CN107419018B - Method and kit for detecting gene mutation based on Blocker primer and ARMS primer - Google Patents
Method and kit for detecting gene mutation based on Blocker primer and ARMS primer Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting gene mutation based on a Blocker primer and an ARMS primer. In the invention, a corresponding Blocker primer is designed according to the annealing temperature, and under the annealing temperature of the Blocker, the Blocker primer is combined with a wild-type template to block the amplification of a wild type, and the amplification of a wild-type background is reduced by using an ARMS technology. According to the invention, a Blocker enrichment technology and an ARMS fluorescent quantitative PCR technology are integrated in a reaction system, so that the enrichment and identification of the mutant gene can be realized in one step, the operation steps are reduced, and the detection sensitivity and specificity are improved.
Description
The application is a divisional application filed on 16/2/2012, and has an application number 201210035665.7 and an invention name of 'a method and a kit for detecting gene mutation based on a Blocker primer and an ARMS primer'.
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method and a kit for detecting gene mutation.
Background
Gene mutation (gene mutation) is a change in the structure of a gene due to the addition, deletion or alteration of base pairs in a DNA molecule. The gene mutations are classified into two types, sexual cell mutation and somatic cell mutation. Sex cell mutation refers to a mutation that occurs in a sex cell and is a heritable type of mutation. Mutations in somatic cells other than sex cells do not cause genetic changes in the offspring, but may cause changes in the genetic structure of certain cells of the present generation. Most somatic mutations have no phenotypic effect. Somatic mutations are rare mutations, which are present in large amounts in wild-type background DNA sequences and are present in small amounts relative to the amount of wild-type background sequence. For example, the tissues and peripheral blood of tumor patients contain a small amount of tumor cell DNA, and the early occurrence of bacterial and viral drug resistance. Somatic mutation is often associated with the onset of disease, and can be used as a marker for disease onset, a main marker for prognosis judgment, and a marker for medication guidance. Therefore, the detection of somatic mutation is of great significance for diagnosis and prognosis evaluation of diseases.
Lung cancer is a common malignant tumor in all countries of the world today and has become the leading cause of cancer death in most countries. Among them, non-small cell lung cancer (NSCLC) is the most common. Currently, targeted therapy has become an important means for clinical treatment of non-small cell lung cancer. Iressa/Gefitinib/Gefitinib, Aslicon and Tarceva (Tarceva/Erlotinib, Roche pharmaceuticals) are the main drugs approved by the FDA in the United states for the targeted treatment of NSCLC as Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKI). However, clinical trials show that iressa and tarceva have significant efficacy in only 10-30% of NSCLC patients. Further research shows that EGFR gene mutation L858R and 19 exon deletion (E746_ A750) has correlation with the curative effect of NSCLC targeted therapy, and most of patients carrying EGFR gene mutation L858R and 19 exon deletion (E746_ A750) have remarkable curative effect. However, almost all patients develop EGFR-TKI resistance sooner or later, and the median progression-free time is only 6-12 months. Whereas EGFR-TKI resistance presents a distinct molecular target, T790M. The EGFR wild type gene sequence is shown in Genbank No. NM-005228.3, and the T790M mutation is that the 2369 th base C in the sequence is mutated into T. The tumor treatment scheme most suitable for NSCLC patients is worked out by combining the detection information of EGFR gene mutation, thereby providing a scientific basis for medication of clinicians, and reducing the treatment risk of doctors and the economic burden of patients.
The detection method of the target precursor cell mutation mainly comprises the technical methods of a DNA sequencing method, mutant enrichment PCR and the like. DNA sequencing is a reliable method for mutation detection and is the most used method. The sequencing method has higher requirements on material obtaining and technology, and more importantly, due to the limitation of the sequencing method, the sensitivity is not high, and only mutant genes with the content of more than 20 percent can be detected. Compared with a sequencing method, the mutant enrichment PCR has two PCR amplifications, so that the sensitivity and the specificity of mutation detection are higher, and one mutant gene can be detected from 104-105 wild-type copies. The main limiting factors of the method are selection of suitable restriction enzyme, complicated operation, long time consumption and easy pollution of two PCR.
Probe Amplification Retardation Mutation System (ARMS) ARMS is also known AS allele specific PCR (allele specific PCR, AS-PCR). The principle is as follows: based on the principle that Taq DNA polymerase lacks 3 ' -5 ' exonuclease activity and the 3 ' end base of PCR primer must be complementary to template DNA for effective amplification, proper primer is designed to detect mutant gene. The method can be at 103～104One mutant gene is detected in each wild type copy, and the detection sensitivity is high. The main limiting factor of this method is that if the site of mutation is a weakly mismatched base sequence, the specificity of the method will be affected.
