CN113373205A - Method for quantitatively detecting site mutation of 19del and L858R of EGFR gene by using digital PCR - Google Patents
Method for quantitatively detecting site mutation of 19del and L858R of EGFR gene by using digital PCR Download PDFInfo
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Abstract
Compared with traditional mutation detection methods such as a gene chip and fluorescence quantitative PCR, the method provided by the invention has the advantages that an indirect quantitative mode of calculating through a standard curve and a Ct value is abandoned through a detection means of discretization treatment and molecular counting of a sample, the absolute copy number of a target gene is directly obtained, and higher detection precision and resolution are provided for research. The method of the invention synchronously detects the 19del and L858R loci of the EGFR gene based on a digital PCR detection platform of a multicolor fluorescence channel, can quantitatively output the information such as the mutation quantity and the mutation proportion in a sample according to the detection result, and can provide reference basis for the selection of subsequent treatment and medication schemes of patients.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a method for quantitatively detecting site mutation of 19del and L858R of an EGFR gene by using digital PCR.
Background
EGFR is one of epidermal growth factor receptor (HER) family members, widely distributed on the cell surfaces of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes and the like, and EGFR signaling pathways play an important role in physiological processes such as growth, proliferation and differentiation of cells. However, when EGFR is mutated, it results in the continuous activation of EGFR signaling pathway, resulting in abnormal cell proliferation. Mutation or abnormal expression of EGFR plays an important role in the growth and development of tumors, especially in lung cancer, with 30% of patients being caused by EGFR mutation. The lung cancer is the first cancer type with morbidity and mortality in China, wherein the percentage of non-small cell lung cancer (NSCLC) is up to 85 percent, so NSCLC patients are the main population for the drug administration of the lung cancer. And because the mutation ratio of the EGFR is different from species to species and the mutation ratio of NSCLC patients in Asia is the highest and is about 50%, the EGFR targeting drug is one of the main drug choices when NSCLC patients in China are treated.
Among all EGFR mutations, the exon 19 deletion mutation (19 del mutation) and the exon 21L 858R point mutation (L858R mutation) accounted for approximately 90%. Moreover, both of these mutations are highly sensitive to EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor-tyrosine kinase inhibitor), and thus are collectively referred to as EGFR-sensitive mutations. The existing research shows that both the 19del mutation and the 21L 858R mutation can make EGFR kinase in an activated state, but the activation mechanism and the activation state of the kinase region at different mutation sites are different. The 21L 858R mutation occurs in the lower half of the binding to the α C helix, and when leucine is mutated to arginine (L → R), the binding domain hardly forms a hydrophobic core region, so that the α C helix segment can be seen in an activated state, but only in an activated state, and not in the highest activated state. Whereas the 19del mutein shortens the binding of the upper half to the α C helix, activating the kinase configuration by α C helix rotation. The shortened 19del mutein is more compact in structure and thus the EGFR kinase is in the highest active state. This is to some extent a determination that the 19del mutant patients with the EGFR kinase in the most active state achieve better efficacy when using EGFR TKI inhibition. Just because of the great difference between the 19del mutation and the L858R mutation, the treatment regimens for both should be subdivided. The current clinical trial data also demonstrates this view. It was found that the individual EGFR TKI drugs were not as effective in patients with the 19del mutation and the L858R mutation. The third generation of the FLAURA study of EGFR TKI axitinib was stratified at the time of enrollment by type of susceptible mutation, with a total population of 18.9 months of PFS, 21.4 months for the 19del mutant and 14.4 months for the L858R mutant. In contrast, the CTONG 1509 study found that patients with the L858R mutant used the A + T pattern, and their PFS could reach 19 months, i.e., the L858R mutant had substantially the same therapeutic effect in the A + T pattern as the 19del mutant patients. Therefore, at present, strict distinction between EGFR 19del and L858R mutation types in EGFR TKI treatment is important for accurate selection of treatment and medication.
