CN111206099A - Kit for detecting L858R site mutation of EGFR gene - Google Patents
Kit for detecting L858R site mutation of EGFR gene Download PDFInfo
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Abstract
The invention discloses a kit for detecting mutation of an L858R site of an EGFR gene, and relates to the technical field of nucleic acid detection. The kit comprises: a primer pair for detecting L858R site mutation, a mutant fluorescent probe, a wild fluorescent probe, a positive quality control substance, a negative quality control substance and a reaction premix. The kit designs a primer and a locked nucleic acid probe with a specific sequence aiming at the L858R locus of the EGFR gene, optimizes a reaction system, realizes high-sensitivity detection on the L858R locus mutation of the EGFR gene by using a digital PCR platform, can effectively distinguish the mutation as low as 1:3000 on a low-concentration DNA sample, has the advantages of simple operation, stable result, high accuracy and good data amplification effect, and can be widely applied to early screening of the EGFR mutation type of the non-small cell lung cancer and drug resistance monitoring in the treatment process.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a kit for detecting mutation of an L858R locus of an EGFR gene.
Background
In recent years, there are up to 429 ten thousand new cases of cancer each year, among which the lung cancer has the first prevalence and mortality rate accounting for 13% and 18% of the new cases of malignant tumor, respectively, and despite the increasing diagnostic methods and therapeutic approaches for lung cancer, the mortality rate of lung cancer has not been effectively controlled. Among all lung cancers, Non-Small Cell lung cancer (NSCLC) accounts for over 80%, and the most common pathogenic mutation in Non-Small Cell lung cancer patients in chinese population comes from Epidermal Growth Factor Receptor (EGFR).
Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein, widely distributed on cell membranes of various tissues of the human body, and is one of members of the tyrosine family of transmembrane receptors. Consists of three parts, namely an extracellular ligand binding region, a hydrophobic transmembrane domain and an intracellular kinase region. The EGFR gene can regulate apoptosis, proliferation, differentiation, migration and cell cycle circulation of cells, accelerate proliferation of tumor cells, promote angiogenesis, accelerate tumor metastasis and inhibit apoptosis of tumor cells. EGFR gene mutation mainly occurs on the 19 th to 21 st exons, and comprises deletion mutation (19del) of the 19 th exon, point mutation (T790M) and fragment insertion of the 20 th exon, and point mutation (such as L858R, L861Q and the like) of the 21 st exon. In the treatment of tumors, Tyrosine Kinase Inhibitor (TKI) blocks EGFR signal pathway by competing with EGFR extracellular ligand binding site, thereby playing an anti-tumor role. However, a significant proportion of patients develop resistance, and this acquired resistance is primarily caused by secondary mutations in the gene, with common mutations including: T790M and L858R.
In recent years, a liquid biopsy technique based on blood or the like has been rapidly developed. When tumor tissues are difficult to obtain, noninvasive and easily-collected blood detection is a proper substitute for tissue EGFR gene mutation analysis, can effectively overcome tumor heterogeneity to a certain extent, can realize noninvasive real-time dynamic detection and the like, and is widely applied to drug resistance monitoring in early screening and treatment of lung cancer. Because the content of free DNA in blood is extremely low and the length is about 70-200bp, the content of mutation-related free DNA (cfDNA) is less and accounts for about 1% of the total free DNA content, and the precision and sensitivity of the common kit can not meet the detection requirements.