The detection of mutation sites based on the Blocker technology has been reported in the literature. The Blocker primer is a nucleic acid sequence that is perfectly matched and complementary to the wild type. The existing method for using the Blocker primer mainly comprises the following two methods, one method is that the Blocker primer and an amplification primer are not overlapped, the Blocker primer is positioned in a mutation site region, and the mutation primer blocks wild type DNA and does not block a mutant template, so that the mutant template is amplified; the other is that the Blocker primer and the amplification primer are overlapped, and the Blocker primer is used for blocking the wild type DNA so as to reduce the amplification of the wild type template DNA. The method can effectively reduce the amplification of the wild type template and improve the detection capability of the mutant type DNA template in the wild type background. However, this method has a problem that, in the blocking process of the Blocker primer, Blocker cannot effectively distinguish the mutant template from the wild-type template, and although there is a difference of one base (with respect to the mutation site), the Blocker primer can be bonded to the mutant template at the Tm (transition temperature) of the primer, or the primer can be bonded to the wild-type template, thereby reducing the efficiency of the wild-type blocking and the efficiency of the mutant detection. Therefore, it is an urgent need in clinical practice to provide a simple, rapid, sensitive and highly specific detection method for mutation sites.
Therefore, to further improve the blocking efficiency of the Blocker primer in ARMS + Blocker technology, we designed the Blocker primer annealing temperature to be higher than the ARMS primer annealing temperature. Thus, the Blocker primer preferentially binds to the wild-type template at the annealing temperature of the Blocker primer, and since the Blocker primer differs from the mutant template by one base, the annealing temperature also differs, under these conditions, the Blocker primer preferentially binds to the wild-type, but not to the mutant, and the amplification primer does not bind to the wild-type. Under the annealing temperature condition of the amplification primer, the amplification primer is combined with the mutant template. The method can effectively reduce the amplification of the wild background and improve the detection efficiency of the mutant.
Disclosure of Invention
The invention aims to provide a method for detecting gene mutation based on a Blocker primer and an ARMS primer, which integrates a Blocker technology and an ARMS fluorescent quantitative PCR technology. The types of mutations that can be detected by the present invention mainly include: point mutation, deletion mutation, insertion mutation and the like.
Another object of the present invention is to provide a kit for detecting gene mutation. The types of mutations that can be detected by the present invention mainly include: point mutation, deletion mutation, insertion mutation and the like.
Therefore, according to a first aspect of the present invention, there is provided a Blocker primer for gene mutation detection, which is an oligonucleotide that is fully complementary to a wild-type sequence of a mutation region to be detected and is modified at its 3' end to prevent extension thereof, wherein the Blocker primer has an annealing temperature (Tm value) higher than that of a mutation detection primer. Therefore, after the denatured double strand of the DNA template is opened, the Blocker primer preferentially binds to the wild-type template at the annealing temperature (Tm) of the Blocker primer to block the binding of the mutation detection primer to the wild-type template, and then the mutation detection primer binds to the mutant-type template at the annealing temperature (Tm) of the mutation detection primer to amplify a fragment of the mutation of the gene to be detected in the sample DNA by enrichment by a PCR reaction.
In the invention, the length of the Blocker primer is preferably 15-35 bp.
In the present invention, preferably, the parameters of the Blocker primer may be: the Tm value is 65.0-85.0 ℃, the GC value is 40.0-65.0%, and the size of the primer is 25 +/-10 bp.
In the present invention, it is preferable that the 3' -end of the Blocker primer is subjected to dideoxy base modification, which is complementary to the base of the wild-type sequence of the sequence to be detected, or modification using other chemical groups such as: c3Spacer or phosphorylation modification.
In the present invention, it is preferable that the Blocker primer directly introduces a chemical group into its sequence during synthesis to increase its Tm value, for example, MGB modification.
In the present invention, it is preferable that the 5' -end of the Blocker primer is dephosphorylated and modified to prevent degradation by Taq polymerase.
In the present invention, it is preferable that the annealing temperature of the Blocker primer is 5 to 25 ℃ higher than that of the mutation detection primer. More preferably, the Tm value of the Blocker primer is 20 to 25 ℃ higher than the Tm value of the mutation detection primer.
In one embodiment, the present invention provides a Blocker primer having a sequence of 5'-TGCAGCTCATCACGCAGCTCATG-3' ddC (SEQ ID NO:1) for detecting point mutation T790M of EGFR gene.
In another embodiment, the present invention provides a Blocker primer having a sequence of 5'-CAAGGAATTAAGAGAAGCAACATC-3' ddC (SEQ ID NO: 9) for detecting a deletion of exon 19 (E746_ A750 deletion mutation) of the EGFR gene.
According to a second aspect of the present invention, there is provided a method for detecting gene mutation based on a Blocker primer and an ARMS primer, which sequentially performs a blocking enrichment PCR reaction and an ARMS fluorescent quantitative PCR reaction in the same reaction system including a sample DNA, a Blocker primer, an ARMS primer, a TaqMan probe, a polymerase and a buffer, the method comprising the steps of:
1) firstly, a Blocker primer is preferentially combined with a wild template at the annealing temperature (Tm) of the Blocker primer so as to block the ARMS primer from being combined with the wild template, then the ARMS primer is combined with a mutant template at the annealing temperature (Tm) of the ARMS primer, and a fragment containing a mutation region of a gene to be detected in a sample DNA is enriched and amplified through a PCR reaction, wherein the Blocker primer is an oligonucleotide which is completely complementary with the wild sequence of the mutation region to be detected and the 3 'end of which is modified so as to prevent the extension of the oligonucleotide, the 3' end of the ARMS primer is positioned at the mutation region, the fragment containing the mutation region of the gene to be detected can be amplified and is consistent with the mutant sequence, and the annealing temperature of the Blocker primer is higher than that of the ARMS primer. Wherein the mutant sequence is a nucleotide sequence in which a mutation, deletion or insertion of a base is pointed.
