CN112824535A

CN112824535A - Primer composition for gene mutation multiplex detection and kit thereof

Info

Publication number: CN112824535A
Application number: CN201911146751.3A
Authority: CN
Inventors: 葛志琪; 赵雨航; 李锦�
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2021-05-21
Anticipated expiration: 2039-11-21
Also published as: CN112824535B

Abstract

The invention discloses a primer composition for gene mutation multiplex detection and a kit thereof; the primer composition comprises a second upstream primer, a probe, a downstream primer and at least two different first upstream primers; wherein the first upstream primer comprises an upstream detection zone and a target sequence binding zone: the upstream detection region has a portion (a) having the same sequence as the second upstream primer and a portion (b) having the same sequence as the probe; the 3' end of the target sequence binding region has an amplification decision site which is complementary to a variation detection site on the mutant target sequence and has upstream thereof a mismatch region consisting of one or more bases which is not complementary to the target sequence; the different first forward primers differ for the target sequence binding region. Primer compositions of the invention also include a wild-type blocker. The primer composition or the kit is used for detecting multiple gene mutations, so that the detection sensitivity and specificity can be improved, and the detection cost can be reduced; and the detection readiness is higher.

Description

Primer composition for gene mutation multiplex detection and kit thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a digital PCR primer composition for gene multiple mutation detection and a kit thereof.

Background

The digital PCR (digital PCR, dPCR) technology is an absolute nucleic acid molecule quantitative technology, and a fluorescent quantitative PCR reaction system is distributed into thousands of independent nanoliter-level microreactors by using the principle of limiting dilution, so that each microreactor contains or does not contain 1 or more copies of target nucleic acid molecules (DNA targets), and single-molecule template PCR amplification is carried out simultaneously. Different from the method of acquiring fluorescence during each amplification cycle of the fluorescent quantitative PCR, the digital PCR independently acquires the fluorescence signal of each reaction unit after the amplification is finished, and finally obtains the original copy number or concentration of the target molecule according to the principle of Poisson distribution and the proportion of positive/negative reaction units.

Compared with fluorescent quantitative PCR, the digital PCR can carry out accurate absolute quantitative detection without depending on Ct value and a standard curve, and has the advantages of high sensitivity and high accuracy. Because the digital PCR only judges the 'existence/nonexistence' of two amplification states during result judgment, the intersection point of a fluorescence signal and a set threshold line does not need to be detected, and the method does not depend on the identification of a Ct value completely, so that the influence of the amplification efficiency on the digital PCR reaction and the result judgment is greatly reduced, and the tolerance capability on PCR reaction inhibitors is greatly improved. In addition, the process of allocating the reaction system in the digital PCR experiment can greatly reduce the concentration of the background sequence having competition effect with the target sequence locally. Therefore, digital PCR represents a significant advantage over traditional fluorescent quantitative PCR when quantification and detection of low copy number differential nucleic acid molecules with high sensitivity is required due to its higher sensitivity and accuracy. In particular, rare mutations are detected in a complex background, such as by detecting circulating tumor dna (ctdna) in the peripheral blood of tumor patients, which shows great advantages and is often used in early screening, medication guidance, prognosis and relapse monitoring applications for tumor patients.

The existing gene mutation detection reagent adopts a TaqMan probe method, a primer specificity distinguishing method and a blocked wild type amplification method. The method comprises the steps of firstly, generally adopting competitive probes respectively aiming at a mutant type and a wild type, designing two probes to be competitive probes similar to point mutation detection, or designing a specific probe on a gene wild type template, designing a general probe capable of indicating the wild type template and the mutant type template at the same time in other conserved regions of a gene, and quantifying the concentration of the mutant type template by using the concentration difference measured by the two probes. The primer specificity distinguishing method is to design different mutant primers to distinguish different mutation types specifically, design a probe and a downstream primer in a downstream conserved region, and design a universal probe capable of indicating wild type and mutant template simultaneously and upstream and downstream primers in other conserved regions of the gene. ③ the wild-type amplification blocking method usually adopts Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA) modification to block the amplification of the wild-type template, and at present, these modifications are expensive and not favorable for clinical application. Therefore, a primer probe design method with high sensitivity, good specificity and low cost is needed to meet the requirement of clinical detection by using a digital PCR technology.