Currently, there are many methods for detecting EGFR mutation in peripheral blood, including sequencing, real-time fluorescence quantitative PCR (RT-PCR), Denaturing High Performance Liquid Chromatography (DHPLC), digital PCR (dpcr), and the like. The sequencing method is the most basic and widely applied method for detecting gene mutation at present, but the amplification, purification and sequence analysis of a sample to be sequenced are required, the process is complicated, the time consumption is long, and the sensitivity is low, so that certain limitation exists in clinical application, and the method is not suitable for analyzing a large number of clinical samples. Although the RT-PCR is used for detecting the target gene, the micro mutation can be detected, but the quantitative detection of the technology depends on the Ct value, and the Ct value is influenced by the amplification efficiency, so that the accurate quantitative detection is limited to a certain extent, and the obtained relative content cannot realize absolute quantification. The detection sensitivity of the DHPLC technology is also low, the specific type of mutation cannot be detected, the result interpretation is easy to make mistakes, when a plurality of fragments need to be detected, due to the fact that a plurality of melting temperatures exist, multi-step detection is needed, and the workload is increased. The digital PCR technology does not need to adopt reference genes and standard curves, has high accuracy and good reproducibility, can realize absolute quantitative analysis, and provides possibility for effectively improving the sensitivity and the accuracy of gene mutation detection. The technology divides a sample into dozens to tens of thousands of parts and distributes the parts to different reaction units. And calculating the concentration of the nucleic acid target molecules through Poisson distribution according to the relative proportion of the amplified fluorescence signals in each reaction unit and the volume of the reaction unit. The digital PCR technology greatly improves the detection sensitivity and the detection rate. However, the currently used digital PCR instrument has many and complex operation steps, and due to the relative loss of liquid drops, the titration type digital PCR is inaccurate in detection of ultra-low content target objects and needs an additional quality control system; chip-type digital PCR also needs a plurality of instrument combinations, is expensive and inconvenient to operate, and cannot meet the requirements of convenient clinical use.
The provision of corresponding detection reagents based on digital PCR systems is becoming a necessary trend for detecting gene mutations today. Chinese patent CN108998526A discloses a kit and a method for detecting EGFR gene 21 exon gene mutation, and the kit can detect 0.1% mutation rate based on a dPCR platform. Chinese patent CN108841953A discloses a kit for detecting 22 mutations of EGFR gene by using a digital PCR technology, and the kit can detect a DNA sample of 2 ng/. mu.L. The existing reagent for detecting EGFR gene mutation based on digital PCR has few types and still has some defects, including difficulty in effectively detecting samples with extremely low DNA content, low sensitivity and the like.
Therefore, the invention develops a kit for detecting the L858R site mutation of the EGFR gene based on digital PCR. The L858R mutation of EGFR gene in tumor, blood, saliva and serum is rapidly detected, so that absolute quantification is provided. The method is more convenient to operate, can accurately perform qualitative and quantitative detection on the extremely-low detection target object, has higher sensitivity and stable and reliable data result, and can be more conveniently used for auxiliary diagnosis and clinical treatment guidance.
Disclosure of Invention
The invention aims to provide a kit for detecting mutation of an L858R site of an EGFR gene. The method overcomes the problems in the prior art, can accurately perform qualitative and quantitative detection on the extremely low detection target object, and improves the sensitivity.
The invention provides a kit for detecting mutation of an EGFR gene L858R site, which comprises a primer pair A and a fluorescent probe B;
the primer pair A comprises an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is as follows: 5'-CACCGCAGCATGTCAAGATCA-3', respectively; as shown in SEQ ID NO. 1;
the nucleotide sequence of the downstream primer is as follows: 5'-CTTTGCCTCCTTCTGCATGGTAT-3', respectively; as shown in SEQ ID NO. 2;
the fluorescent probe B comprises a mutant fluorescent probe and a wild fluorescent probe;
the nucleotide sequence of the mutant fluorescent probe is as follows: 5'-TTGGGCGGGCCAAAC-3', respectively; as shown in SEQ ID NO. 3;
the nucleotide sequence of the wild type fluorescent probe is as follows: 5'-TTGGGCTGGCCAAAC-3', respectively; as shown in SEQ ID NO. 4;
furthermore, the 7 th base at the 5' end of the mutant fluorescent probe and the wild fluorescent probe is a base modified by a locked nucleic acid.
Further, the structure of the locked nucleic acid is shown as the formula (1):
preferably, the 5 'end of the fluorescent probe B contains a fluorescent reporter group FAM and/or CY3, and the 3' end contains a fluorescent quencher group BHQ-1 and/or BHQ-2;
further preferably, the 5 'end of the mutant fluorescent probe contains a fluorescence reporter group FAM, and the 3' end of the mutant fluorescent probe contains a fluorescence quencher group BHQ-1; the 5 'end of the wild type fluorescent probe contains a fluorescent reporter group CY3, and the 3' end of the wild type fluorescent probe contains a fluorescent quenching group BHQ-2.
Preferably, the kit also comprises a positive quality control substance and a negative quality control substance; the positive quality control product contains 1% L858R mutant DNA, and the negative quality control product contains EGFR gene wild type DNA.