2) On the basis of the reaction 1), performing fluorescence detection on the mutant gene by an ARMS primer and a Taqman probe marked by FAM at the 5' end through a fluorescent quantitative PCR reaction.
Blocker primer
In the method of the present invention, preferably, the Blocker primer is designed near the mutation region (including the mutation region), and the Blocker primer is perfectly complementary to the wild-type sequence of the mutation region and is modified at the 3' -end to prevent extension thereof.
In the method of the present invention, preferably, the length of the Blocker primer is 15-35 bp.
In the method of the present invention, preferably, the relevant parameters of the Blocker primer may be: the Tm value is 65.0-85.0 ℃, the GC value is 40.0-65.0%, and the size of the primer is 25 +/-10 bp.
In the method of the present invention, preferably, the 3' end of the Blocker primer is subjected to dideoxy base modification, or is modified using other chemical groups such as: c3Spacer or phosphorylation modification.
In the present invention, it is preferable that the Blocker primer directly introduces a chemical group into its sequence during synthesis to increase its Tm value, for example, MGB modification.
In the present invention, it is preferable that the 5' -end of the Blocker primer is dephosphorylated and modified to prevent degradation by Taq polymerase.
ARMS primers
In the method of the present invention, preferably, the ARMS primers can amplify a product of a 60-150bp fragment, and the relevant parameters of the ARMS primers can be: the Tm value is 55.0-60.0 ℃, the GC value is 40.0-60.0%, and the size of the primer is 20 +/-5 bp.
In the method of the present invention, it is preferable that the ARMS primer has a 3' terminal nucleotide identical to a mutant nucleotide of the mutant gene.
In the method of the present invention, preferably, the 3' end of the ARMS primer may be located at the mutation region and coincide with the mutated base sequence of the mutant gene. To improve the specificity, it is preferable to introduce a mismatch base at the 2 nd, 3 rd or 4 th base at the 3' end of the ARMS primer. Specifically, when the mutation is a point mutation, the 3' terminal base of the ARMS primer may be a base at the site of the mutation; when the mutation is a deletion mutation, the 3' terminal base of the ARMS primer may be the next base adjacent to the deleted base; when the mutation is an insertion mutation, the 3' terminal base of the ARMS primer may be an insertion base.
In the method, the annealing temperature of the Blocker primer is preferably 5-25 ℃ higher than that of the ARMS primer. More preferably, the annealing temperature of the Blocker primer is 20-25 ℃ higher than that of the ARMS primer.
Probe needle
Designing probes in the ARMS primer amplification fragments, wherein the related parameters of the probes are as follows: the Tm value is 68.0-70.0 ℃, the GC value is 40.0-70.0%, FAM at the 5 'end of the probe is marked, and the corresponding quenching fluorescent group TAMAR or BHQ is marked at the 3' end.
In one embodiment, the invention provides a one-step detection method for EGFR gene point mutation T790M, which is characterized in that the sequence of the Blocker primer is 5'-TGCAGCTCATCACGCAGCTCATG-3' ddC (SEQ ID NO:1), and the sequence of the upstream ARMS primer is as follows: 5'-CACCGTGCAGCTCATTAT-3' (SEQ ID NO:2), the sequence of the downstream primer is: 5'-CACACACCAGTTGAGCAGGTACT-3' (SEQ ID NO: 3); the sequence of the Taqman probe is as follows: 5'-CCTTCGGCTGCCTCCTGGACTATGT-3' (SEQ ID NO: 4). Preferably, the reaction conditions are: 1) 5 minutes at 95 ℃; 5 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 2) total 40 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, and 60 ℃ for 45 seconds (fluorescence collected).
In another embodiment, the invention provides a one-step detection method for exon 19 deletion (E746_ a750 deletion mutation) of EGFR gene, wherein the sequences of the Blocker primers are as follows: 5'-CAAGGAATTAAGAGAAGCAACATC-3' ddC (SEQ ID NO: 9), the sequence of the upstream ARMS primer is: 5'-AATTCCCGTCGCTATCAAAAC-3' (SEQ ID NO: 10), the sequence of the downstream primer is: 5'-ACCCCCACACAGCAAAGC-3' (SEQ ID NO: 11), the sequence of the Taqman probe is: 5'-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3' (SEQ ID NO: 12). Preferably, the reaction conditions are: 1) 5 minutes at 95 ℃; 5 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 2) total 40 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, and 60 ℃ for 45 seconds (fluorescence collected).
According to a third aspect of the present invention, there is provided a kit for detecting a gene mutation, comprising: the kit comprises a Blocker primer, an ARMS primer and a 5 ' end FAM labeled Taqman probe, wherein the Blocker primer is an oligonucleotide which is completely complementary with a wild type sequence of a detected mutation region and the 3 ' end of the oligonucleotide is modified to prevent the elongation of the oligonucleotide, the 3 ' end of the ARMS primer is positioned at the mutation region, a fragment containing the mutation region of a gene to be detected can be amplified and is consistent with the mutation region, and the annealing temperature of the Blocker primer is higher than that of the ARMS primer. Wherein the mutant sequence is a nucleotide sequence in which a mutation, deletion or insertion of a base is pointed.
