CN112430646A - EGFR gene amplification detection method based on digital PCR platform - Google Patents
EGFR gene amplification detection method based on digital PCR platform Download PDFInfo
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- CN112430646A CN112430646A CN202011458578.3A CN202011458578A CN112430646A CN 112430646 A CN112430646 A CN 112430646A CN 202011458578 A CN202011458578 A CN 202011458578A CN 112430646 A CN112430646 A CN 112430646A
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to the technical field of molecular biology, in particular to an EGFR gene amplification detection method based on a digital PCR platform, which comprises the following steps: s1, extraction of cfDNA: extracting cfDNA of a sample to be detected, wherein the sample to be detected is a whole blood sample; s2, preparing a digital PCR reaction mixture: mixing a cfDNA template of a sample to be detected, forward and reverse amplification primers of an EGFR gene amplification area, a TaqMan probe of the EGFR gene amplification area, forward and reverse amplification primers of an internal reference gene EFTUD2, a TaqMan probe marked by an internal reference gene EFTUD2 and ddPCR reaction premix to prepare a digital PCR reaction mixture, wherein the content of the cfDNA template is 0.5-2 ng/mu L; s3, loading the PCR reaction mixed solution obtained in the step S2 into a digital PCR chip for ddPCR amplification reaction; s4, reading a fluorescent signal of the PCR chip; and S5, analyzing data and counting results. The invention has high sensitivity and simple operation, does not need to collect tissue samples, effectively reduces the pain of patients and is particularly suitable for patients intolerant to tissue sampling.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an EGFR gene amplification detection method based on a digital PCR platform.
Background
Human Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein Receptor with tyrosine kinase activity, controls intracellular signal transduction, and plays an important role in cell Growth, differentiation, proliferation, adhesion, apoptosis or in angiogenesis. Research shows that high expression or abnormal expression of EGFR exists in a plurality of solid tumors, and provides theoretical basis and experimental basis for targeted therapy of EGFR and signal transduction intervention therapy aiming at EGFR signal transduction pathways. Currently clinically used anti-EGFR drugs include EGFR kinase small molecule inhibitors (gefitinib/iressa, erlotinib/tarceva), monoclonal antibodies (victib/panitumumab, erbitux/cetuximab, tamosin/nimotuzumab) and receptor tyrosine kinase small molecule inhibitors (lapatinib).
A large number of clinical studies show that EGFR gene amplification can predict the treatment sensitivity of colorectal cancer, non-small cell lung cancer and other cancers to tyrosine kinase inhibitors such as Iressa (gefitinib), Tarceva (erlotinib) and the like. About 1/3 patients with non-small cell lung cancer, the result of EGFR gene amplification test is positive, while in the positive group of patients, the effective rate of applying Iressa (gefitinib) and Tarceva (erlotinib) is 35%, and the disease control rate is as high as 70%. The method is beneficial to screening sensitive people of anti-EGFR medicines such as Iressa and Tarceva and is beneficial to guiding clinical medication.
The most common method for detecting EGFR gene amplification at present is Fluorescence In Situ Hybridization (FISH), which has the basic principle that Fluorescence signals are distinguished and counted under a Fluorescence microscope by performing in situ hybridization of a Fluorescence-labeled DNA probe and DNA of a sample to be detected, so that chromosomes and cells and tissue samples with gene abnormality are detected and diagnosed. The amplification specificity of the EGFR detected by the FISH technology is high, but the sample processing period is long, the cost is high (the probe is expensive), high-flux detection cannot be realized, and the result interpretation subjectivity and the specialty are strong.