Preferably, the kit further comprises a reaction premix, wherein the reaction premix comprises, but is not limited to, the following reagents: dATP, dCTP, dGTP, dTTP, magnesium ions, BSA (bovine serum albumin), hot start Taq enzyme, single molecule amplification enhancer, etc.;
the single-molecule amplification enhancer comprises 2M betaine, 0.2% TritonX-100 by volume and 0.1U thermostable pyrophosphatase.
Further preferably, the reaction premix comprises dATP, dCTP, dGTP and dTTP at a concentration of 0.4mM, BSA at a concentration of 10. mu.g/. mu.L and hot start Taq enzyme at a concentration of 1U.
The sample detectable by the kit of the invention has various sources, including nucleic acid in tumor, blood, saliva, serum and the like.
The invention also provides application of the kit in preparation of products for detecting whether the EGFR gene of a lung cancer patient has L858R site mutation.
The invention also provides application of the kit in preparation of products for detecting whether the lung cancer patients have drug resistance after taking EGFR-TKI.
The invention also provides a method for detecting the mutation of the L858R site of the EGFR gene by using the kit, which comprises the following steps:
(1) mixing a DNA template to be detected, a primer pair A and a fluorescent probe B in the kit with the reaction premix to prepare a digital PCR reaction system;
the PCR reaction system is as follows:
a: the final concentration of the DNA template is 0.5-1.5 ng/. mu.L, preferably 1 ng/. mu.L;
b: the final concentration of the primer pair A is 0.2-0.6 mu M, and preferably 0.4 mu M;
c: the final concentration of the fluorescent probe B is 0.1-0.4 mu M, and preferably 0.2 mu M;
(2) manufacturing a PCR micro-reaction unit according to the digital PCR reaction system obtained in the step (1), and carrying out PCR amplification reaction to obtain an amplification product;
the amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, extension at 60 ℃ for 30s, and 40 cycles in total; terminating the reaction at 10 ℃;
(3) and (3) collecting fluorescent signals of the amplification products obtained in the step (2), and judging whether the DNA template containing EGFR gene L858R site mutation in the sample to be detected and the quantity and content of the DNA template are contained according to the type of the fluorescent signals.
Specifically, the method for detecting the mutation of the L858R site of the EGFR gene by using the kit comprises the following steps:
(1) mixing a DNA template to be detected, a primer pair A and a fluorescent probe B in the kit with the reaction premix to prepare a digital PCR reaction system;
two DNA fragments with the length of 200bp, which respectively contain the wild type and the mutant type of the mutation site, are artificially synthesized and transferred into a vector plasmid to prepare a DNA template. Double-stranded DNA quantification was performed with a Qubit and the true copy number was calculated according to the following formula:
(6.02×1023)×(ng/μL×10-9) (DNA length. times.660) ═ copies/. mu.L, template DNA concentration 10 ng/. mu.L;
the PCR reaction system is as follows:
the PCR reaction system is as follows:
a: the final concentration of the DNA template is 0.5-1.5 ng/. mu.L, preferably 1 ng/. mu.L;
b: the final concentration of the primer pair A is 0.2-0.6 mu M, and preferably 0.4 mu M;
c: the final concentration of the fluorescent probe B is 0.1-0.4 mu M, and preferably 0.2 mu M;
(2) manufacturing a PCR micro-reaction unit according to the digital PCR reaction system obtained in the step (1), and carrying out PCR amplification reaction to obtain an amplification product;
the amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, extension at 60 ℃ for 30s, and 40 cycles in total; terminating the reaction at 10 ℃;
(3) and (3) collecting fluorescent signals of the amplification products obtained in the step (2), and judging whether the DNA template containing EGFR gene L858R site mutation in the sample to be detected and the quantity and content of the DNA template are contained according to the type of the fluorescent signals.
