CN115851928A - Blocker probe and primer group for amplifying EGFR-T790M gene variation and application thereof - Google Patents

Blocker probe and primer group for amplifying EGFR-T790M gene variation and application thereof Download PDF

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CN115851928A
CN115851928A CN202211027049.7A CN202211027049A CN115851928A CN 115851928 A CN115851928 A CN 115851928A CN 202211027049 A CN202211027049 A CN 202211027049A CN 115851928 A CN115851928 A CN 115851928A
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egfr
gene
blocker probe
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齐盼盼
唐东江
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Zhuhai Livzon Cynvenio Diagnostics Ltd
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract

The invention relates to the technical field of gene detection, in particular to a blocker probe, a primer group and application thereof for amplifying EGFR-T790M gene variation. The length of the blocker probe provided by the invention is less than 20nts, and the thermal stability of the combination of the blocker probe and a wild-type template can be ensured by modifying locked nucleic acid in the range of 1 or 2nts on both sides of a mutation site. In addition, the invention also provides a primer group for amplifying EGFR-T790M gene variation, the primer group comprises the above blocker probe, and also comprises two upstream primers matched with the blocker probe, and the two upstream primers are arranged to avoid the occurrence of false negative results when G & gtA mutation exists at the SNP locus rs 1050171.

Description

Blocker probe and primer group for amplifying EGFR-T790M gene variation and application thereof
Technical Field
The invention relates to the technical field of gene detection, in particular to a blocker probe, a primer group and application thereof for amplifying EGFR-T790M gene variation.
Background
At present, on a qPCR platform, a common method for detecting tumor mutation sites is a mutation amplification system (ARMS) method, an upstream primer and a blocker of the method are in a competitive combination relationship, the difference of Tm values between the primer/blocker and a template is utilized to ensure that the blocker is preferentially combined with a wild template, the primer is preferentially combined with a mutant, the wild amplification is blocked, and the mutant amplification is not blocked, so that the effect of enriching the mutant template is achieved.
Based on the above, the applicant provides a blocker probe for selective amplification of EGFR-T790M gene mutant type in Chinese patent application and multiple homologous invention applications with the application number of CN2019104222174, the application number of which is CN 2019104174 and the invention name of which is 'a non-quenched oligonucleotide probe for amplifying mutant target gene fragment and application thereof' filed in 2019, 5, 21.
However, in the subsequent use process, the applicant finds that (1) the blocker probe is too long, so that the blocker probe is poor in performance in a qPCR reaction system, obviously inhibits mutants and cannot achieve a good effect; (2) A high-frequency SNP locus rs1050171 (G > A) exists in front of the T790M locus, in 19.6% of Asians, the rs1050171 locus is A (G > A), and the mutation state of the rs1050171 influences the sensitivity of an EGFR-T790M detection system based on ARMS, so that a false negative or false positive result is generated.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to further improve a blocker probe on the basis of the existing blocker probe of EGFR-T790M genetic variation of an applicant to obtain an iterative blocker probe and an amplification primer group so as to further improve the detection sensitivity of low-frequency genetic variation, and meanwhile, the blocker probe is expected to achieve the detection effect which is not inferior to that of digital PCR or high-throughput sequencing under the condition of qPCR, so that the advantages of simple operation, low cost and high cost performance are achieved.
In order to solve the above technical problems and achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the invention provides a blocker probe for amplifying EGFR-T790M gene variation, (1) a nucleotide sequence of the blocker probe covers EGFR-T790M gene mutation sites, is completely matched with wild type EGFR-T790M gene segments, and is mismatched with mutant type EGFR-T790M gene segments at the mutation sites; (2) The length of the nucleotide sequence of the blocker probe is less than 20nts, wherein the mutation site and the corresponding base in the range of 1 or 2nts on both sides of the mutation site are modified by locked nucleic acid.
