CN114703288B

CN114703288B - Blocker probe and primer group for amplifying EGFR-T790M gene variation and application thereof

Info

Publication number: CN114703288B
Application number: CN202210631750.3A
Authority: CN
Inventors: 齐盼盼; 唐东江
Original assignee: Zhuhai Livzon Cynvenio Diagnostics Ltd
Current assignee: Zhuhai Livzon Cynvenio Diagnostics Ltd
Priority date: 2022-06-06
Filing date: 2022-06-06
Publication date: 2022-09-13
Anticipated expiration: 2042-06-06
Also published as: CN114703288A; CN115851928A

Abstract

The invention relates to the technical field of gene detection, in particular to a blocker probe, a primer set and application thereof for amplifying EGFR-T790M gene variation. The length of the blocker probe provided by the invention is less than 20nts, and meanwhile, the thermal stability of the blocker probe combined with 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 arrangement of the two upstream primers can avoid the occurrence of false negative results when the 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 set 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, which are filed by Chinese patent office in 2019, 5/21/2019, have the application number of CN2019104222174 and are entitled as 'a non-quenched oligonucleotide probe for amplifying mutant target gene fragment and application thereof'.

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 before 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) the 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 nucleotide sequence length of the blocker probe is less than 20nts, wherein the mutation site and corresponding bases in the range of 1 or 2nts on both sides 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 ）CA C GC（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 n nucleotides which are completely matched with the wild type EGFR-T790M gene segment are positioned at the downstream of the 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 CTCATC CAGCAGCTC, wherein the nucleotide sequence shown in SEQ ID No.2 is CTCATC CAGCAGCTC, wherein the nucleotide sequence shown as SEQ ID No.3 is CTCATCA C GCAGCTCATG, the nucleotide sequence shown in SEQ ID No.4 is CTCATC CAGCAGCTCATGC。

In an alternative embodiment, the 3' end of the blocker probe is modified by extension blocking.

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 the SNP locus rs1050171 of the EGFR-T790M gene, wherein the nucleotide sequence of the first upstream primer is (X) _m1 ）A（X _n1 ) Said X is _m1 M1 matched nucleotides at the upstream of the rs1050171 site and with the EGFR-T790M gene fragment, wherein X is _n1 The nucleotide sequence of the second upstream primer is (X) in the sequence of n1 nucleotides matched with EGFR-T790M gene fragments at the downstream of the rs1050171 site _m2 ）G（X _n2 ) Said X is _m2 M2 matched nucleotides at the upstream of the rs1050171 site and with the EGFR-T790M gene fragment, wherein X is _n2 The gene fragment is characterized in that n2 nucleotides matched with the EGFR-T790M gene fragment are downstream of the rs1050171 site, and 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 1-7.

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; or,

(2) amplification enrichment is carried out before EGFR-T790M gene mutant type detection; or,

(3) and (3) establishing a library before detecting the EGFR-T790M gene mutant type.

In a fourth aspect, the present invention provides a EGFR-T790M gene mutation type selective amplification method, 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 amplification 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, the arrangement of the two upstream primers can avoid the occurrence of false negative result under the condition that G & gtA mutation of SNP locus rs1050171 exists.

The invention also provides a method for selectively amplifying EGFR-T790M gene mutation type by using the above-mentioned blocker probe or primer group, the method is based on the combination of the blocker probe and double upstream primers, through detecting clinical samples and making ROC curve, the result shows that the detection result of the clinical samples is compared with the result of the digital PCR, the positive coincidence rate is 96.3%, the negative coincidence rate is 95.7%, and the T790M locus detection rate is not less than 95% under the condition of 7.5 ng/reaction DNA and 0.125% mutation proportion, and 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 description of the embodiments or the prior art 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 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 results of optimizing the final concentration of a primer numbered 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 results of optimizing the final concentration of the fluorescent probe in Experimental example 5;