Compared with the prior art, the invention has the advantages that:
the kit for detecting the mutation of the L858R site of the EGFR gene provided by the invention designs a primer with a specific sequence and a locked nucleic acid probe aiming at the L858R site of the EGFR gene, and optimizes a reaction system of the kit. The method can effectively detect the low-concentration sample, has high sensitivity, can effectively distinguish the mutation as low as 1:3000, can smoothly complete the technical requirement of liquid biopsy, has simple operation, reduces the manual operation interference, and has stable result, high accuracy and good data amplification effect; in addition, the detectable sample sources are various, including nucleic acid in tumor, blood, saliva and serum, the application range of the kit, the reaction system and the method is expanded, and the kit can be widely applied to early screening of EGFR mutant type non-small cell lung cancer and drug resistance monitoring in the treatment process.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The digital PCR instrument used in the present invention is the nucleic acid amplification instrument of patent CN 201911061352.7. The related sequences in the invention are detailed in a sequence table.
The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, of J. SammBruker et al, Huang Petang et al) or according to product instructions. The various chemicals used were obtained by conventional commercial routes unless otherwise specified.
Example 1: design and synthesis of primer pair and probe for detecting mutation of L858R site of EGFR gene
Designing and synthesizing a primer pair A and a fluorescent probe B for detecting the mutation of the L858R site of the EGFR gene based on the digital PCR technology according to the 858 th mutation of the exon EGFR 21 (namely, the L858R mutation, c2573T > G); the specific sequence is as follows:
the primer pair A comprises an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is as follows: 5'-CACCGCAGCATGTCAAGATCA-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-CTTTGCCTCCTTCTGCATGGTAT-3'
The fluorescent probe B comprises a mutant fluorescent probe and a wild fluorescent probe;
the nucleotide sequence of the mutant fluorescent probe is as follows: 5'-TTGGGCGGGCCAAAC-3', respectively;
the nucleotide sequence of the wild-type fluorescent probe is as follows: 5'-TTGGGCTGGCCAAAC-3', respectively;
and the 7 th base at the 5' end of the mutant fluorescent probe and the wild fluorescent probe is a base modified by locked nucleic acid.
Primers and probes were synthesized by Shanghai Biotech and purified using HPLC grade. The 5 'end of the mutant fluorescent probe contains a fluorescence reporter group FAM, and the 3' end of the mutant fluorescent probe contains a fluorescence quenching group BHQ-1; the 5 'end of the wild type fluorescent probe contains a fluorescent reporter group CY3, and the 3' end of the wild type fluorescent probe contains a fluorescent quenching group BHQ-2. The fluorescent reporter group and the quencher group can be reasonably selected according to a specific platform.
Example 2: kit for detecting L858R site mutation of EGFR gene
The kit provided by the invention comprises: the primer pair A, the fluorescent probe B, the positive quality control material, the negative quality control material and the reaction premix solution provided in the embodiment 1.
The preparation method of the positive quality control product comprises the following steps: two DNA fragments with a length of 200bp, each containing a wild type and a mutant type of a mutation site, were artificially synthesized and loaded into a plasmid vector pET-23d (+) (Promega), respectively. Quantification was performed using Qubit 3.0, and the copy number concentrations of the two types of plasmids were calculated, based on the copy number ratio for the mutants: two plasmids (DNA containing 1% L858R mutation) were mixed at 1:100 wild type, and then the plasmid mixture was broken into fragments of about 180bp by sonication and quantified to 10 ng/. mu.l as a positive quality control.
The negative quality control product is composed of the plasmid containing the wild type (namely only DNA of the wild type of the EGFR gene) alone, and then the plasmid is broken into fragments of about 180bp by the same method, and the fragments are quantified to 10 ng/. mu.L to be used as the negative quality control product.
The reaction premix comprises: dATP, dCTP, dGTP, dTTP, magnesium ions, BSA (bovine serum albumin), hot start Taq enzyme, single molecule amplification enhancer; the single-molecule amplification enhancer comprises 2M betaine, 0.2% TritonX-100 by volume and 0.1U thermostable pyrophosphatase.
Wherein the concentrations of dATP, dCTP, dGTP and dTTP are all 0.4mM, the concentration of BSA is 10. mu.g/. mu.L, and the concentration of hot start Taq enzyme is 1U.
Example 3: method for detecting mutation of L858R site of EGFR gene
Using the kit described in example 2, a PCR reaction system was set up as shown in Table 1, according to ddH2And O, reaction premix, a probe, a primer and template DNA, adding the samples into a PCR tube according to the reaction system in the table 1, uniformly mixing the mixed system for 15s by using soft vortex, and collecting the solution to the bottom of the tube by short-time centrifugation. And loading the prepared reaction system on a PCR chip to form a micro-reaction unit. The chip is placed in a digital PCR instrument, PCR reaction is carried out according to the PCR reaction conditions in the table 2, and FAM and CY3 are selected as channels for fluorescence detection.