Blocker primer
In the kit of the present invention, preferably, the Blocker primer is designed near the mutation region (including the mutation region), and the Blocker primer is completely complementary to the wild-type sequence of the mutation region and is modified at the 3' -end to prevent extension thereof.
In the kit of the invention, preferably, the length of the Blocker primer is 15-35 bp.
In the kit of the present invention, preferably, the parameters of the Blocker primer may be: the Tm value is 65.0-85.0 ℃, the GC value is 40.0-65.0%, and the size of the primer is 25 +/-10 bp.
In the kit of the present invention, preferably, the 3' -end of the Blocker primer is dideoxy-base-modified.
In the kit of the present invention, preferably, the Blocker primer directly introduces a chemical group into its sequence during synthesis to increase its Tm value, for example, MGB modification.
In the kit of the present invention, it is preferable that the 5' -end of the Blocker primer is dephosphorylated and modified to prevent degradation by Taq polymerase.
ARMS primers
In the kit of the present invention, preferably, the ARMS primers can amplify a product of a 60-150bp fragment, and the relevant parameters of the ARMS primers can be: the Tm value is 55.0-60.0 ℃, the GC value is 40.0-60.0%, and the size of the primer is 20 +/-5 bp.
In the kit of the present invention, preferably, the 3' end of the ARMS primer is located at the mutation region and coincides with the sequence of the mutant gene. To improve the specificity, a mismatch base can be introduced at the 2 nd, or 3 rd base or 4 th base of the 3' end of the ARMS primer. Specifically, when the mutation is a point mutation, the 3' terminal base of the ARMS primer may be a base at the site of the mutation; when the mutation is a deletion mutation, the 3' terminal base of the ARMS primer may be the next base adjacent to the deleted base; when the mutation is an insertion mutation, the 3' terminal base of the ARMS primer may be an insertion base.
In the invention, preferably, the annealing temperature of the Blocker primer is 5-25 ℃ higher than that of the ARMS primer. More preferably, the annealing temperature of the Blocker primer is 20-25 ℃ higher than that of the ARMS primer.
Probe needle
Designing probes in the ARMS primer amplification fragments, wherein the related parameters of the probes are as follows: the Tm value is 68.0-70.0 ℃, the GC value is 40.0-70.0%, FAM at the 5 'end of the probe is marked, and the corresponding quenching fluorescent group TAMAR or BHQ is marked at the 3' end.
In one embodiment, the invention provides a one-step detection kit for EGFR gene point mutation T790M, which is characterized in that the sequence of the Blocker primer is 5'-TGCAGCTCATCACGCAGCTCATG-3' ddC (SEQ ID NO:1), and the sequence of the upstream ARMS primer is as follows: 5'-CACCGTGCAGCTCATTAT-3' (SEQ ID NO:2), the sequence of the downstream primer is: 5'-CACACACCAGTTGAGCAGGTACT-3' (SEQ ID NO: 3); the sequence of the Taqman probe is as follows: 5'-CCTTCGGCTGCCTCCTGGACTATGT-3' (SEQ ID NO: 4). Preferably, the reaction conditions are: 1) 5 minutes at 95 ℃; 5 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 2) total 40 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, and 60 ℃ for 45 seconds (fluorescence collected).
In another embodiment, the invention provides a one-step detection kit for exon 19 deletion (E746a750 deletion mutation) of EGFR gene, wherein the sequences of the Blocker primers are: 5'-CAAGGAATTAAGAGAAGCAACATC-3' ddC (SEQ ID NO: 9), the sequence of the upstream ARMS primer is: 5'-AATTCCCGTCGCTATCAAAAC-3' (SEQ ID NO: 10), the sequence of the downstream primer is: 5'-ACCCCCACACAGCAAAGC-3' (SEQ ID NO: 11), the sequence of the Taqman probe is: 5'-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3' (SEQ ID NO: 12). Preferably, the reaction conditions are: 1) 5 minutes at 95 ℃; 5 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 2) total 40 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, and 60 ℃ for 45 seconds (fluorescence collected).
The term "mutant sequence" as used herein refers to a nucleotide sequence in which a base is point-mutated, deleted or inserted.
The term "mutated region" as used herein refers to a site of mutation, sequence deletion or sequence insertion.
The term "identical" as used herein refers to the case where the nucleotide sequence of the mutation region to be detected is identical or complementary, and is listed in the embodiments herein as being identical to the nucleotide sequence of the mutation region to be detected.
In the invention, a corresponding Blocker primer is designed according to the annealing temperature, and under the annealing temperature of the Blocker, the Blocker primer is combined with a wild-type template to block the amplification of a wild type, and the amplification of a wild-type background is reduced by using an ARMS technology.