In alternative embodiments, the nucleotide sequence of the blocker probe is (X) from 5 'end to 3' end m )
Figure BDA0003815942440000021
(X n ) Or a complementary sequence which hybridizes strictly thereto, said X m For M nucleotides which are perfectly matched with the wild type EGFR-T790M gene segment at the upstream of the mutation site, X n The method is characterized in that n nucleotides which are completely matched with wild type EGFR-T790M gene segments are positioned at the downstream of a mutation site, wherein M + n is less than 15, the nucleotide which is marked in an italic and bold font in the blocker probe corresponds to the mutation site, underlining shows that the nucleotide is modified by locked nucleic acid, and at least one nucleotide in CA at the upstream of the mutation site and GC at the downstream of the mutation site is modified by locked nucleic acid.
In alternative embodiments, m ≦ 6.
In an optional embodiment, the nucleotide sequence of the blocker probe is selected from any one of SEQ ID No. 1-4, and the nucleotide sequence shown in SEQ ID No.1 is
Figure BDA0003815942440000022
The nucleotide sequence shown in SEQ ID No.2 is->
Figure BDA0003815942440000023
The nucleotide sequence shown in SEQ ID No.3 is->
Figure BDA0003815942440000024
The nucleotide sequence shown as SEQ ID No.4 is +>
Figure BDA0003815942440000025
In an alternative embodiment, the 3' end of the blocker probe is modified by extension blocking.
In a second aspect, the invention provides a primer set for amplifying an EGFR-T790M gene variation, the primer set comprising a blocker probe according to any one of the preceding embodiments, two upstream primers and at least one downstream primer;
the nucleotide sequences of the two upstream primers cover SNP locus rs1050171 of EGFR-T790M gene, wherein the nucleotide sequence of the first upstream primer is (X) m1 )A(X n1 ) Said X is m1 Matching M1 nucleotides upstream of rs1050171 site with EGFR-T790M gene segment, wherein X is n1 The downstream n1 nucleotides of the rs1050171 site are matched with the EGFR-T790M gene segment, and the nucleotide sequence of the second upstream primer is (X) m2 )G(X n2 ) Said X is m2 Matching M2 nucleotides with EGFR-T790M gene segment at the upstream of rs1050171 site, wherein X is n2 The downstream n2 nucleotides of the rs1050171 site are matched with the EGFR-T790M gene segment, and both n1 and n2 are selected from any number value of 3-7.
In an alternative embodiment, the 3 'ends of the two upstream primers and the 5' end of the blocker probe have nucleotide overlap, and the number of the overlapped nucleotides is 1 to 7.
In alternative embodiments, the primer set further comprises a fluorescent probe that binds to the antisense strand of the EGFR-T790M gene.
In a third aspect, the present invention provides a use of the blocker probe of any one of the preceding embodiments or the primer set of any one of the preceding embodiments, the use comprising:
(1) Preparing a preparation, a PCR reaction system or a kit for selectively amplifying EGFR-T790M gene mutant type; alternatively, the first and second electrodes may be,
(2) Amplification and enrichment before EGFR-T790M gene mutant type detection; alternatively, the first and second liquid crystal display panels may be,
(3) And (3) establishing a library before detecting the EGFR-T790M gene mutant type.
In a fourth aspect, the present invention provides a method for selective amplification of EGFR-T790M gene mutation type, wherein a PCR reaction system is configured using the blocker probe according to any one of the foregoing embodiments or the primer set according to any one of the foregoing embodiments, and after a sample to be amplified is denatured, annealed and extended, the amplified product is analyzed;
the annealing temperature and the extension temperature are the same and are selected from 54-62 ℃;
the sample to be amplified is derived from blood, body fluid, tissue, circulating tumor cells, or cfDNA.
The length of the blocker probe provided by the invention is less than 20nts, the blocker probe is matched with a primer, the specificity and sensitivity of the reaction can be obviously improved, and the thermal stability of the blocker probe combined with the template can be ensured by modifying locked nucleic acid within the range of 1 or 2nts on both sides of a mutation site, so that the specificity of the blocker probe for identifying mutant types and wild types is improved.
The invention also provides a primer group for amplifying EGFR-T790M gene variation, the primer group comprises the above-mentioned blocker probe, and also comprises two upstream primers matched with the blocker probe, and the arrangement of the two upstream primers can avoid the occurrence of false negative results under the condition that G & gtA mutation of the SNP locus rs1050171 exists.