Table 1: reaction system
Table 2: PCR reaction conditions
After the amplification is finished, the effective fluorescence positive points of the two channels are interpreted through computer analysis, and the result is analyzed.
Example 4: detection of standards
The preparation method of the standard substance containing the mutant gene comprises the following steps: two DNA fragments with a length of 200bp, each containing a wild type and a mutant type of a mutation site, were artificially synthesized and loaded into a plasmid vector pET-23d (+) (Promega), respectively. Quantification was performed using the Qubit 3.0 and the copy number concentrations of both types of plasmids were calculated. Mutation types were exemplified by copy number ratios: mixing wild type ratios of 1:10, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000 and 1:3000 (i.e. mutation rates of 10%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05% and 0.033%), and breaking the resulting mixed plasmid into 180bp fragmented DNA close to ctDNA fragments; the preparation method of the Wild type standard (control group, Wild type) comprises the following steps: breaking the DNA plasmid of the wild type of the EGFR gene L858R into 180bp of fragmented DNA close to the ctDNA fragment; no template sample control (NTC).
8 mutant standards, 1 wild-type standard control and no template sample control (NTC) were obtained as described above. The test was carried out by the method described in example 3, and the test was repeated 3 times. The results are shown in Table 3.
Table 3: test results of the standards
As can be seen from Table 3, the kit provided by the invention can effectively detect a sample with a final DNA concentration of 1 ng/. mu.L, can effectively distinguish a mutation standard product as low as 1:3000, can detect the mutation of the L858R site of the EGFR gene with the lowest mutation rate of 0.033%, can smoothly complete the technical requirements of liquid biopsy, and can be widely applied to early screening of EGFR mutants of non-small cell lung cancer and drug resistance detection in the treatment process.
Example 5: effect of template DNA Final concentration on Standard detection
The standard substance in example 4 was detected using the kit in example 2, and the detection method was different from the method described in example 3 only in that the final concentration of the template DNA in the reaction system was 0.5 ng/. mu.L or 1.5 ng/. mu.L.
And (3) detection results: when the final concentration of the template DNA is 0.5 ng/. mu.L and 1.5 ng/. mu.L, the mutant standard substance as low as 1:3000 can be effectively distinguished.
Example 6: influence of final concentrations of upstream and downstream primers on detection of standard substance
The standard substance in example 4 was detected using the kit in example 2, and the detection method was different from the method described in example 3 only in that the final concentration of the upstream and downstream primers in the reaction system was 0.2. mu.M or 0.6. mu.M. The results of the measurements are shown in tables 4 and 5 (only the results of the low concentration measurements are shown here).
Table 4: detection result of primer concentration 0.2. mu.M
Table 5: detection result of primer concentration of 0.6. mu.M
The results show that: when the final concentration of the upstream primer and the downstream primer is 0.2 mu M or 0.6 mu M, the mutant standard substance with the ratio as low as 1:3000 can be effectively distinguished.
Example 7: effect of Final concentration of Probe on Standard detection
The standard substance in example 4 was detected using the kit in example 2, and the detection method was different from the method described in example 3 only in that the final concentration of the mutant-type probe and the wild-type probe in the reaction system was 0.1. mu.M or 0.4. mu.M. The results of the measurements are shown in tables 6 and 7 (only the results of the low concentration measurements are shown here).
Table 6: detection result of 0.1. mu.M probe concentration
Table 7: detection result of 0.4. mu.M probe concentration
The results show that: when the final concentration of the mutant probe and the wild probe is 0.1 mu M or 0.4 mu M, the mutant standard substance with the ratio as low as 1:3000 can be effectively distinguished.
Example 8: sample testing of non-small cell lung cancer patients
The ctDNA in the blood sample was detected by using the kit of example 2 and the method of example 3, and the sampling amount was 10 mL. Samples were derived from 6 clinically confirmed non-small cell lung cancer patients and treated with EGFR-TKI for 6-7 months of blood. The sample was tested by the method of the present invention, and a part of the blood (about 9 mL) was sent to a professional testing company for testing by the NGS method. The NGS method detects the gold standard of the detection item at present, and can directly compare the result with the method of the invention.