Disclosure of Invention
The invention aims to solve the technical problem of providing the EGFR gene amplification detection method based on the digital PCR platform, which has the advantages of high sensitivity, simple operation, no need of collecting tissue specimens, effective reduction of pain of patients and particular suitability for patients intolerant to tissue sampling.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for detecting EGFR gene amplification based on a digital PCR platform comprises the following steps:
s1, extraction of cfDNA: extracting cfDNA of a sample to be detected, wherein the sample to be detected is a whole blood sample;
s2, preparing a digital PCR reaction mixture: mixing a cfDNA template of a sample to be detected, forward and reverse amplification primers of an EGFR gene amplification area, a TaqMan probe of the EGFR gene amplification area, forward and reverse amplification primers of an internal reference gene EFTUD2, a TaqMan probe marked by an internal reference gene EFTUD2 and ddPCR reaction premix to prepare a digital PCR reaction mixture, wherein the content of the cfDNA template is 0.5-2 ng/mu L;
s3, loading the PCR reaction mixed solution obtained in the step S2 into a digital PCR chip for ddPCR amplification reaction;
s4, reading a fluorescent signal of the PCR chip;
and S5, analyzing data and counting results.
Preferably, the more preferable content of the cfDNA template in step S2 is 0.8-1.2 ng/. mu.L.
Preferably, the EGFR gene amplification region forward and reverse amplification primers in step S2 comprise a specific primer pair for amplifying the EGFR gene amplification region, the specific primer pair comprises a forward primer SEQ ID NO. 1 and a reverse primer SEQ ID NO. 2, and the sequence of the forward primer SEQ ID NO. 1 is: 5'-ACCTTTGCAGAGAGGCTTAAT-3', the sequence of the reverse primer SEQ ID NO. 2 is: 5'-CCTAGGCCCAAAGGAATGATAG-3' are provided.
Preferably, the EGFR gene amplification region marker TaqMan probe in step S2 comprises a probe SEQ ID NO. 3 capable of specifically hybridizing to the EGFR gene amplification region, wherein the sequence of SEQ ID NO. 3 is 5'-TGCTCTTAAAGGGATATCCTCTCCTGGT-3'.
Preferably, the probe SEQ ID NO of the EGFR gene amplification region is FAM as a fluorescent group marked at the 5 'end of 3 and BHQ1 as a fluorescence quenching group marked at the 3' end.
Preferably, the reference gene EFTUD2 forward and reverse amplification primers in step S2 include a specific primer pair for amplifying the reference gene, the specific primer pair comprises a forward primer SEQ ID NO. 4 and a reverse primer SEQ ID NO. 5, the sequence of the forward primer SEQ ID NO. 4 is 5'-GGTCTTGCCAGACACCAAAG-3', and the sequence of the reverse primer SEQ ID NO. 5 is 5'-TGAGAGGACACACGCAAAAC-3'.
Preferably, the TaqMan probe labeled with the internal reference gene EFTUD2 in step S2 comprises a probe SEQ ID NO. 6 capable of specifically hybridizing with the amplified region of the internal reference gene EFTUD2, and the sequence of SEQ ID NO. 6 is 5'-GGACATCCTTTGGCTTTTGA-3'.
Preferably, the probe SEQ ID NO of the amplified region of the reference gene EFTUD2 is VIC as a fluorescent group labeled at the 5 'end and QSY as a fluorescent quenching group labeled at the 3' end.
Preferably, the ddPCR amplification procedure in step S3 is: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds, annealing/extension at 57 ℃ for 1 minute, 45 cycles in total, and hold at 10 ℃.
The invention has the beneficial effects that:
1) compared with the traditional method, the detection sensitivity of the detection method based on the EGFR gene amplification of the digital PCR platform is obviously improved, and the minimum detection proportion can reach 0.01%;
2) the detection method based on the EGFR gene amplification of the digital PCR platform does not need to collect a tissue sample, can finish detection only by using peripheral blood, reduces the pain of patients, and is particularly suitable for patients intolerant to tissue sampling;
3) the detection method based on the EGFR gene amplification of the digital PCR platform is simple and convenient to operate, greatly reduces the clinical detection cost and workload, and has high application value. .
Drawings
FIG. 1 shows the specific amplification results of ddPCR of samples 1-1 according to the present invention;
FIG. 2 shows the specific amplification results of ddPCR of samples 1-2 according to the present invention.