The invention is improved on the basis of a common detection method, simultaneously detects a plurality of clinically common mutant genes with higher mutation frequency in one reaction tube, and does not carry out typing treatment on the mutant types. One tube can detect multiple mutation types simultaneously, certain challenge is provided, on one hand, the sensitivity of digital PCR is extremely high, and false positive is easy to occur; on the other hand, since a plurality of primer probes exist in one tube and the mutual interference effect between the primer probes cannot be ignored, the requirement on the specificity of the primer probes is extremely high, and cross reaction needs to be avoided as much as possible. Therefore, when a multiple gene mutation detection system is constructed by using a digital PCR technology, how to improve the specificity of the system, reduce the mutual interference among primers and protect the detection performance of the most common clinical mutation subtype is particularly important.

Disclosure of Invention

In order to solve the above problems, the present invention provides a gene mutation multiplex detection primer composition and a kit based on digital PCR; solves the problems that the accuracy of detection and analysis is influenced and false positive results are caused due to the mutual interference of a plurality of primers of a gene mutation multiple detection system.

In one aspect, a primer composition for multiplex detection of gene mutations, the primer composition comprising a second forward primer, a probe, a reverse primer and at least two different first forward primers;

wherein the first upstream primer comprises an upstream detection region and a target sequence binding region from 5 'end to 3' end in sequence:

(1) the upstream detection zone comprises from 5 'end to 3' end: a portion (a) having the same sequence as the second forward primer, and a portion (b) having the same sequence as the probe, and

(2) the 3' end of the target sequence binding region has an amplification decision site which is complementary to a variation detection site on the mutant target sequence, and the upstream of the amplification decision site has a mismatch region consisting of one or more bases which is not complementary to the target sequence;

said different first upstream primers being different in said target sequence binding region, said upstream detection region being identical or different in part (a), and said upstream detection region being identical in part (b);

in some embodiments, the different first upstream primer, the target sequence binding region, and the portion (a) of the upstream detection region are different from the portion (a), the portion (b), the target sequence binding region, or none of the portions of the upstream detection region.

The second upstream primer has a sequence identical to the portion (a) of the upstream detection region and is not complementary-paired with any target nucleic acid sequence, the first upstream primer and the downstream primer, and the sequence thereof can be freely changed.

The probe has a sequence identical to the portion (b) of the upstream detection region and is not complementary-paired with any target nucleic acid sequence, the first upstream primer and the downstream primer, and the sequence thereof can be freely changed.

In some embodiments, the primer composition further comprises a wild-type blocker (blocker).

The wild-type blocker is a blocker for blocking amplification of a wild-type gene sequence.

In some embodiments, the wild-type blocker has a Tm greater than or equal to the Tm of the first upstream primer > the Tm of the second upstream primer.

The Tm value of the first forward primer refers to the Tm value of the portion of the first forward primer that is paired with the template.

During digital PCR amplification, the wild-type blocker is preferentially combined with the wild-type template, so that the non-specific combination of the first upstream primer and the wild-type template is avoided; then, the first upstream primer is specifically combined with the mutant template to pre-amplify the mutant template; the second upstream primer then amplifies the complementary downstream bound probe to the enriched mutant template, thereby detecting the corresponding target nucleic acid sequence.

The second forward primer and probe pair binds to the pre-amplified product only after the first forward primer pair specifically amplifies the target nucleic acid, thereby initiating the probe and separating the reporter and quencher groups from each other, releasing a detectable signal.

In some embodiments, the wild-type blocker is an oligonucleotide complementary to a wild-type template comprising a common mutation region sequence; the length of the oligonucleotide is 13-30 bp, the Tm value is 50-80 ℃, and the GC content is 40-80%; the 3' terminus of the blocker is modified by a non-hydroxyl group including, but not limited to, dideoxy base modification, C6 spacer, C3 spacer, phosphorylation modification, amino, halo, or other modification; in further embodiments, the 3' terminus of the blocker is dideoxy modified.