FIG. 10 shows the final concentration optimization results of the upstream and downstream primers of the internal reference and the internal reference probe 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 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) the nucleotide sequence of the blocker probe covers the mutation site of EGFR-T790M gene, is completely matched with the wild-type EGFR-T790M gene fragment, and is mismatched with the mutant EGFR-T790M gene fragment at the mutation site; (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 ）CA C GC（X _n ) Or a complementary sequence which hybridizes strictly thereto, said X _m The m nucleotides are completely matched with the wild type EGFR-T790M gene segment at the upstream of the mutation site, and the X is _n The n nucleotides which are completely matched with the wild type EGFR-T790M gene segment are positioned at the downstream of the 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 the present invention, m, n, m1, n1, m2, or n2 all represent the number of nucleotides, and are therefore all 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 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 invention provides a primer set for amplifying an 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 second upstream primerThe nucleotide sequence of an upstream primer is (X) _m1 ）A（X _n1 ) Said X is _m1 M1 matched nucleotides at the upstream of the rs1050171 site and with the EGFR-T790M gene fragment, wherein X is _n1 The nucleotide sequence of the second upstream primer is (X) in the sequence of n1 nucleotides matched with EGFR-T790M gene fragments at the downstream of the rs1050171 site _m2 ）G（X _n2 ) Said X is _m2 M2 matched nucleotides at the upstream of the rs1050171 site and with the EGFR-T790M gene fragment, wherein X is _n2 The gene fragment is characterized in that n2 nucleotides matched with the EGFR-T790M gene fragment are downstream of the rs1050171 site, and n1 and n2 are selected from any number value of 3-7. For m1 and m2 of the two upstream primers, after determining the combined sequence of the upstream primer 3' end on the blocker probe, the conventional selection can be carried out according to the requirements of the actual PCR reaction system.

In alternative embodiments, the 3 'ends of the two forward primers overlap the 5' end of the blocker probe by 1 to 7 nucleotides, such as 1, 2, 3, 4, 5, 6, or 7 nucleotides. The partially overlapped nucleotide sequence 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 the 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 strands and amplified antisense strands of the EGFR-T790M gene is obviously different, and when the template strands of the EGFR-T790M gene combined by the fluorescent probe and the blocker probe are different strands, the specificity and sensitivity of the reaction can be improved.

(2) amplification enrichment before detection of EGFR-T790M gene mutant type; or,

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.

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 provides 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:

number of implementation	Nucleotide sequence of blocker probe (5 '-3')
		Example 1	CTCATC A C G CAGCTC （ SEQ ID No.1 ）
Example 2	CTCATC A C GCAGCTC （ SEQ ID No. 2 ）
		Example 3	CTCATCA C G C AGCTCATG （ SEQ ID No. 3 ）
Example 4	CTCATC A C GCAGCTCATGC （ SEQ ID No. 4 ）
		Comparative example 1	GCTCATCA C GCAGCTCAT （ SEQ ID No. 5 ）
Comparative example 2	CA GCTCATCA C G CAGCTCAT （ SEQ ID No. 6 ）

Examples 5 to 8

In the embodiments of the group, MGB is added to the 3' end of each of the blocker probes described in the embodiments 1-4 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 amplification primers are numbered, and comprise an upstream primer and a downstream primer, and also cover rs1050171 site mutant and wild type, 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 type, and 2 represents rs1050171 site wild type; c represents the specific sequence number of a certain primer:

primer sequence Number (C)	Primers Model (III)	SNP mutant	SNP wild type
				111	Upstream guide Article (A)	CCGTGCAACTCATCA （ SEQ ID No.7 ）
112	Upstream guide Article (A)	ACCGTGCAACTCATCA （ SEQ ID No.8 ）
				113	Upstream guide Article (A)	CACCGTGCAACTCATCA （ SEQ ID No.9 ）
114	Upstream guide Article (A)	CCACCGTGCAACTCATCA （ SEQ ID No.10 ）
				121	Upstream guide Article (A)		CCGTGCAACTCATCA （ SEQ ID No.11 ）
122	Upstream guide Article (A)		CACCGTGCAGCTCAT （ SEQ ID No.12 ）
				123	Upstream guide Article (A)		CCGTGCAGCTCATC （ SEQ ID No.13 ）
211	Downstream guide Article (A)	GCAGGTACTGGGAGCCAATA （ SEQ ID No.14 ）	GCAGGTACTGGGAGCCAATA （ SEQ ID No.14 ）
				212	Downstream guide Article (A)	AGCAGGTACTGGGAGCCAATA （ SEQ ID No.15 ）	AGCAGGTACTGGGAGCCAATA （ SEQ ID No.15 ）
213	Downstream guide Article (A)	GCAGGTACTGGGAGCCAAT A （ SEQ ID No.16 ）	GCAGGTACTGGGAGCCAAT A （ SEQ ID No.16 ）

Example 10

This example provides a kit for detection of the EGFR T790M mutant sequence, the composition of which is shown in the following table:

the detection procedure of the above kit is shown in the following table:

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:

name of reagent	Volume (ul)
		2x Superstart Premix plus	25
T790M upstream primer: primer No. 111	1
		T790M downstream primer: primer No. 211	1
Blocker (example 1)/not add Blocker group Water supplement	1.5
		PROBE	0.5
H ₂ O	6
		DNA	15