Disclosure of Invention
In response to the above actual needs, the present invention provides a method for quantitative detection of site mutations at 19del and L858R of EGFR gene by using digital PCR. The method for quantitatively detecting the 19del and L858R site mutation of the EGFR gene by using the digital PCR has the advantages that the 19del and L858R site mutation of the EGFR gene is synchronously detected by using a digital PCR detection platform based on a multicolor fluorescence channel, the information such as the mutation quantity, the mutation proportion and the like in a sample can be quantitatively output according to the detection result, a reference basis can be provided for the selection of subsequent treatment and medication schemes of a patient, and the method has wide application prospect.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a method for quantitatively detecting site mutations of 19del and L858R of EGFR gene by using digital PCR, which is characterized in that: the method comprises the following steps:
(1) extracting a tissue sample or a plasma free DNA sample;
(2) preparing PCR amplification reaction by using a detection system consisting of dPCR-enzyme reaction solution, 10 multiplied primer probe mixture, a DNA template and deionized water;
(3) adding the reaction mixture into the sample adding holes of the ddPCR nano-microporous plate, and sealing the nano-microporous plate;
(4) the digital PCR reaction system automatically performs distribution, thermal cycle amplification reaction and result interpretation on reaction liquid in the nano-microporous plate;
(5) the thermal cycling amplification reaction is carried out according to the following conditions: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 15 seconds; annealing at 55-63 ℃ for 15 seconds; extension at 72 ℃ for 15 seconds; amplifying for 40 cycles; finishing the reaction;
(6) and acquiring fluorescence signals, analyzing data, obtaining the mutation quantity and mutation proportion results of 19del and L858R in the detection sample, and giving a judgment result.
Preferably, the primer probe mixture consists of specific primers and specific probes for detecting the EGFR 19del site and the EGFR L858R site.
Preferably:
(1) the specific primers for detecting the EGFR 19del site and the EGFR L858R site are as follows:
EGFR 19del forward primer: 5'-ACTCTGGATCCCAGAAGGTG-3', respectively;
EGFR 19del reverse primer: 5'-GAAACTCACATCGAGGATT-3', respectively;
EGFR L858R forward primer: 5'-CGTACTGGTGAAAACACCGCA-3', respectively;
EGFR L858R reverse primer: 5'-CTTTGCCTCCTTCTGCATGG-3', respectively;
(2) the specific probes for detecting the EGFR 19del site and the EGFR L858R site are as follows:
EGFR 19del wild-type probe: 5'-TGCTTCTCTTAATTCCTT-3', respectively;
EGFR 19del1 mutant probes: 5'-CGCTATCAAAACATCTC-3', respectively;
EGFR 19del2 mutant probes: 5'-CGCTATCAAGACATCTC-3', respectively;
EGFR L858R wild-type probe: 5'-AGCAGTTTGGCCAGC-3', respectively;
EGFR L858R mutant probe: 5'-AGCAGTTTGGCCCGC-3' are provided.
Preferably, the 5 'end of the EGFR 19del wild-type probe has a FAM fluorophore modification, the 5' ends of the EGFR 19del1 and EGFR 19del2 mutant probes have a Cy5 fluorophore modification, the 5 'end of the EGFR L858R wild-type probe has a VIC fluorophore modification, and the 5' end of the EGFR L858R mutant probe has a ROX fluorophore modification.
Preferably, the 3' ends of the wild-type probe and the mutant-type probe are subjected to dideoxy modification, amino modification or phosphorylation modification so as to block the probes from extending in the amplification process; preferably, the 3' terminal position of the probe is modified with MGB non-fluorescence quenching group.
Preferably, the nucleotides corresponding to the wild type or the mutant type in the nucleotide sequences of the wild type probe and the mutant type probe are nucleotides modified to enhance the thermal stability of the probes and the complementary strand; preferably, the nucleotide modified to enhance the thermal stability of the probe to the complementary strand is a locked nucleic acid modified nucleotide; more preferably, the nucleotide sequence of the locked nucleic acid modification is:
EGFR L858R wild-type probe: 5 '-AGCAGTTTGGCC/iXNA _ A/GC-3'
EGFR L858R mutant probe: 5 '-AGCAGTTTGGCC/iXNA _ C/GC-3'.