According to the invention, a Blocker enrichment technology and an ARMS fluorescent quantitative PCR technology are integrated in a reaction system, so that the enrichment and identification of the mutant gene can be realized in one step, the operation steps are reduced, and the detection sensitivity and specificity are improved.
Drawings
FIG. 1 shows the amplification curves of the T790 site wild-type plasmid with and without addition of the Blocker primer. A: t790 site wild type plasmid 107 copies/μ l background without added Blocker primer, B: the Blocker primer was added to the T790 site wild type plasmid at 107 copies/. mu.l background.
FIG. 2 shows the amplification curve of T790 site mutant plasmid without addition of Blocker primer. A: t790 site mutant plasmid 104Copies/. mu.l, B: t790 site mutant plasmid 103Copy/. mu.l, C: t790 site mutant plasmid 102 copies/μ l, D: t790 site mutant plasmid 10 copies/. mu.l.
FIG. 3 shows the amplification curve of T790 site mutant plasmid with the addition of Blocker primer. A: t790 site mutant plasmid 104 copies/. mu.l, B: t790 site mutant plasmid 103 copies/. mu.l, C: t790 site mutant plasmid 102 copies/μ l, D: t790 site mutant plasmid 10 copies/. mu.l.
FIG. 4 shows the T790 site mutant plasmid mixed in 107 copies/. mu.l wild type background plasmid amplified with Blocker primerIncreasing the curve. A: t790 site mutant plasmid 104Copies/. mu.l, B: t790 site mutant plasmid 103Copy/. mu.l, C: t790 site mutant plasmid 102 copies/μ l, D: t790 site mutant plasmid 10 copies/. mu.l.
FIG. 5 shows the T790 site mutant plasmid mix at 107One copy/. mu.l of wild type background plasmid was amplified without addition of a Blocker condition. A: t790 site mutant plasmid 104 copies/. mu.l, B: t790 site mutant plasmid 103Copy/. mu.l, C: t790 site mutant plasmid 102Copies/. mu.l, D: t790 site mutant plasmid 10 copies/. mu.l.
FIG. 6 shows the amplification curves of the 19 deletion site wild-type plasmid with and without the addition of the Blocker primer. A: 19 deletion site wild type plasmid 107Individual copies/. mu.l background without addition of Blocker primers, B: 19 deletion site wild type plasmid 107Each copy/. mu.l background was added with Blocker primers.
FIG. 7 shows the amplification curve of the 19 deletion mutant plasmid without addition of the Blocker primer. A: 19 deletion mutant plasmid 104Copies/. mu.l, B: 19 deletion mutant plasmid 103Copy/. mu.l, C: 19 deletion mutant plasmid 102Copies/. mu.l, D: 19 deletion mutant plasmid 10 copies/. mu.l.
FIG. 8 shows the amplification curve of the 19 deletion variant plasmid with the addition of the Blocker primers. A: 19 deletion mutant plasmid 104Copies/. mu.l, B: 19 deletion mutant plasmid 103Copy/. mu.l, C: 19 deletion mutant plasmid 102Copies/. mu.l, D: 19 deletion mutant plasmid 10 copies/. mu.l.
FIG. 9 shows the amplification curve of 19 deletion mutant plasmids mixed in 107 copies/. mu.l of wild type background plasmid under the condition of adding Blocker primers. A: t790 site mutant plasmid 104Copies/. mu.l, B: t790 site mutant plasmid 103 copies/. mu.l, C: t790 site mutant plasmid 102Copies/. mu.l, D: t790 site mutant plasmid 10 copies/. mu.l.
FIG. 10 shows the 19 deletion mutationType plasmids were mixed in 107 copies/. mu.l wild type background plasmid and the amplification curve was performed without addition of Blocker. A: t790 site mutant plasmid 104Copies/. mu.l, B: t790 site mutant plasmid 103Copy/. mu.l, C: t790 site mutant plasmid 102Copies/. mu.l, D: t790 site mutant plasmid 10 copies/. mu.l.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. In the following examples, unless otherwise specified, all methods are conventional.
Examples
Example 1 mutation blocking enrichment ARMS fluorescent quantitative PCR one-step detection method of EGFR gene point mutation T790M
1. Primer and probe design
1) Blocker primer design
The primer is designed near the site of mutation (including the site of mutation), and the 3' end is dideoxy base-modified to prevent extension and raise the annealing temperature. The relevant parameters are: the Tm value is 63.0-80.0 ℃, the GC value is 40.0-65.0%, and the size of the primer is 25 +/-10 bp.
The designed sequences of the Blocker primers are as follows:
blocker primers: 5'-TGCAGCTCATCACGCAGCTCATG-3' ddC (SEQ ID NO:1)
2) ARMS primer design
Designing a primer capable of amplifying a segment of 60-150bp, wherein relevant parameters are as follows: the Tm value is 55.0-60.0 ℃, the GC value is 40.0-60.0%, and the size of the primer is 20 +/-3 bp. The 3 'end of the upstream ARMS primer is positioned at the mutation site and is consistent with the mutant gene, in order to improve the specificity, a mismatched base is introduced at the 3 rd base of the 3' end, and the sequence of the ARMS primer is designed as follows:
upstream ARMS primers: 5'-CACCGTGCAGCTCATTAT-3' (SEQ ID NO:2)
A downstream primer: 5'-CACACACCAGTTGAGCAGGTACT-3' (SEQ ID NO:3)
3) Probe design
Designing probes in ARMS primer amplified fragments, wherein relevant parameters are as follows: the Tm value is 68.0-70.0 ℃, the GC value is 40.0-70.0%, and the 5' end FAM labeling is carried out on the probe, and the sequence is as follows:
and (3) probe: 5'-CCTTCGGCTGCCTCCTGGACTATGT-3' (SEQ ID NO:4)
The Blocker primer, ARMS primer, primer and probe in example 1 were all prepared by Shanghai bioengineering Co., Ltd.