Detailed Description
In order to facilitate understanding of those skilled in the art, the present invention will be further described with reference to the following examples and drawings, which are not intended to limit the present invention.
Example 1
1. Extraction of cfDNA:
the kit is used for extracting cfDNA in a whole blood sample, and the extraction steps are as follows:
1) add 300. mu.L of QIAGEN proteinase K to a 50mL centrifuge tube, add 3mL of plasma and 2.4mL of Buffer ACL, cap, and vortex for 30 s.
2) Incubating at 60 deg.C for 30 min; then taking out, adding 5.4mLBuffer ACB, covering the cover, whirling for 15-30s, and incubating the mixture on ice for 5 min.
3) Then vacuum pump extraction is carried out. The specific operation is described in QIAamp Circulating Nuleacid Kit instruction of QIAGEN company, and the extracted cfDNA is used as a template for PCR detection, wherein the content of the cfDNA template is 1.0 ng/. mu.L.
Example 2
2. Designing a primer:
designing primers aiming at an amplified region of the human EGFR gene, respectively designing a pair of specific PCR primers and a FAM-labeled hybridization probe aiming at the region for hybridizing an amplified product of the EGFR gene, simultaneously designing a pair of amplification primers according to a sequence of an internal reference gene EFTUD2, and labeling the hybridization probe by VIC. The designed PCR primer and probe sequences are shown in Table 1.
TABLE 1
| Primer name | Serial number | 5'-3' |
| EGFR-F | SEQ ID NO:1 | ACCTTTGCAGAGAGGCTTAAT |
| EGFR-R | SEQ ID NO:2 | CCTAGGCCCAAAGGAATGATAG |
| EGFR-P | SEQ ID NO:3 | TGCTCTTAAAGGGATATCCTCTCCTGGT |
| EFTUD2-F | SEQ ID NO:4 | GGTCTTGCCAGACACCAAAG |
| EFTUD2-R | SEQ ID NO:5 | TGAGAGGACACACGCAAAAC |
| EFTUD2-P | SEQ ID NO:6 | GGACATCCTTTGGCTTTTGA |
Example 3
Digital PCR amplification reaction:
amplifying a target fragment containing an EGFR gene amplification region by utilizing a specific PCR technology, preparing an amplification reaction according to the reaction method of the table 2, and amplifying according to the reaction program of the table 3 after the preparation of the reaction method is finished.
TABLE 2
| Reagent | Dosage of |
| 2×ddPCR Master Mix | 10μL |
| cfDNA template | 1μL |
| Each primer | 0.8μM |
| Each probe | 0.25μM |
| Supplying DEPC water to | 20μL |
TABLE 3
Example 4
And (3) reading a fluorescence signal of the PCR chip: and (3) collecting signals of the products after the PCR amplification reaction, and judging whether the EGFR gene amplification is contained in the sample to be detected according to the type of the fluorescent signals.
Example 5
Data analysis was performed using QuantaSoft (Bio-Rad) software, and copy numbers of two genes in the sample were calculated, and then copy number results were obtained by removing the copy number of the internal reference gene with the copy number of the EGFR gene. And if the ratio is less than 3, judging the sample to be EGFR negative, and if the ratio is more than 3, judging the sample to be EGFR positive.
Example 6
The analysis results of this example are shown in table 4, in which sample 1 and sample 2 of this example are respectively subjected to two times of repeated experiments, and are marked as samples 1-1 and 1-2 and samples 2-1 and 2-2; the copy number ratios of samples 1-1 and 1-2 were 1.34 and 1.25, respectively, indicating that sample 1 was EGFR negative; the copy number ratio of samples 2-1 and 2-2 is 6.80 and 7.10, respectively, indicating that sample 2 is positive for EGFR, and the specific amplification results of samples 1-1 and 1-2 are shown in FIG. 1 and FIG. 2, respectively.