Preferably, the concentrations of the specific primers and the specific probes for detecting the EGFR 19del site and the EGFR L858R site are respectively as follows:
the concentration of the EGFR 19del forward primer is 3.0-8.0 mu mol/L;
the concentration of EGFR 19del reverse primer is 3.0-8.0 mu mol/L;
the concentration of the forward primer of EGFR L858R is 5.0-9.5 mu mol/L;
the concentration of the EGFR L858R reverse primer is 5.0-9.5 mu mol/L;
the concentration of the EGFR 19del wild-type probe is 2.5-4.5 mu mol/L;
the concentration of the EGFR 19del1 mutant probe is 1.5-3.0 mu mol/L;
the concentration of the EGFR 19del2 mutant probe is 1.5-3.0 mu mol/L;
the concentration of the EGFR L858R wild-type probe is 4.5-6.0 mu mol/L;
the concentration of the EGFR L858R mutant probe was 4.5-6.0. mu. mol/L.
Preferably, if the average length of the detected sample DNA is more than 20kb, a restriction enzyme is added during the preparation of the PCR amplification reaction, the reaction system is kept at room temperature of 15-25 ℃ for 10 minutes before the PCR amplification reaction, and digestion is carried out, wherein the restriction enzyme selected in the digestion reaction is any one of EcoRI, PvuII, Xba I, Alu I, CviQ I and Hae III.
Preferably, the number of mutations and the mutation ratio of the 19del site in the sample are calculated by using the signals of the wild-type probe and the sum of the signals of the two mutant probes of the EGFR 19 del; the wild type probe signal and the mutant type probe signal of the EGFR L858R are used for calculating the mutation number and the mutation ratio of the L858R locus in the sample.
Preferably, according to the read FAM, Cy5, VIC and ROX signals, the 19del and L858R site mutation situations of the EGFR gene of the sample are quantitatively detected; wherein, FAM and Cy5 signals can be judged as 19del site mutation, and then calculation analysis of mutation ratio is carried out; VIC and ROX signals can be judged as EGFR L858R mutation, and then calculation analysis of mutation ratio is carried out.
In another aspect, the present invention provides a digital PCR reaction kit for quantitatively detecting site mutations of 19del and L858R of EGFR gene, comprising a detection system consisting of dPCR-enzyme reaction solution, 10 × primer probe mixture, DNA template and deionized water as described above.
Compared with the prior art, the invention has the following beneficial effects:
(1) compared with traditional mutation detection methods such as a gene chip and fluorescent quantitative PCR, the method for quantitatively detecting the 19del and L858R site mutation of the EGFR gene by using the digital PCR provided by the invention has the advantages that the digital PCR directly obtains the absolute copy number of the target gene by using an indirect quantitative mode calculated by a standard curve and a Ct value through a detection means of discretization treatment and molecular counting of a sample, and provides higher detection precision and resolution for research.
(2) The digital PCR technology is adopted, and the absolute quantification of the target gene can be realized no matter the traditional liquid drop type digital PCR or chip type digital PCR. The detection platform-QIAcuity Digital PCR System used in the invention is a more unique and accurate nano-micropore plate type Digital PCR System. In the detection system, the reaction system of each sample is stably distributed to 26000 nano-scale micropores, multiple detection channels are configured, the droplet reaction is accurately and efficiently controlled, the detection result deviation is avoided to the maximum extent, and the accuracy of the data result is improved.
Drawings
FIGS. 1-2 show the results of genotyping two sites of EGFR 19del/L858R by detecting DNA-1 of the cell line according to the present invention;
FIGS. 3-4 show the results of genotyping two sites of EGFR 19del/L858R by detecting DNA-2 of the cell line according to the present invention;
FIGS. 5-6 show the results of genotyping at two sites of EGFR 19del/L858R by detecting FFPE DNA-1 according to the present invention;
FIGS. 7-8 are the results of genotyping at two sites of EGFR 19del/L858R using FFPE DNA-2 of the present invention;
FIGS. 9 to 10 are the results of genotyping at two sites of EGFR 19del/L858R using the detection of cfDNA-1 according to the present invention, respectively;
FIGS. 11 to 12 are the results of genotyping at two sites of EGFR 19del/L858R using the detection of cfDNA-2 according to the present invention, respectively.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
Example 1 design and establishment of digital PCR reaction System for quantitative detection of site mutation of 19del and L858R of EGFR Gene
1. Design of primer-probe combination
First, referring to the GeneBank sequence number of EGFR (epidermal growth factor receptor) gene reported in the literature, the gene sequences corresponding to the respective sites were found from the NCBI database, and specific amplification primer pairs were designed upstream and downstream of the 19del and L858R mutation sites confirmed by the study, and wild-type and mutant-type specific probes were designed while covering the mutation site nucleotides.
