CN112210606A - Digital PCR detection system for EGFR T790M locus and application thereof - Google Patents
Digital PCR detection system for EGFR T790M locus and application thereof Download PDFInfo
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Abstract
The invention provides a digital PCR detection system of EGFR T790M locus and application thereof. The detection system comprises a specific amplification primer of EGFR T790M locus, a wild type probe and a mutant type probe, and the sensitivity and the stability of the whole detection system are greatly improved by optimizing the concentration ratio of the primer probe in the PCR amplification system. Meanwhile, the detection efficiency of the EGFR T790M locus is enhanced by introducing a specific probe modification technology, particularly the combination of wild type and mutant type probes and corresponding locus nucleotides is enhanced by using a locked nucleic acid technology, and the detection efficiency of a low-concentration plasma free nucleic acid (cfDNA) sample is remarkably improved.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a digital PCR detection system of EGFR T790M locus and application thereof.
Background
Studies have shown that cancer is the result of the interaction of environmental factors with the genetic material of cells, multifactorial, multistage and polygenic effects, and the accumulation of gene mutations. Among the numerous genes in humans, protooncogenes and tumor suppressor genes are closely related to the development and progression of cancer. Mutations in proto-oncogenes and or suppressor genes may cause cellular carcinogenesis. Cancer a minute amount of tumor DNA has been released into the circulating blood before solid masses are formed. The mutation of the genes in the circulating blood is detected by gene detection technologies such as sequencing, PCR and the like, so that early or super-early cancers can be screened, the curative effect of radiotherapy and chemotherapy can be evaluated, the curative effect of targeted drugs can be predicted, and early relapse after operation and the like can be monitored; the gene mutation detection is carried out on the tumor tissue, which is helpful for understanding the generation and development processes of the tumor and judging the type and prognosis of the tumor; is helpful for predicting the curative effect of the tumor drug related to the protooncogene.
Currently clinically used targeted drugs against EGFR (epidermal growth factor receptor) are EGFR tyrosine kinase inhibitors (EGFR-TKI). The EGFR-TKI blocks an EGFR signal conduction path by inhibiting EGFR autophosphorylation, thereby inhibiting proliferation and differentiation of tumor cells and realizing targeted therapy. The curative effect of EGFR-TKI is closely related to the mutation condition of EGFR gene. The EGFR gene is located in the short arm of the human chromosome 7 and consists of 28 exons, and the mature EGFR gene contains 1186 amino acids with the relative molecular mass of 170KD and belongs to the receptor Tyrosine Kinase (TKI) family. After being combined with ligand, EGFR can lead to dimerization and intracellular region phosphorylation of the EGFR, and further activates a series of downstream signal pathways of the EGFR, wherein the downstream signal pathways mainly comprise a Src/STAT pathway, a PI3K/Akt pathway, a Ras-Raf-MEK-Erk/MAPK pathway and the like, so that a plurality of physiological processes of cell growth, differentiation, invasion, anti-apoptosis and the like are regulated and controlled. The T790M site mutation of the EGFR gene is uniformly considered to be the main reason for the secondary drug resistance of EGFR-TKI and is the first gene discovered to be related to drug resistance. EGFR wild-type patients are not recommended for EGFR-TKI treatment because patients with EGFR gene mutations benefit from treatment with EGFR-TKI.
Plasma free DNA (cfDNA, also called blood circulation DNA) refers to DNA free outside cells in blood, and its sources are mainly three: DNA released by leukocyte disintegration, DNA released into blood from necrotic tissue, especially tumor tissue, and DNA infected by virus and bacteria. In recent years, with the development of gene diagnosis technology, the application value of free nucleic acid is increasing, such as eugenic screening, early diagnosis of tumor, genetic disease detection, pathogen infection gene detection and the like. However, the content of free DNA in blood is generally low, the fragments are small and difficult to extract, which greatly influences the gene detection of free nucleic acid and the development of various researches.