TABLE 4
| Sample numbering | Copy number ratio |
| Sample 1-1 | 0.17 |
| |
0.18 |
| Sample 2-1 | 6.80 |
| Sample 2-2 | 7.10 |
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Sequence listing
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Claims (9)
1. A detection method for EGFR gene amplification based on a digital PCR platform is characterized in that: the method comprises the following steps:
s1, extraction of cfDNA: extracting cfDNA of a sample to be detected, wherein the sample to be detected is a whole blood sample;
s2, preparing a digital PCR reaction mixture: mixing a cfDNA template of a sample to be detected, forward and reverse amplification primers of an EGFR gene amplification area, a TaqMan probe of the EGFR gene amplification area, forward and reverse amplification primers of an internal reference gene EFTUD2, a TaqMan probe marked by an internal reference gene EFTUD2 and ddPCR reaction premix to prepare a digital PCR reaction mixture, wherein the content of the cfDNA template is 0.5-2 ng/mu L;
s3, loading the PCR reaction mixed solution obtained in the step S2 into a digital PCR chip for ddPCR amplification reaction;
s4, reading a fluorescent signal of the PCR chip;
and S5, analyzing data and counting results.
2. The method for detecting EGFR gene amplification based on digital PCR platform of claim 1, wherein: more preferably, the cfDNA template in the step S2 is contained in an amount of 0.8 to 1.2 ng/. mu.L.
3. The method for detecting EGFR gene amplification based on digital PCR platform of claim 1, wherein: the EGFR gene amplification region forward and reverse amplification primers in step S2 comprise a specific primer pair for amplifying the EGFR gene amplification region, the specific primer pair comprises a forward primer SEQ ID NO. 1 and a reverse primer SEQ ID NO. 2, and the sequence of the forward primer SEQ ID NO. 1 is as follows: 5'-ACCTTTGCAGAGAGGCTTAAT-3', the sequence of the reverse primer SEQ ID NO. 2 is: 5'-CCTAGGCCCAAAGGAATGATAG-3' are provided.
4. The method for detecting EGFR gene amplification based on digital PCR platform of claim 1, wherein: the EGFR gene amplification region marker TaqMan probe in the step S2 comprises a probe SEQ ID NO. 3 capable of specifically hybridizing the EGFR gene amplification region, wherein the sequence of the SEQ ID NO. 3 is 5'-TGCTCTTAAAGGGATATCCTCTCCTGGT-3'.
5. The method for detecting EGFR gene amplification based on digital PCR platform of claim 4, wherein: 3, the fluorescent group marked at the 5 'end of the probe SEQ ID NO of the EGFR gene amplification region is FAM, and the fluorescent quenching group marked at the 3' end is BHQ 1.
6. The method for detecting EGFR gene amplification based on digital PCR platform of claim 1, wherein: the internal reference gene EFTUD2 forward and reverse amplification primers in the step S2 comprise a specific primer pair for amplifying the internal reference gene, wherein the specific primer pair comprises a forward primer SEQ ID NO. 4 and a reverse primer SEQ ID NO. 5, the sequence of the forward primer SEQ ID NO. 4 is 5'-GGTCTTGCCAGACACCAAAG-3', and the sequence of the reverse primer SEQ ID NO. 5 is 5'-TGAGAGGACACACGCAAAAC-3'.
7. The method for detecting EGFR gene amplification based on digital PCR platform of claim 1, wherein: the TaqMan probe labeled by the internal reference gene EFTUD2 in the step S2 comprises a probe SEQ ID NO. 6 capable of specifically hybridizing with the amplification region of the internal reference gene EFTUD2, and the sequence of the SEQ ID NO. 6 is 5'-GGACATCCTTTGGCTTTTGA-3'.
8. The method for detecting EGFR gene amplification based on digital PCR platform of claim 7, wherein: 6, the 5 'end marked fluorescent group is VIC, and the 3' end marked fluorescent quenching group is QSY.
9. The method for detecting EGFR gene amplification based on digital PCR platform of claim 1, wherein: the ddPCR amplification procedure in step S3 is: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds, annealing/extension at 57 ℃ for 1 minute, 45 cycles in total, and hold at 10 ℃.
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