The PCR amplification procedure was as follows:

the amplification results are shown in fig. 1 and fig. 2, and the results show that compared with the method without the addition of the blocker probe set, the wild-type amplification can be obviously inhibited after the addition of the blocker probe, and meanwhile, the mutant-type 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

In this experimental example, the detection results of 4 different primer groups consisting of the different blocker probes provided in examples 5 to 8 and the different SNP mutation type primers provided in example 9 were randomly selected, and specifically the following were obtained:

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 experimental results are as follows:

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 have no obvious inhibition on mutant types and wild type templates, and the minimum delta Ct values are all less than-10, so that the requirements are met, wherein the best effect is shown in 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 rs1050171 wild type, and MB231 gDNA and H1975 gDNA are rs1050171 mutant type. H441 gDNA and MB231 gDNA are T790M wild type, H1975 gDNA and plasmid 002 are T790M mutant.

According to the experimental results, the primer number 123 had the best effect, the minimum Δ Ct value was the largest in absolute value, no inhibition was observed for the mutant type, and the wild type was inhibited well.

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 a rs1050171 wild type T790M wild type DNA template, and synthesized plasmid 002 is used as a rs1050171 wild type T790M mutant template; DNA extracted from MDA-MB231 cells is used as a wild type DNA template of rs1050171 mutant T790M, and DNA extracted from NCI-H1975 cells is used as a mutant template of rs1050171 mutant T790M; each template was 15 ng/reaction. Four templates were tested using three combinations of primers, primer No. 111 (rs1050171 mutant primer), primer No. 123 (rs1050171 wild-type primer), and two primer combinations (T790M FP-1+ T790M FP-2).

The single primer combination is prepared into a system according to the following formula:

	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:

	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
		H ₂ O	5.65
DNA	15

The PCR reaction procedure was as follows:

the amplification results were as follows:

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 the 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 concentration

Selecting 3-4 concentrations of each reaction component, using 4 templates (DNA extracted from H441 cells is used as rs1050171 wild type T790M wild type DNA template, H441 genomic DNA and plasmid 002 are proportionally configured into 0.25% T790M mutant type template as rs1050171 wild type T790M mutant type template, DNA extracted from MDA-MB231 cells is used as rs1050171 mutant type T790M wild type DNA template, DNA extracted from MDA-MB231 cells and DNA extracted from NCI-H1975 cells are proportionally configured into 0.25% T790M mutant type template as rs 1050171T 790M mutant type template, and each template is 15 ng/reaction), testing the mutant type DNA, detecting the specificity under each condition, and selecting the concentration with the best specificity.

The optimization results of the concentrations of the primers in each reaction are shown in fig. 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 same template 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-MB231 gDNA 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 0.25% mutation frequency, 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 optimized mainly, and the reaction conditions with the best specificity are selected. The conditions to be tested were as follows: firstly, 58 ℃ and 60 s; ② 56 ℃ for 60 s; ③ 58 ℃ for 40 s; 56 ℃ for 40 s. 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-MB231 gDNA 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 0.25% mutation frequency, 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 60s, respectively, and the reaction specificity was better and the performance was better.

Application example 1

Determination of positive judgment values was performed using 142 clinical blood samples (samples from shengmei gene testing technologies ltd, bazai). 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:

	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:

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.

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.

SEQUENCE LISTING

<110> Zhuhaishengmei bio-diagnostic technology Co., Ltd

<120> blocker probe and primer set for amplifying EGFR-T790M gene variation and application thereof

<160> 21

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（7）...（9）

<223> blocker 1

<400> 1

ctcatcacgc agctc 15

<210> 2

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（7）...（8）

<223> blocker 2

<400> 2

ctcatcacgc agctc 15

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（8）and（10）

<223> blocker 3

<400> 3

ctcatcacgc agctcatg 18

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（7）...（8）

<223> blocker 4

<400> 4

ctcatcacgc agctcatgc 19

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（9）

<223> blocker 5

<400> 5

gctcatcacg cagctcat 18

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（11）...（12）

<223> blocker 6

<400> 6

cagctcatcacg cagctcat 20

<210> 7

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP mutant type upstream primer 1

<400> 7

ccgtgcaact catca 15

<210> 8

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP mutant type upstream primer 2

<400> 8

accgtgcaac tcatca 16

<210> 9

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP mutant type upstream primer 3

<400> 9

caccgtgcaa ctcatca 17

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP mutant type upstream primer 4

<400> 10

ccaccgtgca actcatca 18

<210> 11

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP wild-type forward primer 1

<400> 11

ccgtgcaact catca 15

<210> 12

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP wild-type forward primer 2

<400> 12

caccgtgcag ctcat 15

<210> 13

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> SNP wild-type forward primer 3