The extraction method of ctDNA in the sample comprises the following steps: 1mL of blood sample is sucked from a blood collection tube, ctDNA in the sample is extracted by using a ctDNA/RNA extraction kit (purchased from Aline Biosciences, Inc., the product number is CFD-6001-50), and high-quality circulating ctDNA can be extracted by carrying out DNA combination, washing, selection and elution on ctDNA fragments after the sample is digested by protease based on a magnetic bead method. The total amount of ctDNA is determined to be not less than 20ng after quality inspection and quantification by the Qubit, the kit in the embodiment 2 is adopted to carry out detection according to the method in the embodiment 3, and the detection results are shown in Table 8: the mutation rate of the No. 1 sample is 4.5%, the mutation rate of the No. 2 sample is 0.9%, and the mutation rate of the No. 5 sample is 1.1% in 3 out of 6 samples. No mutations were detected in the remaining 3 samples.
Table 8: test results of 6 clinical specimens
| Sample numbering | 1 | 2 | 3 | 4 | 5 | 6 |
| Mutation Rate test results (%) | 4.5 | 0.9 | 0 | 0 | 1.1 | 0 |
The above results are consistent with NGS detection results. The experimental result shows that the kit provided by the invention can meet the requirement of clinical liquid biopsy, and greatly reduces the detection time and cost compared with the detection method of NGS.
The inventor of the application also carries out detection of EGFR gene L858R site mutation on DNA from serum, plasma, peripheral blood, pleural effusion, body fluid or tissue, the detection limit can reach 1:3000, and the repeatability is good.
Comparative example 1:
the standard substance in example 4 was detected using the kit in example 2, and the detection method was different from the method described in example 3 only in that the final concentration of the upstream and downstream primers in the reaction system was 0.1. mu.M.
And (3) detection results: when the final concentration of the upstream and downstream primers in the reaction system is 0.1 mu M, the fluorescence intensity of the positive hole is analyzed in real time, and the cycle number that the fluorescence intensity of the positive hole reaches the detectable lower limit is obviously increased, the number of positive points of two channels is also reduced, so that the reaction efficiency is obviously reduced, the final quantitative result is influenced, and the mutation standard product as low as 1:1000 can be effectively distinguished.
Comparative example 2:
the standard substance in example 4 was detected using the kit in example 2, and the detection method was different from the method described in example 3 only in that the final concentration of the mutant-type probe and the wild-type probe in the reaction system was 0.05. mu.M.
And (3) detection results: when the final concentration of the mutation type probe and the wild type probe in the reaction system is 0.05 mu M, the fluorescence intensity of the positive hole is analyzed in real time, and the cycle number that the fluorescence intensity of the positive hole reaches the detectable lower limit is obviously increased, the fluorescence intensity of the positive hole does not reach the plateau stage when the reaction is completed, the number of positive points of two channels is also reduced, the condition that the number of the probes in the system is insufficient, the result detection is influenced, and the mutation standard substance as low as 1:500 can be effectively distinguished.
Comparative example 3: kit for detecting L858R site mutation of EGFR gene
This kit differs from the kit described in example 2 only in that the mutant fluorescent probe and the wild-type fluorescent probe are not modified by locked nucleic acid. The kit is used for detecting the standard substance in the example 4, and the detection method is the method described in the example 3.
And (3) detection results: when the final concentration of the mutation type probe and the wild type probe in the reaction system is 0.05 mu M, the fluorescence intensity of the positive hole is analyzed in real time, and the cycle number that the fluorescence intensity of the positive hole reaches the detectable lower limit is obviously increased, the number of positive points of two channels is also reduced, so that the reaction efficiency is obviously reduced, the final quantitative result is influenced, and the mutation standard substance as low as 1:500 can be effectively distinguished.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Ningbo offspring Rui biomedical instruments, Limited liability company
<120> kit for detecting L858R site mutation of EGFR gene
<130>2020
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<170>SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial sequence)
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caccgcagca tgtcaagatc a 21
<210>2
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<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>2
ctttgcctcc ttctgcatgg tat 23
<210>3
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<212>DNA
<213> Artificial sequence (Artificial sequence)
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ttgggcgggc caaac 15
<210>4
<211>15
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<213> Artificial sequence (artificalsequence)
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Claims (10)
1. A kit for detecting mutation of an L858R site of an EGFR gene is characterized by comprising a primer pair A and a fluorescent probe B;
the primer pair A comprises an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is as follows: 5'-CACCGCAGCATGTCAAGATCA-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-CTTTGCCTCCTTCTGCATGGTAT-3', respectively;
the fluorescent probe B comprises a mutant fluorescent probe and a wild fluorescent probe;
the nucleotide sequence of the mutant fluorescent probe is as follows: 5'-TTGGGCGGGCCAAAC-3', respectively;
the nucleotide sequence of the wild type fluorescent probe is as follows: 5'-TTGGGCTGGCCAAAC-3', respectively;
and the 7 th base at the 5' end of the mutant fluorescent probe and the wild fluorescent probe is a base modified by locked nucleic acid.