The invention also provides a method for EGFR-T790M gene mutation type selective amplification by using the blocker probe or the primer group, the method is based on the combination of the blocker probe and the double upstream primers, a clinical sample is detected, an ROC curve is made, and the result shows that the detection result of the clinical sample is compared with the result of the digital PCR, the positive coincidence rate is 96.3 percent, the negative coincidence rate is 95.7 percent, and the T790M site detection rate is not less than 95 percent under the conditions of 7.5 ng/reaction DNA and 0.125 percent mutation proportion, so that the detection level of the digital PCR is achieved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an amplification curve obtained in the absence of a blocker probe in Experimental example 1;
FIG. 2 is an amplification curve when a blocker probe is added in Experimental example 1 of the present invention;
FIG. 3 is a graph showing an amplification curve when the NCI-H1975 template is used in Experimental example 4 of the present invention;
FIG. 4 is a graph showing an amplification curve when a plasmid 002 template was used in Experimental example 4 of the present invention;
FIG. 5 shows the result of optimizing the final concentration of the primer No. 123 in Experimental example 5 of the present invention;
FIG. 6 shows the results of optimizing the final concentration of the primer No. 111 in Experimental example 5;
FIG. 7 shows the results of optimizing the final concentration of the primer No. 211 in Experimental example 5 of the present invention;
FIG. 8 shows the results of optimizing the final concentration of the probe provided in example 1 of Experimental example 5 of the present invention;
FIG. 9 shows the result of optimizing the final concentration of the fluorescent probe in Experimental example 5 according to the present invention;
FIG. 10 shows the final concentration optimization results of the upstream and downstream primers and the internal reference probe of the internal reference in Experimental example 5;
FIG. 11 shows the results of optimizing the reaction volume in Experimental example 5 of the present invention;
FIG. 12 shows the results of the optimization of the number of reaction cycles in Experimental example 5 of the present invention;
FIG. 13 shows the results of the annealing/elongation temperature and time optimization in Experimental example 5 of the present invention;
FIG. 14 shows the ROC curve obtained in application example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures. Furthermore, the terms "first," "second," "third," and the like are used solely to distinguish one from another and are not to be construed as indicating or implying relative importance.
In a specific embodiment, in a first aspect, the invention provides a blocker probe for amplifying EGFR-T790M gene variation, (1) a nucleotide sequence of the blocker probe covers EGFR-T790M gene mutation sites, is completely matched with a wild-type EGFR-T790M gene segment, and is mismatched with a mutant-type EGFR-T790M gene segment at the mutation sites; (2) The length of the nucleotide sequence of the blocker probe is less than 20nts, wherein the mutation site and the corresponding base in the range of 1 or 2nts on both sides of the mutation site are modified by locked nucleic acid.
In alternative embodiments, the nucleotide sequence of the blocker probe is (X) from 5 'end to 3' end m )
Figure BDA0003815942440000041
(X n ) Or a complementary sequence which hybridizes strictly thereto, said X m The M nucleotides which are completely matched with the wild type EGFR-T790M gene segment are arranged at the upstream of the mutation site, and the X is n And (2) completely matching nucleotides with the wild-type EGFR-T790M gene segment by n nucleotides at the downstream of the mutation site, wherein M + n is less than 15, the mutation site corresponds to the nucleotides marked by italics and bold font in the blocker probe, underlining indicates that the nucleotide is modified by locked nucleic acid, and at least one nucleotide in CA at the upstream of the mutation site and GC at the downstream of the mutation site is modified by locked nucleic acid.
In the present invention, m, n, m1, n1, m2, or n2 represents the number of nucleotides, and thus all represent natural numbers. Thus, the above m + n may be selected from 0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
The above-mentioned complementary sequence which strictly hybridizes refers to each nucleotide and the sequence (X) in the complementary sequence m )CACGC(X n ) The corresponding nucleotides all satisfy the base complementary pairing principle.
In alternative embodiments, m ≦ 6.
m is less than or equal to 6, the rs1050171 locus can be prevented from being covered by the blocker probe, so that the Tm values of the combination of the blocker probe and the rs1050171 locus wild type template and the mutant type template are consistent, the Tm values are consistent in the two types of templates, the blocking effect difference cannot occur, the reaction is more stable, and the probability of false negative or false positive results is reduced. Said m may be selected from 0, 1, 2, 3, 4, 5 or 6.
