in FIG. 4 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 from the position of the fluorescence value of each mutation signal in the graph.
As can be seen from FIG. 4, when the working concentrations of the three sets of primers (50nM) and the three wild-type probes (20nM) are determined to be constant, the fluorescence values continuously rise with the continuous increase of the working concentration of the mutant-type probes, and then reach the plateau phase, and the fluorescence values do not rise with the increase of the concentration, and the fluorescence concentration with the maximum difference of the end point signals of the mutation sites is selected as ddPCR to perform the subsequent detection experiments and kit development according to the difference of the fluorescence signals of the probes at different concentrations. Based on the above principle, the most suitable probe concentration for each mutant probe is selected as follows: 50nM of the PIK3CA gene E545K mutation probe MP-E545K; the PIK3CA gene H1047R mutant MP-H1047R is 30 nM; the concentration of the BRAF gene V600E mutation detection probe MP-V600E is 10 nM. When the three probe concentrations are combined, the mutual influence of mutation signal values is minimum, so that the false positive can be greatly reduced, and the detection sensitivity can be improved.
According to the experimental conclusion, the PIK3CA gene and BRAF gene mutation multiple detection kit of the embodiment carries out the next step of ddPCR reaction, verifies the effectiveness of the system, firstly prepares all primer probes into MIX2 according to the conclusion, then adds the public reagent components and the reaction template to carry out the subsequent experiment, and the ddPCR reaction system is shown in Table 6.
TABLE 6 Table of components of ddPCR reaction system
Figure BDA0003436764730000091
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 mutant templates for ddPCR reactions
Figure BDA0003436764730000092
The dPCR reaction was performed and the ddPCR reaction procedure is shown in Table 8.
TABLE 8 ddPCR reaction schedule
Figure BDA0003436764730000093
After the reaction is finished, the subsequent analysis is carried out by adopting desktop software RainDropAnalyst II (V1.0.0) carried by the instrument of the company RainDance, and the steps are as follows:
1. double-clicking opens RainDropAnalyst II software, and clicking the 'Add sample' machine by a left button automatically reads fcs data files and imports the fcs data files into the software to prepare for subsequent analysis.
2. In the first step, sample data with mutation frequency of 0.5 is selected for analysis, and at this time, a negative spot set, a wild-type probe VIC fluorescence spot set and a mutant spot set with FAM fluorescence can be roughly selected, and an "Apply spectral compensation" function button is used, wherein the ordinate is set as FAM fluorescence signal value, and the abscissa is set as Vic as signal value.
3. The software interface 'Elliptical Gate' function box can select and adjust the area range of the aggregation points, the selected aggregation points are concentrated as much as possible, the positive mutation frequency of the aggregation points is close to the mutation frequency of 50% of a theoretical value as much as possible, the selection area range of the standard is used as the standard selection range standard, and the standard selection range standard is applied to analysis of other samples.
When data analysis was performed using the reaction system of this example, a standard sample in which each site was mutated to 0.5 was used together with the same experiment system and experiment conditions. Firstly, analyzing data obtained by a standard substance experiment with the mutation frequency of 0.5, determining the wild type aggregation site region and the optimal region of each mutation site aggregation region, and providing the optimal selection region for sample analysis. Meanwhile, a proportionality constant k, k is Tt/Na, which is calculated for each wild mutation site from a standard substance with a mutation frequency of 0.5, and the constant k is used for calculating the number of wild type sites of samples with different mutation frequencies in the batch of reactions, as shown in FIG. 2, E545K, k is 1323679/5970176 is 0.221; H1047R, k 1409329/5970176 ═ 0.236; V600E: k is 1012225/5970176 is 0.169.
In the process of using the PIK3CA gene and BRAF gene mutation multiplex detection kit of this embodiment, a whole wild type standard needs to be set to perform a concomitant experiment by using the same system and method, and the number of positive sites in a mutation region obtained by analyzing the whole wild type standard is a false positive background value (Tf) of the same batch of samples, as shown in fig. 1, E545K:6, H1047R:7, and V600E: 9. True positive site values (Tt) ═ total site values (Tn) — false positive background values (Tf) are shown in fig. 3, E545K:161-6 ═ 155, H1047R:171-7 ═ 164, V600E:126-9 ═ 117.
Using the reaction system and reaction program 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 the site, Nt ═ k × Na, N a is the total number of wild-type sites, and k is calculated from a standard experimental system with a mutation frequency of 0.5. As shown in FIG. 3, the kit has the advantages of one ten thousandth of sensitivity, good specificity and high signal-to-noise ratio because E545K: 0.011%, H1047R: 0.011% and V600E: 0.011%.
The PIK3CA gene and BRAF gene mutation multiple 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, chest, abdomen and tissue samples.
The invention provides a multiple reaction system of PIK3CA gene and BRAF gene multiple point mutation based on digital PCR system, which improves the detection sensitivity and accuracy of micro tumor DNA (ctDNA) in plasma free DNA (cfDNA) PIK3CA gene E545K, H1047R and BRAF gene V600E mutation, an independent ddPCR reaction system simultaneously detects 3 EGFR gene multiple point mutation, improves the utilization efficiency of plasma free DNA, reduces the detection cost, and develops a kit through an optimization system at 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 medium mutation frequency standard substance from the mutant template and the wild type template according to the copy number ratio of 1: 1, firstly carrying out full PCR reaction on a common fluorescence quantitative qPCR instrument, wherein the reaction templates are the template with the mutation frequency of 0.5 and the pure wild type template, the concentration of the probe is set at each level of 10nM, 20nM, 30nM, 40nM, 50nM and 60nM, collecting the end-point fluorescence value generated by the mutation probe after the PCR reaction, and collecting the difference of the fluorescence value corresponding to each end-point fluorescence probe. The fluorescence values for the mutated probes at each concentration are plotted in the coordinate system, and the appropriate configuration of the concentration for each probe in the following multiplex dPCR is selected. As the reaction is carried out in the uniform reaction tube, the fluorescence terminal values generated by the wild-type probes are consistent, so that the difference of the fluorescence signal values corresponding to the mutant-type probes is maximized, namely, the reasonable probe matching is selected. And (3) adopting digital PCR (polymerase chain reaction) to react by taking the medium-abrupt-frequency standard product as a template, preparing a data statistical graph according to reaction data, and selecting a wild-type fluorescence region and a mutant-type fluorescence region, wherein the ratio of the copy number of the fluorescence signal in the mutant-type fluorescence region to that in the wild-type fluorescence region is the same as the copy number ratio of the mutant-type template to the wild-type template in the medium-abrupt-frequency standard product.
And (3) carrying out reaction by adopting a digital PCR (polymerase chain reaction) with a wild-type template as a template, wherein the copy number of a fluorescence signal appearing in a mutant fluorescence region is the background threshold value of the detection system.
Preparing the mutant template and the wild template into a low mutation frequency standard substance according to the copy number proportion of 1: 1 and 1: 10000, adopting digital PCR to react by taking the low mutation frequency standard substance as the template, and if the copy number of a fluorescence signal appearing in a mutant fluorescence area is more than 2.5 times of the background threshold value, ensuring that the sensitivity of the detection system reaches the value with the same copy number proportion of the low mutation frequency standard substance.
It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.
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Claims (7)

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