2. Preparing a sample to be detected:
wild-type plasmid construction: PCR amplification was performed using the human genome as a template using the upstream primer sequence 1 (5'-TTCACAGCCCTGCGTAAAC-3' (SEQ ID NO: 5)) and the downstream primer sequence 1 (5'-TTTCCACATGCAGATGGGAC-3' (SEQ ID NO: 6)) containing the T790 site, respectively (PCR reaction conditions: 1) at 95 ℃ for 5 minutes; 35 cycles of 95 ℃ for 15 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 72 ℃ for 10 minutes), the product was cloned into the Takara pMD19 vector and samples were extracted for use after sequencing to verify correctness. The reaction system is shown in Table 1.
TABLE 1
Construction of mutant plasmids: the product 1 was recovered after PCR amplification using the upstream primer sequence 1 (5'-TTCACAGCCCTGCGTAAAC-3' (SEQ ID NO: 5)) and the downstream primer sequence 2 (5'-CATGAGCTGCaTGATGAGCTGCA-3' (SEQ ID NO: 8)) containing the T790 site, respectively, using a wild-type plasmid as a template. Then, product 2 was recovered after PCR amplification using the forward primer sequence 2 (5'-TGCAGCTCATCAtGCAGCTCATG-3' (SEQ ID NO: 7)) using the wild type plasmid as a template. Using product 1 and product 2 as templates, PCR amplification was performed using the upstream primer sequence 1 (5'-TTCACAGCCCTGCGTAAAC-3' (SEQ ID NO: 5)) and the downstream primer sequence 1 (5'-TTTCCACATGCAGATGGGAC-3' (SEQ ID NO: 6)) containing the T790 site. The product was cloned into Takara pMD19 vector, and the sample was extracted for use after the sequencing was verified to be correct (lower case letters in the upstream primer sequence 2 and the downstream primer sequence 1 represent the mutant site 790). The PCR reaction conditions and reaction system for constructing the mutant plasmid are the same as those for constructing the wild plasmid.
3. The detection method comprises the following steps: the mutation blocking enrichment one-step detection method applies a Blocker technology to mutation site PCR blocking enrichment and ARMS fluorescence quantitative PCR, and sequentially performs PCR blocking enrichment and ARMS fluorescence quantitative PCR identification in a reaction system, wherein the specific process is that 20 mu l of the reaction system (containing a DNA template of a sample to be detected) comprising the components in the following table is firstly subjected to high-temperature denaturation on a fluorescence PCR instrument to open a genome double chain; at the annealing temperature of 80 ℃, the Blocker primer is combined with a wild type gene template to block the ARMS primer from being combined with the wild type gene template; the ARMS primer can not amplify the wild type template because the combination of the ARMS primer and the wild type template is blocked when annealing is carried out at 60 ℃; and the combination of the mutant template and the ARMS primer, and extension at 72 ℃ enable the part to be detected containing the mutant base to be selectively amplified and enriched. Then, the detection reaction of the mutation site is operated, and the amplification of the wild type gene is reduced and the interference of the wild type gene on the mutant type gene is reduced due to the blocking effect of the Blocker primer on the wild type gene; extension at 60 ℃ for 45 seconds, and collection of the mutation-amplified fluorescent signal. The reaction system is shown in Table 2.
TABLE 2
The reaction conditions were as follows: 1) 5 minutes at 95 ℃; 5 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 2) total 40 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, and 60 ℃ for 45 seconds (fluorescence collected). The fluorescent quantitative PCR instrument used was ABISTENE (applied biosystems).
In the experimental process, wild type plasmid templates are respectively provided with experimental groups without a Blocker primer and with a Blocker primer, mutant plasmid simulation mutation type samples with different concentrations are respectively added under wild type background (107 copies/. mu.l) for detection, and experimental groups without a Blocker primer and with a Blocker primer are respectively provided; and setting corresponding mutant plasmid templates, and detecting by using a primer system without adding Blocker.
And (4) analyzing results: as shown in FIG. 1, the wild-type sample of the group to which no Blocker primer was added had an amplification curve, and after addition of the Blocker primer, the amplification curve of the wild-type plasmid was closed and no amplification curve was observed. FIG. 2, FIG. 3, FIG. 4 and FIG. 5 show that the background signal of wild-type plasmid can be effectively blocked after the primer Blocker is added, and mutant plasmid in the mixed sample can be effectively detected; wild type plasmid samples and mutant type plasmid samples which cannot be effectively distinguished by a reaction system without adding a Blocker primer; meanwhile, the detection capability of the reaction system on wild-type plasmids is not changed after the Blocker primers are added.