In an optional embodiment, the nucleotide sequence of the blocker probe is selected from any one of SEQ ID No. 1-4, and the nucleotide sequence shown in SEQ ID No.1 is
Figure BDA0003815942440000051
The nucleotide sequence shown in SEQ ID No.2 is->
Figure BDA0003815942440000052
The nucleotide sequence shown in SEQ ID No.3 is->
Figure BDA0003815942440000053
The nucleotide sequence shown in SEQ ID No.4 is->
Figure BDA0003815942440000054
In an alternative embodiment, the 3' end of the blocker probe is modified by extension blocking. The extension blocking modification is a modification performed on the 3 'end of the blocker probe aiming at preventing the 3' end of the blocker probe from extending in the amplification process, and the specific modification includes but is not limited to MGB modification, dideoxy modification, amino modification, phosphorylation modification or spacer C3.
In a second aspect, the present invention provides a primer set for amplifying EGFR-T790M gene variation, the primer set comprising the blocker probe of any one of the preceding embodiments, two upstream primers and at least one downstream primer;
the nucleotide sequences of the two upstream primers cover rs1050171 site of EGFR-T790M gene, wherein the nucleotide sequence of the first upstream primer is (X) m1 )A(X n1 ) Said X is m1 Matching M1 nucleotides upstream of rs1050171 site with EGFR-T790M gene segment, wherein X is n1 The downstream n1 nucleotides of the rs1050171 site are matched with the EGFR-T790M gene segment, and the nucleotide sequence of the second upstream primer is (X) m2 )G(X n2 ) Said X is m2 Matching M2 nucleotides with EGFR-T790M gene segment at the upstream of rs1050171 site, wherein X is n2 The downstream n2 nucleotides of the rs1050171 site are matched with the EGFR-T790M gene segment, and both n1 and n2 are selected from any number value of 3-7. For m1 and m2 of the two upstream primers, after a combined sequence of the 3' end of the upstream primer on the blocker probe is determined, conventional selection can be performed according to the requirements of an actual PCR reaction system.
In alternative embodiments, the 3 'ends of the two upstream primers overlap the 5' end of the blocker probe by nucleotides from 1 to 7, for example 1, 2, 3, 4, 5, 6 or 7. The partially overlapped nucleotide sequences can ensure that the upstream primer is directly prevented from being combined with the template after the blocker probe is combined with the wild-type template, and compared with the conventional method for preventing wild-type extension, the blocking effect is more obvious.
In alternative embodiments, the primer set further comprises a fluorescent probe that binds to the antisense strand of the EGFR-T790M gene. Due to the blocking effect of the blocker probe, the number of amplified sense chains and amplified antisense chains of the EGFR-T790M gene is obviously different, and when the template chains of the EGFR-T790M gene combined by the fluorescent probe and the blocker probe are different chains, the specificity and the sensitivity of the reaction can be improved.
In a third aspect, the present invention provides a use of the blocker probe of any one of the preceding embodiments or the primer set of any one of the preceding embodiments, the use comprising:
(1) Preparing a preparation, a PCR reaction system or a kit for selectively amplifying EGFR-T790M gene mutant; alternatively, the first and second liquid crystal display panels may be,
(2) Amplification and enrichment before EGFR-T790M gene mutant type detection; alternatively, the first and second liquid crystal display panels may be,
(3) And (3) building a library before EGFR-T790M gene mutant type detection.
It should be noted that, for other auxiliary reagents or consumables used in the above preparation, PCR reaction system or kit, those skilled in the art can make routine selections according to actual needs, including but not limited to adding internal reference primers, to verify the amplification quality and assist in interpretation of the result.
In a fourth aspect, the present invention provides a method for selective amplification of EGFR-T790M gene mutation type, wherein a PCR reaction system is configured using the blocker probe according to any one of the foregoing embodiments or the primer set according to any one of the foregoing embodiments, and after a sample to be amplified is denatured, annealed and extended, the amplified product is analyzed;
the annealing temperature and the extension temperature are the same and are selected from 54-62 ℃, such as 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃,60 ℃, 61 ℃ or 62 ℃;
the sample to be amplified is derived from blood, body fluid, tissue, circulating tumor cells or cfDNA.
Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments described below and the features of the embodiments can be combined with each other without conflict.
Examples 1 to 4 and comparative examples 1 and 2
The group of embodiments respectively provide a blocker probe for amplifying EGFR-T790M gene variation, and the details are shown in the following table, wherein underlining indicates that the nucleotide is modified by a nucleic acid:
Figure BDA0003815942440000061
Figure BDA0003815942440000071
examples 5 to 8
In the embodiments of the group, MGB is added to the 3' end of the blocker probe described in embodiments 1 to 4 for modification.
Comparative examples 3 and 4
Comparative examples 3 and 4 MGB modification was added to the 3' end of the blocker probe described in comparative examples 1 and 2, respectively.
Example 9
The embodiment provides a group of amplification primers aiming at EGFR-T790M gene, and the number of the amplification primers is numbered, the amplification primers comprise an upstream primer and a downstream primer, and the rs1050171 site mutant type and wild type are covered at the same time, the specific primer information is as follows, in the sequence number "abc" of the primers in the table, a is selected from 1 or 2, wherein 1 represents the upstream primer, and 2 represents the downstream primer; b is selected from 1 or 2, wherein 1 represents rs1050171 site mutant, and 2 represents rs1050171 site wild type; c represents a specific sequence number of a certain primer:
Figure BDA0003815942440000072
example 10
This example provides a kit for detection of EGFR T790M mutant sequence, the composition of which is shown in the following table:
Figure BDA0003815942440000081
the detection procedure of the above kit is shown in the following table:
Figure BDA0003815942440000082
experimental example 1
In the experimental example, the detection results of adding the blocker probe and not adding the blocker probe are compared under the condition of using one upstream primer, and the specific steps are as follows:
DNA extracted from MDA-MB231 cells was used as a wild-type DNA template, and DNA extracted from NCI-H1975 cells was used as a mutant template. The PCR reaction system was prepared according to the following formula:
Figure BDA0003815942440000083
/>
Figure BDA0003815942440000091
the PCR amplification procedure was as follows:
Figure BDA0003815942440000092
the amplification results are shown in fig. 1 and fig. 2, and the results show that the amplification of the wild type can be obviously inhibited after the blocker probe is added compared with the method without the blocker probe set, and the mutant template is not inhibited. This demonstrates that the specificity of the reaction can be significantly increased by the addition of the blocker probe.
Experimental example 2
The experimental example randomly selects the detection results of 4 different primer groups consisting of different blocker probes provided in examples 5 to 8 and different SNP mutation type primers provided in example 9, and specifically includes the following steps:
DNA extracted from MDA-MB231 cells was used as a wild-type DNA template, and DNA extracted from NCI-H1975 cells was used as a mutant template, each of which was 15 ng/reaction. And (3) performing combined screening by using different primers and the blocker, and setting a control group without the blocker. The combination and sequence of the primers and the blocker are shown in the following table. Remarking: minimum Δ Ct = wild-type template Ct value minimum-mutant template Ct value maximum).
The primer group composition and the experimental results are as follows:
Figure BDA0003815942440000093
Figure BDA0003815942440000094
/>
Figure BDA0003815942440000101
Figure BDA0003815942440000102
Figure BDA0003815942440000103
/>
Figure BDA0003815942440000111
according to the experimental result, the comparative example 3 has a weak inhibition effect on wild type and cannot meet the requirement; comparative example 4 strongly inhibits the mutant template, which lowers the sensitivity of the kit and fails to satisfy the requirements. Examples 5 to 8 all have no obvious inhibition on mutant and wild type template, and the minimum delta Ct values are all less than-10, which meets the requirements, wherein the best effect is example 5.
Experimental example 3
In this experimental example, the detection results of the 3 different SNP wild-type upstream primers provided in example 9 were compared with the blocker probe provided in example 5, and the details are as follows:
DNA extracted from H441 cells was used as a wild-type DNA template, plasmid 002 (SEQ ID No. 21) was used as a mutant template, the amount of template used for H441 genomic DNA was 15 ng/reaction, and plasmid 002 was 4000 copy/reaction.
Wherein, H441 gDNA and plasmid 002 are both rs1050171 wild type, and MB231gDNA and H1975 gDNA are both rs1050171 mutant type. H441 gDNA and MB231gDNA were T790M wild type, H1975 gDNA and plasmid 002 were T790M mutant.