The principle of primer design: the Primer pair for quantitatively detecting the EGFR gene mutation sites 19del and L858R is designed by Primer5 and NCBI Blast software; the length of the primer is 18-23 nucleotides, the GC content of the primer is 40-60%, and the Tm value of the primer is more than or equal to 60 ℃. The Tm values of the primers are approximately close to each other to ensure efficient amplification of primer pairs at the same annealing temperature. After the design is finished, each primer is compared by NCBI Blast software, and the primers can be ensured to be specifically compared to a target region in a proper range. The amplification product size of the primer is in the range of 90-110 bp. The designed primers were analyzed for primer Dimer by Auto Dimer software to ensure specificity and avoid the occurrence of dimers.
Principle of probe design: the wild type and mutant specific probes for quantitatively detecting EGFR gene mutation sites 19del and L858R are modified at the 3 'end to prevent the probes from extending in the amplification process, and the 3' end is modified with MGB non-fluorescence quenching groups. The 5 'end of the EGFR 19del wild-type probe is modified by a FAM fluorescent group, the 5' end of the EGFR 19del mutant probe is modified by a Cy5 fluorescent group, the 5 'end of the EGFR L858R wild-type probe is modified by a VIC fluorescent group, and the 5' end of the EGFR L858R mutant probe is modified by a ROX fluorescent group. Wherein, the wild type and mutant nucleotides of the EGFR L858R use the locked nucleic acid technology to enhance the specific binding of the probe and the target product, thereby achieving the effects of enhancing the binding efficiency and enhancing the fluorescent signal.
The sequences of specific primers and probes for EGFR 19del and L858R site amplification are shown in Table 1:
TABLE 1
2. Detection system set-up
2.1 preparing 10 × primer probe mixture by using the designed primer probes according to different concentration ratios, wherein the specific concentrations of each primer and probe are shown in table 2:
TABLE 2
2.2 preparing an amplification reaction according to a digital PCR reaction system: wherein the volume of the 4 XDPCR-enzyme reaction solution is 10. mu.L, the volume of the 10 XDPCR-enzyme reaction solution is 4. mu.L, the volume of the restriction enzyme (if necessary) is 10U, the volume of the template DNA is 1-25. mu.L, and the volume of the deionized water is filled to 40. mu.L.
2.3 the reaction mixture is added to the dPCR microplate addition wells and the microplate is subsequently sealed.
2.4 if the average length of the detected sample DNA is more than 20kb, carrying out restriction enzyme digestion reaction, and placing the reaction system at room temperature (15-25 ℃) for 10 minutes for carrying out enzyme digestion; if the plasma free DNA sample is detected, the PCR amplification reaction is directly carried out without enzyme digestion reaction.
The restriction enzyme selected in this example was EcoRI, and the cell line DNA was digested.
2.5 QIAcuity system digital PCR reaction system automatically performs distribution, thermal cycle amplification reaction and result interpretation on the reaction solution in the nano-microplate.
2.6 thermal cycling amplification reactions were carried out as follows: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 15 seconds; annealing at 55-63 ℃ for 15 seconds; extension at 72 ℃ for 15 seconds; amplifying for 40 cycles; the reaction was complete.
2.7 acquiring fluorescence signals, analyzing data, obtaining the results of the number and the proportion of the 19del and L858R mutations in the detected sample, and giving a judgment result.