<400> 13

ccgtgcagct catc 14

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> downstream primer 1

<400> 14

gcaggtactg ggagccaata 20

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> downstream primer 2

<400> 15

agcaggtact gggagccaat a 21

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<221> locked nucleic acid modification

<222>（20）

<223> downstream primer 3

<400> 16

gcaggtactg ggagccaata 20

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> fluorescent probe

<400> 17

tgtctttgtg ttcccggaca tagt 24

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> internal reference upstream primer

<400> 18

acaagacttc cagccacaga a 21

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> internal reference downstream primer

<400> 19

ggtgccaagg agataacaga ga 22

<210> 20

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> internal reference probe

<400> 20

tctaagatct gcttcctggt gtgt 24

<210> 21

<211> 892

<212> DNA

<213> Artificial Sequence

<220>

<223> plasmid 002 sequence

<400> 21

cacaattgcc agttaacgtc ttccttctct ctctgtcata gggactctgg atcccagaag 60

gtgagaaagt taaaattccc gtcgctatca aaacatctcc gaaagccaac aaggaaatcc 120

tcgatgtgag tttctgcttt gctgtatgcg aagccacact gacgtgcctc tccctccctc 180

caggaagcct acgtgatggc cagcgtggac aacccccacg tgtgccgcct gctgggcatc 240

tgcctcacct ccaccgtgca gctcatcatg cagctcatgc ccttcggctg cctcctggac 300

tatgtccggg aacacaaaga caatattggc tcccagtacc tgctcaactg gtgtgtgcag 360

atcgcaaagg taatcaggga agggagatac ggggagggca gagcttcttc ccatgatgat 420

ctgtccctca cagcagggtc ttctctgttt cagggcatga actacttgga ggaccgtcgc 480

ttggtgcacc gcgacctggc agccaggaac gtactggtga aaacaccgca gcatgtcaag 540

atcacagatt ttgggcgggc caaactgctg ggtgcggaag agaaagaata ccatgcagaa 600

ggaggcaaag taaggaggtg gctttaggtc agccagcatt ttcctgacac cagggaccag 660

taacctgact ggttaacagc agtcctttgt aaacagtgtt ttaaactctc ctagtcaata 720

tccaccccat ccaatttatc aaggaagaaa tggttcagaa aatattttca gcctacagtt 780

atgttcagtc acacacacat acaaaatgtt ccttttgctt ttaaagtaat ttttgactcc 840

cagatcagtc agagccccta cagcattgtt aagaaagtat ttgatttttg tc 892

Claims

1. The blocker probe for amplifying EGFR-T790M gene variation is characterized in that 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 CTCATC CAGCAGCTC, wherein the nucleotide sequence shown in SEQ ID No.2 is CTCATC CAGCAGCTC, wherein the nucleotide sequence shown as SEQ ID No.3 is CTCATCA C GCAGCTCATG, the nucleotide sequence shown in SEQ ID No.4 is CTCATC CAGCAGCTCATGC, respectively; the 3' end of the blocker probe is subjected to extension blocking modification, the nucleotide which is in an italic font and is marked in a bold manner in the blocker probe corresponds to a mutation site, and underlining indicates that the nucleotide is subjected to nucleic acid locking modification.

2. A primer set for amplifying EGFR-T790M gene variation, wherein the primer set comprises the blocker probe of claim 1, 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 M1 matched nucleotides at the upstream of the rs1050171 site and with the EGFR-T790M gene fragment, wherein X is _n1 The nucleotide sequence of the second upstream primer is X, wherein n1 downstream nucleotides of the rs1050171 site are matched with the EGFR-T790M gene fragment _m2 GX _n2 Said X is _m2 Matching m2 nucleotides with EGFR-T790M gene fragment at upstream of rs1050171 site, wherein X is _n2 The gene fragment is characterized in that n2 nucleotides matched with the EGFR-T790M gene fragment downstream of the rs1050171 locus, wherein n1 and n2 are selected from any numerical value of 3-7;

the 3 'ends of the two upstream primers are overlapped with the 5' end of the blocker probe by nucleotides, and the number of the overlapped nucleotides is 1-7.

3. The primer set of claim 2, further comprising a fluorescent probe that binds to the antisense strand of the EGFR-T790M gene.

4. Use of the blocker probe of claim 1 or the primer set of claim 2 or 3, comprising:

5. The EGFR-T790M gene mutation type selective amplification method which is not used for diagnosis is characterized in that a PCR reaction system is configured by using the blocker probe of claim 1 or the primer group of claim 2 or 3, and an amplification product is analyzed after an amplification sample is denatured, annealed and extended;

the annealing temperature and the extension temperature are the same and are selected from 54-62 ℃.