Figure BDA0003815942440000112
According to the experimental results, the primer number 123 had the best effect, the minimum Δ Ct value had the largest absolute value, no inhibition was observed on the mutant type, and the wild type was well inhibited.
Experimental example 4
The experimental example examines the amplification conditions of the single upstream primer and the double upstream primer, and concretely comprises the following steps:
DNA extracted from H441 cells is used as an rs1050171 wild type T790M DNA template, and synthesized plasmid 002 is used as an rs1050171 wild type T790M mutant template; DNA extracted from MDA-MB231 cells is used as an rs1050171 mutant T790M wild type DNA template, and DNA extracted from NCI-H1975 cells is used as an rs1050171 mutant T790M mutant template; each template was 15 ng/reaction. Four templates were tested using three combinations of primers, primer No. 111 (rs 1050171 mutant primer), primer No. 123 (rs 1050171 wild-type primer), and two primer combinations (T790M FP-1+ T790M FP-2).
The single primer combination is prepared according to the following formula:
components Volume (ul)
2x Superstart Premix plus 25
T790M FP-1/T790M FP-2(111/123) 1
T790M RP(211) 1
Blocker (example 5) 1.5
Fluorescent probe (SEQ ID No. 17) 0.5
H 2 O 6
DNA 15
The double-primer combination is prepared according to the following formula:
components 1X
2x Superstart Premix plus 25
T790M FP-1(111) 0.75
T790M FP-2(123) 0.6
T790M RP(211) 1
Blocker (example 5) 1.5
FluorescenceProbe (SEQ ID No. 17) 0.5
H 2 O 5.65
DNA 15
The PCR reaction procedure was as follows:
Figure BDA0003815942440000121
the amplification results were as follows:
Figure BDA0003815942440000122
remarking: minimum Δ Ct value = T790M mutant maximum Ct value-T790M wild type minimum Ct value.
As shown in FIGS. 3 and 4, it was found that when rs1050171 mutant primer No. 111 was used for amplification and the template was plasmid 002, the amplification efficiency of the template was significantly reduced, and the specificity was also reduced. When rs1050171 wild type primer No. 123 was used for amplification, the amplification efficiency was significantly reduced when the template was NCI-H1975. When the two templates are used in combination, the two types of templates have higher amplification efficiency and specificity and perform best.
Experimental example 5
In this example, parameters of the amplification system are optimized based on the double upstream primer system obtained in example 4, and specifically include the concentration of reaction components, the reaction volume, and the reaction procedure.
5.1 reaction component concentrations
3 to 4 concentrations of each reaction component were selected, and tested using 4 templates (using DNA extracted from H441 cells as the rs1050171 wild-type T790M wild-type DNA template, H441 genomic DNA and plasmid 002 in proportion as 0.25% T790M mutant-type template as the rs1050171 wild-type T790M mutant-type template, DNA extracted from MDA-MB231 cells as the rs1050171 mutant-type T790M wild-type DNA template, DNA extracted from MDA-MB231 cells and DNA extracted from NCI-H1975 cells in proportion as 0.25% T790M mutant-type template as the rs1050171 mutant-type T790M mutant-type template, each template being 15 ng/reaction), and specificity under each condition was examined, and the concentration with the best specificity was selected.
The optimization results of the primer concentrations for each reaction are shown in FIGS. 5 to 10, and the specific results are as follows:
primer sequence number Optimization conditions (final concentration of the Components) Conclusion (final concentration of the components)
123 150nM、120nM、100nM、180nM 120nM (FIG. 5)
111 150nM、100nM、200nM 150nM (FIG. 6)
211 200nM、140nM、260nM 200nM (FIG. 7)
Block of example 1 300nM、200nM、400nM 300nM (FIG. 8)
Fluorescent probe 100nM、70nM、130nM 100nM (FIG. 9)
Internal reference upstream primer 50nM、70nM、30nM 30nM (FIG. 10)
Internal reference downstream primer 50nM、70nM、30nM 30nM (FIG. 10)
Internal reference probe 50nM、70nM、30nM 30nM (FIG. 10)
5.2 reaction volume optimization
The reaction volume was selected in both cases of 50ul/40ul, and it was tested using 4 kinds of templates (using the templates as above), and the results are shown in FIG. 11, where WT MIN-MT MAX Δ Ct (FAM-VIC) was detected in each condition, and the larger the value, the better the reaction specificity.