In some embodiments, the first upstream primer has a length of 50-90 bp, a Tm value of 50-80 ℃, and a GC content of 40-80%.

In some embodiments, the length of the second upstream primer is 13-30 bp, the Tm value is 50-80 ℃, and the GC content is 40-80%.

In some specific embodiments, the length of the downstream primer is 15-30 bp, the Tm value is 55-75 ℃, and the GC content is 40-80%.

In the present invention, the same downstream primer is preferably used for the first upstream primer and the second upstream primer.

In some embodiments, the Tm value of the wild-type blocker is 5 ℃ to 20 ℃ higher than the Tm value of the first upstream primer; alternatively, the Tm value of the wild-type blocker is the same as the Tm value of the first upstream primer. In a further preferred embodiment, the Tm value of the wild-type blocker is 10 ℃ to 15 ℃ higher than the Tm value of the first upstream primer.

In some embodiments, the Tm value of the first forward primer is 5 ℃ to 20 ℃ higher than the Tm value of the second forward primer; in a further preferred embodiment, the Tm value of the first upstream primer is 10 ℃ to 15 ℃ higher than the Tm value of the second upstream primer.

In some embodiments, on the different first upstream primers, portion (a) of the upstream detection region is the same or different, and portion (b) of the upstream detection region is the same or different.

When parts (a) and (b) of the upstream detection region on the first upstream primer are changed, the corresponding second upstream primer and probe are also changed accordingly.

In some embodiments, the downstream primer is complementary to the target sequence at a position 1-150 bp downstream of the mutation detection site.

In some embodiments, the mismatch region in the target sequence binding region on the first forward primer is 1 to 15 bases in length; the distance between the amplification determining site and the mismatch region on the first upstream primer is 1-15 bases.

In some embodiments, the probe is modified with a reporter group that is detectable only after the probe is hydrolyzed. In further embodiments, the probe carries a reporter group and a quencher group. In still further embodiments, the reporter group can be a fluorophore selected from the group consisting of: : FAM, HEX, VIC, ROX, Cy5, Cy3, etc.; the quencher group may be selected from the group consisting of: TAMRA, BHQ1, BHQ2, BHQ3, DABCYL, QXL, DDQI, etc. In some embodiments, the probe does not carry any other modifications besides the reporter and quencher, e.g., MGB, LNA, PNA, BNA, SuperBase, etc. In a preferred embodiment, the probe of the invention is a Taqman probe. In a preferred embodiment, the reporter is located at the 5 'end of the probe and the quencher is located at the 3' end of the probe.

In a second aspect, a gene mutation multiplex detection kit comprises the primer composition.

In some embodiments, the kit further comprises a reference primer composition, wherein the reference primer composition is a reference upstream primer rF, a reference downstream primer rR and a reference probe rP which are designed to be complementary to a conserved sequence at a non-mutated and conserved site of the target sequence.

The reference primer composition is used to detect the total amount of template, including wild type and mutant.

In some embodiments, the rF and rR primers have a length of 5-30 bp, a Tm value of 55-75 ℃ and a GC content of 40-80%.

The 5 'end of the probe rP is modified by a fluorescent group, the 3' end of the probe rP is modified by a quenching group, the length of the probe is 15-30 bp, the Tm value is 55-75 ℃, and the GC content is 40-80%.

In a third aspect, a primer composition for detecting a mutation in an exon 19 of the EGFR gene, the primer composition comprising:

a first upstream primer SEQ ID NO. 15, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6;

a second upstream primer SEQ ID NO. 9;

probe SEQ ID NO 10;

the downstream primer is SEQ ID NO. 11.

In some embodiments, a primer composition for detecting a mutation in an exon 19 of the EGFR gene, the primer composition comprising:

the first upstream primer is SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6;

the second upstream primer SEQ ID NO. 8, SEQ ID NO. 9;

probe SEQ ID NO 10;

the downstream primer is SEQ ID NO. 11.