Therefore, the digital PCR detection system for the EGFR T790M locus is provided to improve the sensitivity and specificity of a detection result, and has wide application prospect and huge market value particularly in the detection of plasma free DNA (cfDNA).
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a method for detecting plasma free DNA gene mutation by using a digital PCR technology, the system enhances the detection efficiency of EGFR T790M locus by introducing a specific probe modification technology, optimizes the detection conditions and primer concentration, finally realizes the high-efficiency, accurate and sensitive detection of the plasma free DNA sample, and has wide application prospect and great market value.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an EGFR T790M site digital PCR detection system, comprising a primer probe mixture, wherein the primer probe mixture consists of a pair of specific primers for detecting EGFR T790M site and a pair of specific probes for detecting EGFR T790M site:
(1) the specific primer comprises:
EGFR T790M forward primer: 5'-GCCTGCTGGGCATCTG-3' the flow of the air in the air conditioner,
EGFR T790M reverse primer: 5'-TCTTTGTGTTCCCGGACATAGTC-3', respectively;
(2) the specific probe comprises:
EGFR T790M wild-type probe: 5' -TGAGCTGCGTGATGAG-3’，
EGFR T790M mutant probe: 5' -TGAGCTGCATGATGAG-3’。
Preferably, the wild-type probe has a VIC fluorophore modification at the 5 'end, and the mutant-type probe has a FAM fluorophore modification at the 5' end.
Preferably, the 3' ends of the wild-type probe and the mutant-type probe are subjected to dideoxy modification, amino modification or phosphorylation modification to block the probes from extending during amplification.
Preferably, the 3' terminal position of the probe is modified with MGB non-fluorescence quenching group.
Preferably, the underlined nucleotides in the nucleotide sequences of the wild-type probe and the mutant-type probe are mutation sites.
Preferably, the underlined nucleotides 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 to 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 or a peptide nucleic acid modified nucleotide.
Preferably, the nucleotide modified to enhance the thermal stability of the probe to the complementary strand is a locked nucleic acid modified nucleotide:
EGFR T790M wild-type probe: 5 '-TGAGCTGC/iXNA _ G/TGATAG-3',
EGFR T790M mutant probe: 5 '-TGAGCTGC/iXNA _ A/TGATAG-3'.
Preferably, the detection system consists of 10 primer probe mixture, ddPCR-enzyme reaction solution, template DNA and deionized water.
For example, in the present invention, when the total volume of the detection system is 20. mu.L, 10 × primer probe mixture is 2.0. mu. L, ddPCR-10. mu.L of the enzyme reaction solution, 1-8. mu.L of the template DNA, and deionized water is added to 20. mu.L.
Preferably, the concentrations of the primer probe mixture are respectively:
the concentration of the forward primer of EGFR T790M is 4.5-10.0 mu mol/L;
the concentration of the EGFR T790M reverse primer is 4.5-10.0 mu mol/L;
the concentration of the wild-type probe of EGFR T790M is 3.5-6.0 mu mol/L;
the concentration of the EGFR T790M mutant probe was 3.5-6.0. mu. mol/L.
In a second aspect, the invention provides an application of a digital PCR detection system of EGFR T790M locus, wherein the detection system is used for detecting mutation of EGFR T790M locus in plasma free DNA.
Preferably, the method for detecting the mutation at the EGFR T790M site of the free plasma DNA comprises the following steps:
(1) extracting free DNA of a plasma sample;
(2) preparing an amplification reaction by adopting a detection system consisting of a 10-star primer probe mixture, ddPCR-enzyme reaction liquid, template DNA and deionized water;
(3) the reaction was carried out under the following amplification conditions: hot start at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; annealing at 60-62 deg.C for 60 s; amplifying for 40 cycles; enzyme inactivation at 98 ℃ for 10 minutes; preserving the heat at 4 ℃ and finishing the reaction;
(4) and (3) collecting fluorescent signals by a digital PCR instrument, analyzing the mutation condition of the EGFR T790M locus in the plasma free DNA, and calculating mutation ratio information.