5.3 reaction cycle number optimization
Experiments were performed using 40/45/50 cycles, respectively, and the number of reaction cycles with the best specificity was selected. Using a template: rs1050171 mutant: T790M wild-type template (genomic DNA extracted from MDA-MB231 cells) and T790M mutant template with a mutation frequency of 0.25% (template configured with MDA-MB231gDNA and NCI-H1975 gDNA); rs1050171 wild type: T790M wild-type template (genomic DNA extracted from H441 cells), T790M mutant template (template configured with plasmid 002 and H441 gDNA) with a mutation frequency of 0.25%, each 15 ng/reaction. The above 4 templates were used to test them, and WT MIN-MT MAX Δ Ct (FAM-VIC) was detected under each condition, and the larger the value, the better the reaction specificity. As shown in FIG. 12, the reaction cycle number was 50, and the specificity of the reaction was better and the performance was better.
5.4 annealing/extension temperature and time optimization
The annealing/extension temperature and time of the reaction are mainly optimized, and the reaction conditions with the best specificity are selected. The conditions to be tested were as follows: (1) at 58 ℃ for 60s; (2) 56 ℃ for 60s; (3) at 58 ℃ for 40s; (4) 56 ℃ for 40s. Using a template: rs1050171 mutant: T790M wild-type template (genomic DNA extracted from MDA-MB231 cells) and T790M mutant template with 0.25% mutation frequency (template configured with MDA-MB231gDNA and NCI-H1975 gDNA); rs1050171 wild type: T790M wild-type template (genomic DNA extracted from H441 cells), T790M mutant template (template configured with plasmid 002 and H441 gDNA) with a mutation frequency of 0.25%, each 15 ng/reaction. The above 4 templates were used to test them, and WT MIN-MT MAX Δ Ct (FAM-VIC) was detected under each condition, and the larger the value, the better the reaction specificity. As shown in FIG. 13, the annealing/extension temperature and time were 58 ℃ and 60 seconds, respectively, and the reaction specificity was better and the performance was better.
Application example 1
Determination of positive judgment was carried out using 142 clinical blood samples (samples from san mei gene testing technology ltd, zhahi). Clinical samples are all non-small cell lung cancer patients in advanced stage, and the patients with drug resistance are treated by the first generation/second generation EGFR TKI drugs. Plasma was obtained and cfDNA was extracted after clinical sample collection. All cfdnas were tested by digital PCR to confirm the information of the T790M mutation site. By using the kit to detect clinical samples, an ROC curve was generated, and as shown in FIG. 14, a positive judgment value of 12 was determined. The positive match rate of the sample was 96.3% and the negative match rate was 95.7%, and sensitivity comparable to digital PCR has been achieved.
Application example 2
cfDNA extracted from blood plasma of healthy people is used as a wild type DNA template, DNA extracted after DNA extracted from NCI-H1975 cells passes through a blood plasma matrix is used as a mutant template, samples with different mutation ratios are prepared, and the prepared samples are quantitatively detected by using a digital PCR method. The mutation ratios were 0.5%,0.25%, and 0.125%, respectively. And simultaneously detecting the sample by using different sample loading amounts.
The system was formulated according to the following formula:
components 1X
2x Superstart Premix plus 25
T790M FP-1(111) 0.75
T790M FP-2(123) 0.6
T790M RP(211) 1
Blocker (example 5) 1.5
Fluorescent probe (SEQ ID NO: 17) 0.5
Internal reference upstream primer (sequence 18) 0.15
Internal reference downstream primer (sequence 19) 0.15
Internal reference probe (sequence 20) 0.15
H 2 O 5.2
DNA 15
The PCR reaction procedure was as follows:
Figure BDA0003815942440000141
the results are as follows: and judging according to a threshold value 12, wherein the lowest detection limit of the site which is not less than 95 percent of detection rate is 0.125 percent of mutation rate under 7.5 ng/reaction DNA concentration.