The 6 first upstream primers are respectively used for amplifying 19 clinically common EGFR gene 19 exon deletion mutants with higher mutation frequency, and the mutation types are not subjected to typing treatment. One tube detects 19 mutants, and other 5 first upstream primers share another second upstream primer except for the first upstream primer of the mutation type with the mutation frequency higher than 50% or the highest mutation frequency.

In some embodiments, the primer composition for detecting an exon 19 mutation in the EGFR gene further comprises a wild-type blocker.

The wild-type blocker is an oligonucleotide which is complementary to a wild-type template and comprises a common mutation region sequence; the length of the oligonucleotide is 13-30 bp, the Tm value is 50-80 ℃, and the GC content is 40-80%; the 3' terminus of the blocker is modified by a non-hydroxyl group including, but not limited to, dideoxy base modification, C6 spacer, C3 spacer, phosphorylation modification, amino, halo, or other modification; in further embodiments, the 3' terminus of the blocker is dideoxy modified.

In a fourth aspect, the primer composition is used for preparing a kit for detecting the mutation of the 19 exon of the EGFR gene.

Compared with the prior art, the technical scheme of the invention has the advantages that:

(1) the false positive is less: the blocker oligonucleotide introduced into the system is completely complementary with the antisense strand of the specific region (common mutation deletion region) of the wild-type template, and is preferentially and closely combined with the wild-type template in the PCR reaction process to competitively inhibit the non-specific combination of the mutant first upstream primer F1 and the wild-type template, so that the generation of false positive fluorescent signals is reduced, and the method is particularly suitable for mutation detection by using a digital PCR method.

(2) The specificity is high: in order to reduce cross reaction and protect the detection of the most common clinical mutant subtype p.E746_ A750del (64.6%) from being interfered by other mutant type first upstream primers F1, a second upstream primer F2 for the type is additionally added, namely 2 different F2 primers are added into the system, and 5F 1 primers share the F2 primer except that one F2 primer is used for the F1 primer of the mutant subtype p.E746_ A750del (64.6%). The scheme can improve the amplification efficiency when detecting the mutant subtype p.E746_ A750del (64.6 percent), and can ensure that the detection of other mutant subtypes is not influenced, the threshold division is clear, and the detection result is accurate and reliable.

(3) The cost is low: the invention uses 6 mutant F1 primers to detect 19 mutant subtypes, different mutant subtypes use one probe P together, multiple mutant subtypes can be detected simultaneously without adding multiple probes P, in addition, the primer probes do not need expensive PNA modification or LNA modification, the use cost of the primer probes is greatly reduced, and the invention is beneficial to clinical use.

(4) The target nucleic acid sequence is short: the primer probe design method has the advantages that the length of the target nucleic acid to be detected is short, and because the probe P can be matched and combined with the complementary sequence at the 5' end of the first upstream primer F1 after the pre-amplification is finished, the actually matched and combined part with the target nucleic acid sequence only has two parts: the sequence of the complementary pair of the 3' end of F1 with the target nucleic acid and the sequence of the downstream primer R. Compared with the primer probe design method, the TaqMan probe method and the ARMS method are more limited by the sequence of the target nucleic acid fragment to be detected, because the two methods both need at least three parts to be matched with the target nucleic acid sequence: an upstream primer, a probe and a downstream primer. Therefore, compared with the TaqMan probe method and the ARMS method, the primer probe design method provided by the invention has the advantage that the length of the target nucleic acid to-be-detected fragment is shorter. In the highly fragmented free DNA detection, because the fragmentation of the DNA is random, a shorter detection fragment can detect more DNA targets, thereby greatly improving the detection sensitivity.

(5) The requirements on the target nucleic acid sequence are low: similar to the above advantages, in the primer probe design method of the present invention, since the probe P can be coupled to the complementary sequence at the 5' end of the first upstream primer F1 after the pre-amplification is completed, the portion actually coupled to the target nucleic acid sequence has only two parts: the 3' end sequence of F1 and the sequence of the downstream primer R. Therefore, the design difficulty of the primer probe is lower compared with that of a TaqMan probe method and ARMS when a complex target nucleic acid sequence is detected.