According to the read FAM, Cy5, VIC and ROX signals, the 19del and L858R site mutation conditions of the EGFR gene can be analyzed. Wherein, the signals of wild type (FAM) and mutant type (Cy 5) at the EGFR 19del site can be judged as 19del site mutation (positive), and the mutation ratio is calculated and analyzed; signals of wild type (VIC) and mutant type (ROX) at EGFR L858R locus can be judged as EGFR L858R mutation (positive), and calculation analysis of mutation ratio is carried out.
As can be seen from FIGS. 1 to 4, according to the digital PCR result map, the mutation condition of the sample to be detected at the sites EGFR 19del and L858R can be relatively intuitively observed, and the mutation ratio can be calculated.
The result of the detection of the cell line DNA-1 in this example was positive for EGFR 19del, and the mutation rate was 54.26%, wherein the mutant signal copies detected by Cy5 was 1338 copies/40. mu.L (FIG. 1, the Y-axis coordinate data is the Cy5 signal of the EGFR 19del mutant probe); wild type signal copies detected by FAM is 1128 copies/40 μ L (FIG. 1X-axis coordinate data is EGFR 19del wild type probe FAM signal); EGFR L858R negative, mutation rate of 0.04%, wherein the ROX detection mutation signal copies is 1 copies/40 μ L (FIG. 2, Y axis coordinate data is ROX signal of EGFR L858R mutant probe); the wild type signal copies detected by VIC was 2503 copies/40. mu.L (FIG. 2X-axis coordinate data is EGFR L858R wild type probe VIC signal).
The detection result of the cell line DNA-2 in this example is EGFR 19del negative, the mutation ratio is 0.00%, wherein the mutant signal copies detected by Cy5 is 0 copies/40 μ L (FIG. 3, Y-axis coordinate data is Cy5 signal of EGFR 19del mutant probe); the wild type signal copies detected by FAM is 2960 copies/40 mu L (the X-axis coordinate data in FIG. 3 is EGFR 19del wild type probe FAM signal); EGFR L858R positive, mutation rate is 46.54%, wherein the ROX detection mutation signal copies is 1400 copies/40 μ L (FIG. 4Y axis coordinate data is the ROX signal of EGFR L858R mutant probe); the wild type signal copies detected by VIC was 1608 copies/40. mu.L (FIG. 4X-axis coordinate data is EGFR L858R wild type probe VIC signal).
EXAMPLE 2 detection of tissue samples from patients
2 non-small cell lung cancer FFPE samples are selected, the detection system is adopted to detect the DNA of the cell line, and the specific operation steps are as follows:
1. non-small cell lung cancer FFPE sample DNA extraction
The FFPE sample of the non-small cell lung cancer is subjected to DNA extraction by using a QIAamp DNA FFPE Tissue Kit of QIAGEN company, and DNA concentration and quality detection are carried out by using Qubit, and finally the FFPE sample is diluted to a concentration of 10 ng/. mu.L for later use.
2.PCR amplification
2.1 preparation of dPCR amplification System
The amplification reaction was prepared according to the QIAcuity system digital PCR reaction system: wherein the volume of the 4 XDPCR-enzyme reaction solution is 10. mu.L, the volume of the 10 XDPCR-enzyme reaction solution is 4. mu.L, the volume of the FFPE DNA is 2. mu.L, and the volume of the deionized water is fully filled to 40. mu.L.
2.2 the reaction mixture is added to the dPCR microplate addition wells and the microplate is subsequently sealed.
2.3 QIAcuity system digital PCR reaction system automatically carries out distribution, thermal cycle amplification reaction and result interpretation on reaction liquid in the nano-microplate.
2.4 thermal cycling amplification reactions were carried out as follows: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 15 seconds; annealing at 60 ℃ for 15 seconds; extension at 72 ℃ for 15 seconds; amplifying for 40 cycles; the reaction was complete.
2.5, acquiring fluorescence signals, analyzing data, obtaining the mutation quantity and mutation proportion results of 19del and L858R in the detection sample, and giving a judgment result.
As can be seen from FIGS. 5 to 8, according to the digital PCR result map, the mutation condition of the sample to be detected at the sites EGFR 19del and L858R can be relatively intuitively observed, and the mutation ratio can be calculated.