Compared with the prior art, the invention has the following beneficial effects:
(1) the digital PCR detection system of the EGFR T790M locus provided by the invention has higher specificity and sensitivity, and has higher improvement in the aspects of sensitivity and accuracy in specificity detection compared with products of other companies in the market; particularly, a probe locked nucleic acid technology is introduced, so that the false positive rate of EGFR T790M locus detection is greatly reduced, and the detection result is accurate and credible;
(2) the digital PCR detection system for the EGFR T790M locus, provided by the invention, has high detection success rate for low-concentration sample types such as plasma free DNA samples, avoids practical problems of repeated detection, repeated sampling and the like, greatly shortens the detection period, and has higher application value in clinical examination.
(3) The amplification system provided by the invention is simple to operate, raw materials are saved, the time required by the whole amplification process is short, the operation is convenient, the result is visual, the analysis is convenient, and the detection result is more accurate and sensitive; the detection system provided by the invention can be used for analyzing clinical samples in a large scale and providing accurate and stable detection results.
Drawings
FIG. 1 shows the results of genotyping at EGFR T790M site in plasma-free DNA samples tested using the present invention;
FIG. 2 shows the result of genotyping at site EGFR T790M in DNA sample 1 of a cell line examined by the present invention;
FIG. 3 shows the result of genotyping at site EGFR T790M in DNA sample 2 of a cell line examined by the present invention;
FIG. 4 shows the result of genotyping of EGFR T790M locus in the plasma free DNA sample 1 of lung cancer detected by the present invention;
FIG. 5 shows the result of genotyping at EGFR T790M site of lung cancer plasma free DNA sample 2 detected by the present invention;
FIG. 6 shows the result of genotyping at EGFR T790M site in the lung cancer plasma free DNA sample 3 detected by the present invention.
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 detection System for EGFR T790M site
1. Design of primer-probe combination
Firstly, referring to GeneBank sequence numbers of genes of EGFR (epidermal growth factor receptor) T790M sites reported in the literature, gene sequences corresponding to the sites are found from NCBI nucleic acid database, specific amplification primer pairs are designed near T790M mutation sites confirmed by research, and wild type and mutant type specific probes are respectively designed under the condition of covering mutation site nucleotides.
The principle of primer design: the Primer pair for detecting the EGFR T790M locus is designed by Primer5 and NCBI Blast software; the length of the primer is between 15 and 25 nucleotides, the GC content of the primer is between 40 and 60 percent, and the Tm value of the primer is more than or equal to 60 ℃. The Tm value of each primer is controlled to the same level as much as possible, and the primer pair can be effectively amplified at the same annealing temperature. After the design is finished, each primer is compared by NCBI Blast software, and the condition that each primer can be compared to obtain a single result in a proper range is ensured. The size of the amplification product of the primer is in the range of 75-100 bp. The designed primers were analyzed for primer Dimer by Auto Dimer software to ensure specificity and prevent the occurrence of dimers.
Principle of probe design: the wild type and mutant type specific probes for detecting EGFR T790M locus in the invention are modified at the 3 'end to block the extension of the probes in the amplification process, and the MGB non-fluorescence quenching group is modified at the 3' end. The 5 'end of the wild type probe is modified by VIC fluorescent group, and the 5' end of the mutant type probe is modified by FAM fluorescent group. The mutation site nucleotides covered by the probe enhance the specific binding of the probe and a target product by using a locked nucleic acid technology, thereby achieving the effects of enhancing the binding efficiency and enhancing a fluorescent signal. The wild probe SEQ ID NO:3 shows:
the mutation site of the 5 '-VIC-TGAGCTGC/iXNA _ G/TGATAG-MGB-3' probe is modified by using a locked nucleic acid technology; mutant probe SEQ ID NO 4:
the mutation site of the 5 '-FAM-TGAGCTGC/iXNA _ A/TGATAG-MGB-3' probe is modified by using a locked nucleic acid technology.