Figure BDA0003815942440000151
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims (8)

1. The blocker probe for amplifying EGFR-T790M gene variation is characterized in that (1) the nucleotide sequence of the blocker probe covers EGFR-T790M gene mutation sites, is completely matched with wild type EGFR-T790M gene segments, and is mismatched with mutant type EGFR-T790M gene segments at the mutation sites; (2) The length of the nucleotide sequence of the blocker probe is less than 20nts, wherein the mutation site and the corresponding base in the range of 1 or 2nts on both sides of the mutation site are modified by locked nucleic acid;
the nucleotide sequence of the blocker probe is from 5 'end to 3' end
Figure FDA0003815942430000015
Or a complementary sequence which hybridizes strictly thereto, said X m The M nucleotides which are completely matched with the wild type EGFR-T790M gene segment are arranged at the upstream of the mutation site, and the X is n The method comprises the steps that n nucleotides which are completely matched with wild type EGFR-T790M gene segments are arranged at the downstream of a mutation site, wherein M + n is less than 15, the mutation site corresponds to the nucleotides which are marked in italics and bold fonts in a blocker probe, underlining indicates that the nucleotides are modified by locked nucleic acid, and at least one nucleotide in CA at the upstream of the mutation site and GC at the downstream of the mutation site is modified by locked nucleic acid;
m is less than or equal to 5.
2. The blocker probe of claim 1, wherein the nucleotide sequence of the blocker probe is selected from any one of SEQ ID Nos. 1 to 4, and the nucleotide sequence shown in SEQ ID No.1 is
Figure FDA0003815942430000011
The nucleotide sequence shown in SEQ ID No.2 is->
Figure FDA0003815942430000012
The nucleotide sequence shown as SEQ ID No.3 is
Figure FDA0003815942430000013
The nucleotide sequence shown as SEQ ID No.4 is +>
Figure FDA0003815942430000014
3. The blocker probe of claim 1 or 2, wherein the 3' end of the blocker probe is modified by extension blocking.
4. A primer set for amplifying EGFR-T790M gene variation, wherein the primer set comprises the blocker probe of any one of claims 1 to 3, two upstream primers and at least one downstream primer;
the nucleotide sequences of the two upstream primers cover SNP locus rs1050171 in front of EGFR-T790M gene, wherein the nucleotide sequence of the first upstream primer is X m1 AX n1 Said X is m1 Matching M1 nucleotides upstream of rs1050171 site with EGFR-T790M gene segment, wherein X is n1 The downstream n1 nucleotides of the rs1050171 site are matched with the EGFR-T790M gene segment, and the nucleotide sequence of the second upstream primer is X m2 GX n2 Said X is m2 Matching M2 nucleotides with EGFR-T790M gene segment at the upstream of rs1050171 site, wherein X is n2 The downstream n2 nucleotides of the rs1050171 site are matched with the EGFR-T790M gene segment, and both n1 and n2 are selected from any number value of 3-7.
5. The primer group of claim 4, wherein the 3 'ends of the two upstream primers overlap with the 5' end of the blocker probe by nucleotides, and the number of the overlapping nucleotides is 1 to 7.
6. The primer set of claim 4 or 5, wherein the primer set further comprises a fluorescent probe that binds to the antisense strand of the EGFR-T790M gene.
7. Use of the blocker probe of any one of claims 1 to 3 or the primer set of any one of claims 4 to 6, comprising:
(1) Preparing a preparation, a PCR reaction system or a kit for selectively amplifying EGFR-T790M gene mutant type; alternatively, the first and second electrodes may be,
(2) Amplification enrichment before EGFR-T790M gene mutant type detection; alternatively, the first and second electrodes may be,
(3) And (3) establishing a library before detecting the EGFR-T790M gene mutant type.
8. The EGFR-T790M gene mutation type selective amplification method without diagnosis purpose is characterized in that a PCR reaction system is configured by using the blocker probe of any claim 1 to 3 or the primer group of any claim 4 to 6, and after an amplified sample is denatured, annealed and extended, the amplified product is analyzed;
the annealing temperature and the extension temperature are the same and are selected from 54-62 ℃;
the sample to be amplified is derived from blood, body fluid, tissue, circulating tumor cells or cfDNA.
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