In this example, the FFPE DNA-1 was negative for EGFR 19del and the mutation rate was 0.00%, wherein the mutant signal copies detected by Cy5 was 0 copies/40. mu.L (FIG. 5, Y-axis coordinate data is Cy5 signal of EGFR 19del mutant probe); the wild type signal copies detected by FAM was 2243 copies/40. mu.L (FIG. 5, X-axis coordinate data is EGFR 19del wild type probe FAM signal); EGFR L858R negative, mutation rate of 0.00%, wherein the ROX detection mutation signal copies is 0 copies/40 μ L (FIG. 6, Y axis coordinate data is ROX signal of EGFR L858R mutant probe); the wild type signal copies detected by VIC was 2794 copies/40. mu.L (FIG. 6X-axis coordinate data is EGFR L858R wild type probe VIC signal).
In this example, the FFPE DNA-2 was negative for EGFR 19del and the mutation rate was 0.00%, wherein the mutant signal copies detected by Cy5 was 0 copies/40. mu.L (FIG. 7, the Y-axis coordinate data is Cy5 signal of EGFR 19del mutant probe); wild type signal copies detected by FAM is 832 copies/40 μ L (FIG. 7X-axis coordinate data is EGFR 19del wild type probe FAM signal); EGFR L858R positive, mutation rate is 3.42%, wherein the ROX detection mutation signal copies is 34 copies/40 μ L (FIG. 8Y axis coordinate data is the ROX signal of EGFR L858R mutant probe); the wild type signal copies detected by VIC was 959 copies/40. mu.L (FIG. 8X-axis coordinate data is EGFR L858R wild type probe VIC signal).
Example 3 detection of plasma free nucleic acid samples
Plasma samples of 2 lung cancer patients are selected, and the detection system is adopted to detect plasma free nucleic acid (cfDNA) of the lung cancer patients, and the specific operation steps are as follows:
1. plasma free nucleic acid (cfDNA) extraction
Plasma samples of cfDNA of 3 lung cancer patients were extracted using the MagMAX Cell-Free DNA Isolation Kit from Applied Biosystems, respectively, and then tested for cfDNA concentration and quality using Qubit.
PCR amplification
2.1 preparation of dPCR amplification System
The amplification reaction was prepared according to the QIAcuity system digital PCR reaction system: wherein the volume of the 4 XDPCR-enzyme reaction solution was 10. mu.L, the volume of the 10 XDPCR-enzyme reaction solution was 4. mu. L, cfDNA 20. mu.L, and the volume of deionized water was 40. mu.L.
2.2 the reaction mixture is added to the dPCR microplate addition wells and the microplate is subsequently sealed.
2.3 QIAcuity system digital PCR reaction system automatically carries out distribution, thermal cycle amplification reaction and result interpretation on reaction liquid in the nano-microplate.
2.4 thermal cycling amplification reactions were carried out as follows: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 15 seconds; annealing at 60 ℃ for 15 seconds; extension at 72 ℃ for 15 seconds; amplifying for 40 cycles; the reaction was complete.
2.5, acquiring fluorescence signals, analyzing data, obtaining the mutation quantity and mutation proportion results of 19del and L858R in the detection sample, and giving a judgment result.
As can be seen from FIGS. 9 to 12, according to the digital PCR result map, the mutation condition of the sample to be detected at the sites EGFR 19del and L858R can be relatively intuitively observed, and the mutation ratio can be calculated.
The cfDNA-1 of this example was positive for EGFR 19del with a mutation rate of 3.78%, wherein the mutant signal copies detected by Cy5 was 48 copies/40. mu.L (FIG. 9, the Y-axis coordinate data is Cy5 signal of EGFR 19del mutant probe); the wild type signal copies detected by FAM is 1221 copies/40 μ L (FIG. 9X-axis coordinate data is EGFR 19del wild type probe FAM signal); EGFR L858R negative, mutation rate of 0.00%, wherein the ROX detection mutation signal copies is 0 copies/40 μ L (FIG. 10, Y axis coordinate data is ROX signal of EGFR L858R mutant probe); the wild type signal copies detected by VIC was 1525 copies/40. mu.L (FIG. 10X-axis coordinate data is EGFR L858R wild type probe VIC signal).