The sequences of specific primers and probes for amplification of EGFR T790M site are shown in Table 1:
TABLE 1
| primer/Probe name | Sequence of | SEQ No. |
| T790M-F | 5’-GCCTGCTGGGCATCTG-3’ | SEQ ID NO:1 |
| T790M-R | 5’-TCTTTGTGTTCCCGGACATAGTC-3’ | SEQ ID NO:2 |
| T790M-W | 5’-VIC-TGAGCTGC/iXNA_G/TGATGAG-MGB-3’ | SEQ ID NO:3 |
| T790M-M | 5’-FAM-TGAGCTGC/iXNA_A/TGATGAG-MGB-3’ | SEQ ID NO:4 |
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
| primer/Probe name | Concentration of |
| T790M-F | 4.5-10.0μmol/L |
| T790M-R | 4.5-10.0μmol/L |
| T790M-W | 3.5-6.0μmol/L |
| T790M-M | 3.5-6.0μmol/L |
2.2 preparing an amplification reaction according to a digital PCR reaction system: wherein the volume of ddPCR-enzyme reaction solution is 10. mu.L, the volume of primer probe mixture is 10. mu.L, the volume of template DNA 1-8. mu.L and deionized water are supplemented to 20. mu.L.
2.3 the Automated Droplet Generator drop Generator in Bio-Rad QX200 AutoDG Droplet Digital PCR system is used to generate water-in-oil droplets for the ddPCR system, and the next PCR reaction can be performed when the Droplet system is completed.
2.4 placing the microdroplet system on a PCR instrument for amplification reaction, wherein the amplification condition parameters are as follows: hot start at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 60 seconds; amplifying for 40 cycles; enzyme inactivation at 98 ℃ for 10 minutes; and (4) preserving the temperature, and finishing the reaction.
2.5, placing the amplified product in a digital PCR instrument for reading a fluorescence signal, counting the quantity of the EGFR T790M wild type template and the mutant type template in the sample according to the actually read FAM and VIC signal values, and finally calculating the actual detection copy number and mutation proportion result of the sample so as to judge the negative and positive conditions of the detected sample.
As can be seen from FIG. 1, according to the map of the digital PCR results, the mutation condition of the sample to be detected at the EGFR T790M site can be observed relatively intuitively. The result of the detection of plasma free DNA (cfDNA) in the example is positive to EGFR T790M, the mutation rate is 48.5 percent, wherein the mutant signal copies detected by FAM is 126.46copies/20 mu L; the wild type signal copies detected by VIC was 134.28 copies/20. mu.L.
According to the digital PCR detection system for the EGFR T790M locus, provided by the invention, the wild type and mutant type loci of a sample are detected by using the double-color fluorescent markers, whether the sample has a mutation condition can be intuitively observed in a result map, and the information of the actual mutation proportion and the mutation copy number in the sample is obtained according to the statistical analysis of the digital PCR system, so that the positive and negative results of the detected sample can be intuitively judged.
EXAMPLE 2 detection of cell line samples
2 cell line samples are selected, the detection system is adopted to detect the cell line DNA, and the specific operation steps are as follows:
1. cell line DNA extraction
DNA was extracted from 2 cell lines using QIAamp DNA Blood Mini Kit from QIAGEN, and DNA concentration and quality were measured using Qubit, and finally diluted to a concentration of 10 ng/. mu.L for use.
PCR amplification
2.1 preparation of ddPCR amplification System
Preparing an amplification reaction according to a digital PCR reaction system: wherein, the volume of ddPCR-enzyme reaction solution is 10 muL, the volume of primer probe mixture is 10 muL, the volume of cell line DNA template is 1 muL, the volume of deionized water is 7 muL, and the volume is filled to 20 muL.
2.2 the Automated Droplet Generator drop Generator in the Bio-Rad QX200 AutoDG Droplet Digital PCR system is used to generate water-in-oil droplets for the ddPCR system, and the next PCR reaction can be carried out when the Droplet system is completed.