As described above, the reaction system containing the Blocker primer enables efficient detection of the mutant type at background concentration of the wild-type plasmid.
Example 2 mutation blocking enrichment ARMS fluorescent quantitative PCR one-step detection method of EGFR gene 19 exon deletion E746_ A750 deletion mutation
1. Primer and probe design
1) Blocker primer design
Primers were designed near the deletion mutant region (including the deleted sequence) and a dideoxy base modification was made at the 3' end to prevent extension and raise the annealing temperature. The relevant parameters are: the Tm value is 65.0-80.0 ℃, the GC value is 40.0-65.0%, and the size of the primer is 25 +/-10 bp.
The designed sequences of the Blocker primers are as follows:
blocker primers: 5'-CAAGGAATTAAGAGAAGCAACATC-3' ddC (SEQ ID NO: 9)
2) ARMS primer design
Designing a primer capable of amplifying a segment of 60-150bp, wherein relevant parameters are as follows: the Tm value is 55.0-60.0 ℃, the GC value is 40.0-60.0%, and the size of the primer is 20 +/-3 bp. The 3 'end of the upstream ARMS primer is positioned at the mutation site and is consistent with the mutant gene, in order to improve the specificity, a mismatched base is introduced at the 3 rd base position of the 3' end, and the designed ARMS
The primer sequences are as follows:
upstream ARMS primers: 5'-AATTCCCGTCGCTATCAAAAC-3' (SEQ ID NO: 10)
A downstream primer: 5'-ACCCCCACACAGCAAAGC-3' (SEQ ID NO: 11)
3) Probe design
Designing probes in ARMS primer amplified fragments, wherein relevant parameters are as follows: the Tm value is 68.0-70.0 ℃, the GC value is 40.0-70.0%, and the 5' end FAM labeling is carried out on the probe, and the sequence is as follows:
and (3) probe: 5'-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3' (SEQ ID NO: 12)
The above Blocker primers, ARMS primers, primers and probes were obtained from Shanghai bioengineering, Inc.
2. Sample preparation:
wild-type plasmid construction: PCR amplification using the human genome as a template (PCR reaction conditions: 1) using the upstream primer sequence 1 (5'-GCCTAGACGCAGCATCATTA-3' (SEQ ID NO: 13)) and the downstream primer sequence 1 (5'-ATGCCTCCATTTCTTCATCC-3' (SEQ ID NO: 14)) containing the E746_ A750 deletion site, respectively, for 5 minutes at 95 ℃; 35 cycles of 95 ℃ for 15 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 72 ℃ for 10 minutes), the product was cloned into the Takara pMD19 vector and samples were extracted for use after sequencing to verify correctness. The reaction system is shown in Table 3.
TABLE 3
Construction of mutant plasmids: product 1 was recovered after PCR amplification using the upstream primer sequence 1 (5'-GCCTAGACGCAGCATCATTA-3' (SEQ ID NO: 13)) and the downstream primer sequence 2 (5'-CGGAGATGTttTGATAGCGACGGGAATT-3' (SEQ ID NO: 16)) containing the E746_ A750 deletion site, respectively, using the wild-type plasmid as a template. Then, product 2 was recovered after PCR amplification using the forward primer sequence 2 (5'-TCGCTATCAaaACATCTCCGAAAGCCAAC-3' (SEQ ID NO: 15)) and the reverse primer sequence 1 (5'-ATGCCTCCATTTCTTCATCC-3' (SEQ ID NO: 14)) using the wild-type plasmid as a template.
Using product 1 and product 2 as templates, PCR amplification was performed using the upstream primer sequence 1 (5'-GCCTAGACGCAGCATCATTA-3' (SEQ ID NO: 13)) and the downstream primer sequence 1 (5'-ATGCCTCCATTTCTTCATCC-3' (SEQ ID NO: 14)) containing the E746_ A750 deletion site. The product was cloned into Takara pMD19 vector, and the sample was extracted for use after the sequencing was verified to be correct (lower case letters in the upstream primer sequence 2 and the downstream primer sequence 1 represent E746_ A750 deletion site).
The PCR reaction conditions and reaction system for constructing the mutant plasmid are the same as those for constructing the wild plasmid.
3. The detection method comprises the following steps: the mutation blocking enrichment one-step detection method applies a Blocker technology to mutation site PCR blocking enrichment and ARMS fluorescence quantitative PCR, and sequentially performs PCR blocking enrichment and ARMS fluorescence quantitative PCR identification in a reaction system, wherein the specific process is that 20 mu l of the reaction system (containing a DNA template of a sample to be detected) comprising the components in the following table is firstly subjected to high-temperature denaturation on a fluorescence PCR instrument to open a genome double chain; at the annealing temperature of 80 ℃, the Blocker primer is combined with a wild type gene template to block the ARMS primer from being combined with the wild type gene template; the ARMS primer can not amplify the wild type template because the combination of the ARMS primer and the wild type template is blocked when annealing is carried out at 60 ℃; and the combination of the mutant template and the ARMS primer, and extension at 72 ℃ enable the part to be detected containing the mutant base to be selectively amplified and enriched. Then, the detection reaction of the mutation site is operated, and the amplification of the wild type gene is reduced and the interference of the wild type gene on the mutant type gene is reduced due to the blocking effect of the Blocker primer on the wild type gene; extension at 60 ℃ for 45 seconds, and collection of the mutation-amplified fluorescent signal. The reaction system is shown in Table 4.