The detection result of cf DNA-2 in this example is positive for EGFR 19del, and the mutation rate is 5.31%, wherein the mutant signal copies detected by Cy5 is 72 copies/40 μ L (FIG. 11, the coordinate data of Y axis is Cy5 signal of EGFR 19del mutant probe); the wild type signal copies detected by FAM is 1285 copies/40 mu L (FIG. 11X-axis coordinate data is EGFR 19del wild type probe FAM signal); EGFR L858R positive, mutation rate is 2.44%, wherein the ROX detection mutation signal copies is 39 copies/40 μ L (FIG. 12, Y axis coordinate data is the ROX signal of EGFR L858R mutant probe); the wild type signal copies detected by VIC was 1557 copies/40. mu.L (FIG. 12X-axis coordinate data is EGFR L858R wild type probe VIC signal).
In conclusion, the invention provides a digital PCR reaction system for quantitatively detecting the site mutation of 19del and L858R of EGFR gene aiming at the actual requirement of clinical treatment. Compared with traditional mutation detection methods such as gene chips, fluorescent quantitative PCR and the like, the digital PCR reaction system provided by the invention has the advantages that an indirect quantitative mode calculated through a standard curve and a Ct value is abandoned through a detection means of discretization treatment and molecular counting of a sample, the absolute copy number of a target gene is directly obtained, and higher detection precision and resolution are provided for research. Meanwhile, the digital PCR detection platform used by the invention is a more unique and accurate nano-micropore plate type digital PCR system. In the detection system, the reaction system of each sample is stably distributed to 26000 nano-scale micropores, multiple detection channels are configured, the droplet reaction is accurately and efficiently controlled, the detection result deviation is avoided to the maximum extent, and the accuracy of the data result is improved.
Sequence listing
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<120> method for quantitatively detecting site mutation of 19del and L858R of EGFR gene by using digital PCR
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Claims (10)
1. A method for quantitatively detecting the 19del and L858R site mutation of EGFR gene by using digital PCR is characterized in that: the method comprises the following steps:
(1) extracting a tissue sample or a plasma free DNA sample;
(2) preparing PCR amplification reaction by using a detection system consisting of dPCR-enzyme reaction solution, 10 multiplied primer probe mixture, a DNA template and deionized water;
(3) adding the reaction mixture into a sample adding hole of a dPCR nano-microporous plate, and sealing the nano-microporous plate;
(4) the digital PCR reaction system automatically performs distribution, thermal cycle amplification reaction and result interpretation on reaction liquid in the nano-microporous plate;
(5) the thermal cycling amplification reaction is carried out according to the following conditions: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 15 seconds; annealing at 55-63 ℃ for 15 seconds; extension at 72 ℃ for 15 seconds; amplifying for 40 cycles; finishing the reaction;
(6) and acquiring fluorescence signals, analyzing data, obtaining the mutation quantity and mutation proportion results of 19del and L858R in the detection sample, and giving a judgment result.
2. The method for quantitatively detecting the 19del and L858R site mutations of EGFR gene according to claim 1 by using digital PCR, wherein: the primer probe mixture consists of specific primers and specific probes for detecting EGFR 19del site and EGFR L858R site.
3. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 2, wherein:
(1) the specific primers for detecting the EGFR 19del site and the EGFR L858R site are as follows:
EGFR 19del forward primer: 5'-ACTCTGGATCCCAGAAGGTG-3', respectively;
EGFR 19del reverse primer: 5'-GAAACTCACATCGAGGATT-3', respectively;
EGFR L858R forward primer: 5'-CGTACTGGTGAAAACACCGCA-3', respectively;
EGFR L858R reverse primer: 5'-CTTTGCCTCCTTCTGCATGG-3', respectively;
(2) the specific probes for detecting the EGFR 19del site and the EGFR L858R site are as follows:
EGFR 19del wild-type probe: 5'-TGCTTCTCTTAATTCCTT-3', respectively;
EGFR 19del1 mutant probes: 5'-CGCTATCAAAACATCTC-3', respectively;
EGFR 19del2 mutant probes: 5'-CGCTATCAAGACATCTC-3', respectively;
EGFR L858R wild-type probe: 5'-AGCAGTTTGGCCAGC-3', respectively;
EGFR L858R mutant probe: 5'-AGCAGTTTGGCCCGC-3' are provided.
4. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 3, wherein: the 5 'end of the EGFR 19del wild-type probe is modified by a FAM fluorescent group, the 5' ends of the EGFR 19del1 and EGFR 19del2 mutant probes are modified by a Cy5 fluorescent group, the 5 'end of the EGFR L858R wild-type probe is modified by a VIC fluorescent group, and the 5' end of the EGFR L858R mutant probe is modified by a ROX fluorescent group.
5. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 3 or 4, wherein: the 3' ends of the wild type probe and the mutant type probe are subjected to dideoxy modification, amino modification or phosphorylation modification so as to block the probes from extending in the amplification process; preferably, the 3' terminal position of the probe is modified with MGB non-fluorescence quenching group.
6. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 3 or 4, wherein: the nucleotides corresponding to the wild type or the mutant type in the nucleotide sequences of the wild type probe and the mutant type probe are nucleotides modified to enhance the thermal stability of the probe and a complementary strand; preferably, the nucleotide modified to enhance the thermal stability of the probe to the complementary strand is a locked nucleic acid modified nucleotide; more preferably, the nucleotide sequence of the locked nucleic acid modification is:
EGFR L858R wild-type probe: 5 '-AGCAGTTTGGCC/iXNA _ A/GC-3'
EGFR L858R mutant probe: 5 '-AGCAGTTTGGCC/iXNA _ C/GC-3'.
7. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 3, wherein: the concentrations of the specific primers and the specific probes for detecting the EGFR 19del site and the EGFR L858R site are respectively as follows:
the concentration of the EGFR 19del forward primer is 3.0-8.0 mu mol/L;
the concentration of EGFR 19del reverse primer is 3.0-8.0 mu mol/L;
the concentration of the forward primer of EGFR L858R is 5.0-9.5 mu mol/L;
the concentration of the EGFR L858R reverse primer is 5.0-9.5 mu mol/L;
the concentration of the EGFR 19del wild-type probe is 2.5-4.5 mu mol/L;
the concentration of the EGFR 19del1 mutant probe is 1.5-3.0 mu mol/L;
the concentration of the EGFR 19del2 mutant probe is 1.5-3.0 mu mol/L;
the concentration of the EGFR L858R wild-type probe is 4.5-6.0 mu mol/L;
the concentration of the EGFR L858R mutant probe was 4.5-6.0. mu. mol/L.
8. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 1, wherein: if the average length of the detected sample DNA is more than 20kb, adding restriction enzyme in the PCR amplification reaction, placing the reaction system at room temperature of 15-25 ℃ for 10 minutes before the PCR amplification reaction, and carrying out enzyme digestion, wherein the restriction enzyme selected in the enzyme digestion reaction is any one of EcoRI, PvuII, XbaI, AluI, CviQ I and HaeIII.
9. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 3 or 4, wherein: calculating the number and the proportion of the mutations at the 19del site in the sample by using the signals of the wild-type probe and the sum of the signals of the two mutant probes of the EGFR 19 del; the wild type probe signal and the mutant type probe signal of the EGFR L858R are used for calculating the mutation number and the mutation ratio of the L858R locus in the sample.
10. The method for quantitatively detecting the 19del and L858R site mutations of the EGFR gene by using digital PCR as claimed in claim 9, wherein: according to the read FAM, Cy5, VIC and ROX signals, carrying out quantitative detection on the 19del and L858R site mutation conditions of the EGFR gene of the sample; wherein, FAM and Cy5 signals can be judged as 19del site mutation, and then calculation analysis of mutation ratio is carried out; VIC and ROX signals can be judged as EGFR L858R mutation, and then calculation analysis of mutation ratio is carried out.
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