2.3 placing the microdroplet system on a PCR instrument for amplification reaction, wherein the amplification condition parameters are as follows: hot start at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 60 seconds; amplifying for 40 cycles; enzyme inactivation at 98 ℃ for 10 minutes; and (4) preserving the temperature, and finishing the reaction.
2.4 the amplification product is placed in a digital PCR instrument for reading the fluorescence signal. And performing signal statistics on 2 samples according to the actually read FAM and VIC signal values, and finally calculating the actual detection copy number and mutation proportion result of the sample so as to judge the positive and negative conditions of the detection sample.
As can be seen from FIGS. 2 and 3, according to the digital PCR result map, in the two cases of cell line DNA detected, the EGFR T790M site of the cell line DNA sample 1 (FIG. 2) is negative, wherein the mutant signal copies detected by FAM is 0copies/20 μ L; the wild type signal copies detected by VIC was 3780 copies/20. mu.L. The EGFR T790M site of the cell line DNA sample 2 (figure 3) is positive, the mutation rate is 8.60 percent, and the mutation type signal copies detected by FAM is 394copies/20 mu L; the wild type signal copies detected by VIC was 4560 copies/20. mu.L.
Example 3 detection of plasma free nucleic acid samples
Plasma samples of 3 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
MagMAX from Applied Biosystems was usedTMCell-Free DNA Isolation Kit extracts cfDNA from plasma samples of 3 lung cancer patients, respectively, and then performs cfDNA concentration and quality measurements using Qubit.
PCR amplification
2.1 preparation of ddPCR amplification System
Preparing an amplification reaction according to a digital PCR reaction system: the total volume of the ddPCR-enzyme reaction solution was 20. mu.L, and the mixture of 10 × primer and probe was 2.0. mu. L, cfDNA, and the template was 8. mu.L.
2.2 the Automated Droplet Generator drop Generator in the Bio-Rad QX200 AutoDG Droplet Digital PCR system is used to generate water-in-oil droplets for the ddPCR system, and the next PCR reaction can be carried out when the Droplet system is completed.
2.3 placing the microdroplet system on a PCR instrument for amplification reaction, wherein the amplification condition parameters are as follows: hot start at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; annealing at 60 ℃ for 60 seconds; amplifying for 40 cycles; enzyme inactivation at 98 ℃ for 10 minutes; and (4) preserving the temperature, and finishing the reaction.
2.4 the amplification product is placed in a digital PCR instrument for reading the fluorescence signal.
As can be seen from FIGS. 4, 5 and 6, according to the digital PCR result map, in the plasma free DNA of 3 lung cancer patients, the EGFR T790M site of the lung cancer plasma free DNA sample 1 (FIG. 4) is positive, the mutation rate is 29.46%, wherein the mutant signal copies detected by FAM is 42.85copies/20 μ L; the wild type signal copies detected by VIC was 102.6 copies/20. mu.L.
The EGFR T790M site of the lung cancer plasma free DNA sample 2 (figure 5) is positive, the mutation rate is 28.19 percent, and the mutation type signal copies detected by FAM is 17.54copies/20 mu L; the wild type signal copies detected by VIC was 44.67 copies/20. mu.L.
EGFR T790M site of lung cancer plasma free DNA sample 3 (FIG. 6) is negative, wherein mutant signal copies detected by FAM is 0copies/20 μ L; the wild type signal copies detected by VIC was 88.71 copies/20. mu.L.