2. The kit according to claim 1, wherein the fluorescent probe B comprises a fluorescent reporter group FAM and/or CY3 at the 5 'end and a fluorescent quencher group BHQ-1 and/or BHQ-2 at the 3' end.
3. The kit according to claim 1, wherein the 5 'end of the mutant fluorescent probe comprises a fluorescence reporter group FAM, and the 3' end of the mutant fluorescent probe comprises a fluorescence quencher group BHQ-1;
the 5 'end of the wild type fluorescent probe contains a fluorescent reporter group CY3, and the 3' end of the wild type fluorescent probe contains a fluorescent quenching group BHQ-2.
4. The kit of claim 1, wherein the kit further comprises a positive quality control material and a negative quality control material;
the positive quality control product contains 1% L858R mutant DNA, and the negative quality control product contains EGFR gene wild type DNA.
5. The kit of claim 1, further comprising a reaction premix comprising: dATP, dCTP, dGTP, dTTP, magnesium ions, BSA, hot start Taq enzyme and a single molecule amplification enhancer;
the single-molecule amplification enhancer comprises 2M betaine, 0.2% TritonX-100 by volume and 0.1U thermostable pyrophosphatase.
6. The kit of claim 5, wherein the reaction premix comprises dATP, dCTP, dGTP and dTTP each at a concentration of 0.4mM, BSA at a concentration of 10. mu.g/. mu.L and hot start Taq enzyme at a concentration of 1U.
7. Use of a kit according to any one of claims 1 to 6 for the preparation of a product for detecting the occurrence of a mutation at the L858R site in the EGFR gene of a patient with lung cancer.
8. Use of a kit according to any one of claims 1 to 6 in the manufacture of a product for detecting the occurrence of drug resistance of an EGFR-TKI administered to a patient with lung cancer.
9. A method for detecting mutation at the L858R site of EGFR gene using the kit of any of claims 1-6, wherein the method comprises the following steps:
(1) preparing a digital PCR reaction system: mixing a DNA template to be detected with the primer pair A, the fluorescent probe B and the reaction premix in the kit of any one of claims 1 to 6 to obtain a digital PCR reaction system;
the PCR reaction system is as follows:
a: the final concentration of the DNA template is 0.5-1.5 ng/. mu.L;
b: the final concentration of the primer pair A is 0.2-0.6 mu M;
c: the final concentration of the fluorescent probe B is 0.1-0.4 mu M;
(2) manufacturing a PCR micro-reaction unit according to the digital PCR reaction system obtained in the step (1), and carrying out PCR amplification reaction to obtain an amplification product;
the amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, extension at 60 ℃ for 30s, and 40 cycles in total; terminating the reaction at 10 ℃;
(3) and (3) collecting fluorescent signals of the amplification products obtained in the step (2), and judging whether the DNA template containing EGFR gene L858R site mutation in the sample to be detected and the quantity and content of the DNA template are contained according to the type of the fluorescent signals.
10. The method of claim 9, wherein the PCR reaction system of step (1) is as follows:
a: the final concentration of the DNA template is 1.0 ng/. mu.L;
b: the final concentration of primer pair A was 0.4. mu.M;
c: the final concentration of fluorescent probe B was 0.2. mu.M.
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| CN119662788A (en) * | 2024-12-30 | 2025-03-21 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | A crRNA for detecting EGFR gene L858R single base mutation and a kit using the crRNA |
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