TABLE 4
The reaction conditions were as follows: 1) 5 minutes at 95 ℃; 5 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 20 seconds; 2) total 40 cycles of 95 ℃ for 15 seconds, 80 ℃ for 20 seconds, and 60 ℃ for 45 seconds (fluorescence collected). The fluorescent quantitative PCR instrument used was ABISTENE (applied biosystems).
In the experimental process, wild type plasmid templates are respectively provided with experimental groups without a Blocker primer and with a Blocker primer, mutant plasmid simulation mutation type samples with different concentrations are respectively added under wild type background (107 copies/. mu.l) for detection, and experimental groups without a Blocker primer and with a Blocker primer are respectively provided; and setting corresponding mutant plasmid templates, and detecting by using a primer system without adding Blocker.
And (4) analyzing results: as shown in FIG. 6, the wild-type sample of the set to which no Blocker primer was added had an amplification curve, and after addition of the Blocker primer, the amplification curve of the wild-type plasmid was closed and no amplification curve was observed. FIG. 7, FIG. 8, FIG. 9 and FIG. 10 show that the background signal of wild-type plasmid can be effectively blocked after adding the Blocker primer, and mutant plasmid in the mixed sample can be effectively detected; wild type plasmid samples and mutant type plasmid samples which cannot be effectively distinguished by a reaction system without adding a Blocker primer; meanwhile, the detection capability of the reaction system on wild-type plasmids is not changed after the Blocker primers are added.
As described above, the reaction system containing the Blocker primer enables efficient detection of the mutant type at background concentration of the wild-type plasmid.
Sequence listing
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Claims (3)
1. A kit for detecting gene mutation based on a Blocker primer and an ARMS primer comprises a sample DNA, a Blocker primer, an ARMS primer, a TaqMan probe, a polymerase and a buffer solution, is used for sequentially carrying out enrichment PCR reaction and ARMS fluorescent quantitative PCR reaction, under the annealing temperature of the Blocker primer, the Blocker primer is preferentially combined with a wild template so as to block the combination of the ARMS primer and the wild template, then under the annealing temperature of the ARMS primer, the ARMS primer is combined with a mutant template, and a fragment containing a gene mutation region to be detected in the sample DNA is enriched and amplified through the PCR reaction, wherein the Blocker primer is oligonucleotide which is completely complementary with the wild sequence of the detected mutation region and the 3 'end of which is modified so as to prevent the extension of the wild sequence, the 3' end of the ARMS primer is positioned at the mutation region, can amplify the fragment containing the gene mutation region to be detected and is consistent with the mutant sequence, wherein, a mismatched base is introduced at the 2 nd, 3 rd or 4 th base of the 3' end of the ARMS primer, wherein, a chemical group is directly introduced into the sequence of the Blocker primer in the synthesis process to improve the Tm value of the Blocker primer, and the annealing temperature of the Blocker primer is 20-25 ℃ higher than that of the ARMS primer; performing fluorescence detection on the mutant gene by an ARMS primer and a Taqman probe marked by FAM at the 5' end through a fluorescent quantitative PCR reaction; wherein the gene mutation is EGFR gene point mutation T790M, wherein the sequence of the Blocker primer is 5'-TGCAGCTCATCACGCAGCTCATG-3' ddC (SEQ ID NO:1), and the sequence of the upstream ARMS primer is as follows: 5'-CACCGTGCAGCTCATTAT-3' (SEQ ID NO:2), the sequence of the downstream primer is: 5'-CACACACCAGTTGAGCAGGTACT-3' (SEQ ID NO: 3); the sequence of the Taqman probe is as follows: 5'-CCTTCGGCTGCCTCCTGGACTATGT-3' (SEQ ID NO: 4).
2. The kit of claim 1, wherein a dideoxy base modification is performed at the 3' end of the Blocker primer; and/or, dephosphorylation modification is carried out on the 5' end of the Blocker primer.
3. The kit of claim 1, wherein the gene mutation is an EGFR gene 19 exon deletion E746_ a750 deletion mutation, wherein the sequences of the Blocker primers are: 5'-CAAGGAATTAAGAGAAGCAACATC-3' ddC (SEQ ID NO: 9), the sequence of the upstream ARMS primer is: 5'-AATTCCCGTCGCTATCAAAAC-3' (SEQ ID NO: 10), the sequence of the downstream primer is: 5'-ACCCCCACACAGCAAAGC-3' (SEQ ID NO: 11), the sequence of the Taqman probe is: 5'-CCAACAAGGAAATCCTCGATGTGAGTTTCTG-3' (SEQ ID NO: 12).
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| CN107419018A (en) | 2017-12-01 |
| CN103255201A (en) | 2013-08-21 |
| CN103255201B (en) | 2017-07-14 |
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