In conclusion, the digital PCR detection system for the EGFR T790M locus provided by the invention has higher specificity and sensitivity, and has higher improvement in sensitivity and accuracy in specificity detection compared with products of other companies in the market; particularly, a probe locked nucleic acid technology is introduced, so that the false positive rate of EGFR T790M locus detection is greatly reduced, and the detection result is accurate and credible. Meanwhile, the detection system has high detection success rate for low-concentration sample types such as plasma free DNA samples, avoids practical problems of repeated detection, repeated sampling and the like, greatly shortens the detection period and has higher application value in clinical examination.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Yuan Chen Biotechnology (Suzhou) Ltd
<120> EGFR T790M locus digital PCR detection system and application thereof
<130> 20201117
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Claims (10)
1. A digital PCR detection system for EGFR T790M site comprises a primer probe mixture, and is characterized in that the primer probe mixture consists of a pair of specific primers for detecting EGFR T790M site and a pair of specific probes for detecting EGFR T790M site:
(1) the specific primer comprises:
EGFR T790M forward primer: 5'-GCCTGCTGGGCATCTG-3' the flow of the air in the air conditioner,
EGFR T790M reverse primer: 5'-TCTTTGTGTTCCCGGACATAGTC-3', respectively;
(2) the specific probe comprises:
EGFR T790M wild-type probe: 5' -TGAGCTGCGTGATGAG-3’，
EGFR T790M mutant probe: 5' -TGAGCTGCATGATGAG-3’。
2. The digital PCR detection system for EGFR T790M locus according to claim 1, wherein the wild-type probe has VIC fluorophore modification at 5 'end, and the mutant-type probe has FAM fluorophore modification at 5' end.
3. The digital PCR detection system for EGFR T790M locus according to claim 1, wherein the 3' ends of the wild type probe and the mutant type probe are dideoxy modified, amino modified or phosphorylation modified to block the probes from extending during the amplification process; preferably, the 3' terminal position of the probe is modified with MGB non-fluorescence quenching group.
4. The digital PCR detection system for EGFR T790M locus according to claim 1, wherein the underlined nucleotides 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 with the complementary strand.
5. The digital PCR detection system for EGFR T790M locus according to claim 4, wherein the nucleotides modified to enhance the thermal stability of the probe to the complementary strand are locked nucleic acid modified nucleotides or peptide nucleic acid modified nucleotides.
6. The digital PCR detection system for EGFR T790M site according to claim 5, wherein the nucleotides modified to enhance the thermal stability of the probe to the complementary strand are locked nucleic acid modified nucleotides:
EGFR T790M wild-type probe: 5 '-TGAGCTGC/iXNA _ G/TGATAG-3',
EGFR T790M mutant probe: 5 '-TGAGCTGC/iXNA _ A/TGATAG-3'.
7. The digital PCR detection system for EGFR T790M locus according to claim 1, wherein the detection system is composed of 10-primer probe mixture, ddPCR-enzyme reaction solution, template DNA and deionized water.
8. The digital PCR detection system for EGFR T790M locus according to claim 7, wherein the concentrations of the primer probe mixture are respectively:
the concentration of the forward primer of EGFR T790M is 4.5-10.0 mu mol/L;
the concentration of the EGFR T790M reverse primer is 4.5-10.0 mu mol/L;
the concentration of the wild-type probe of EGFR T790M is 3.5-6.0 mu mol/L;
the concentration of the EGFR T790M mutant probe was 3.5-6.0. mu. mol/L.
9. Use of the digital PCR detection system for EGFR T790M site according to claim 7 or 8, wherein the detection system is used for detecting mutation at EGFR T790M site in plasma free DNA.
10. The use of the digital PCR detection system for EGFR T790M locus as claimed in claim 9, wherein the method for detecting the mutation at EGFR T790M locus in plasma free DNA comprises the following steps:
(1) extracting free DNA of a plasma sample;
(2) preparing an amplification reaction by adopting a detection system consisting of a 10-star primer probe mixture, ddPCR-enzyme reaction liquid, template DNA and deionized water;
(3) the reaction was carried out under the following amplification conditions: hot start at 95 ℃ for 10 minutes; denaturation at 94 ℃ for 30 seconds; annealing at 60-62 deg.C for 60 s; amplifying for 40 cycles; enzyme inactivation at 98 ℃ for 10 minutes; preserving the heat at 4 ℃ and finishing the reaction;
(4) and (3) collecting fluorescent signals by a digital PCR instrument, analyzing the mutation condition of the EGFR T790M locus in the plasma free DNA, and calculating mutation ratio information.
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