(6) The algorithm requirement on software is reduced: the primer probe design method of the invention makes the division of the wild type and the mutant threshold more clear and obvious, so that software can automatically divide the threshold more easily to obtain the mutation ratio.

(7) The application range is wide: the reaction system can detect short-fragment DNA smaller than 100bp, has good tolerance to PCR inhibitors, and can be suitable for nucleic acid detection of various sample types, including formalin-fixed paraffin-embedded tissue (FFPE) samples, fresh tissue samples, peripheral blood samples, urine samples, lavage fluid samples, cerebrospinal fluid samples, artificially cultured cell line samples, artificially synthesized plasmid samples and the like.

Drawings

FIG. 1 shows a method for designing a primer probe according to the present invention; wherein, FIG. 1A is a structural diagram of a first upstream primer F1, which comprises an upstream detection region and a target sequence binding region from 5 ' end to 3 ' end in sequence, wherein, the 3 ' end near the target sequence binding region can be added with mismatch; the upstream detection zone comprises from the 5 'to the 3' end: the same sequence as the second forward primer F2 and probe P. FIG. 1B shows the detection principle of the scheme of the present invention, first, the blocker oligonucleotide specifically binds to the wild-type template, the mutant type first upstream primer F1 and the reverse primer R specifically enrich the mutant type target nucleic acid sequence to be detected, and a new segment of sequence complementary to the first upstream primer "upstream detection zone" is added for the subsequent pairing identification of the second upstream primer F2 and the probe P. After the target nucleic acid template is enriched, the second upstream primer F2 and the probe P identify a sequence complementary to the 'upstream detection zone' of the first upstream primer on the pre-amplification product, then form a primer pair with the reverse primer R and perform template amplification, and release a fluorescent signal by the TaqMan probe principle. In the whole PCR reaction process, a primer pair amplification template (mutant type and wild type) is formed by the reference primer rF and the reference primer rR, and a fluorescent signal is released by a TaqMan probe rP;

FIG. 2 is a two-dimensional graph of a negative control sample detected in example 1; the first channel signal is a FAM signal, and the second channel signal is a HEX signal;

FIG. 3 is a two-dimensional diagram of a sample for detecting a mutation (p.E746_ A750del deletion mutation) in example 1;

FIG. 4 is a graph of the concentration and abundance of mutations detected in the mutant sample (p.E746_ A750del deletion mutation) and the negative control sample in example 1;

FIG. 5 is a one-dimensional graph of the detection of different signal channels of a mock clinical sample (clinically common subtype) in example 2; 5A is FAM signal indicating the number of mutants detected; 5B is a HEX signal indicating the number of wild type detected;

FIG. 6 is a two-dimensional graph of a negative control sample detected in example 3;

FIG. 7 is a two-dimensional graph of a sample for detecting mutations (p.E746_ A750del deletion mutations) in example 3;

FIG. 8 shows a primer probe design method described in example 4; first, a first upstream primer F1 and a reverse primer R are used for specifically enriching a target nucleic acid sequence (mutant/wild type) to be detected, and a sequence complementary to an "upstream detection zone" of the first upstream primer is added for the subsequent pairing identification of a second upstream primer F2 and a probe P. After the target nucleic acid template is enriched, a second upstream primer F2 and a probe P are enabled to recognize a sequence complementary to an upstream detection area of a first upstream primer on a pre-amplification product by utilizing the change of annealing temperature, then a primer pair is formed with a reverse primer R for template amplification, and a fluorescent signal is released by the TaqMan probe principle;

FIG. 9 is a two-dimensional graph of a negative control sample for the test of example 4;

FIG. 10 is a two-dimensional graph of a sample for detecting mutations (p.E746_ A750del deletion mutations) in example 4;

FIG. 11 is a two-dimensional graph of a negative control sample for a comparative example test;

FIG. 12 is a two-dimensional graph of a mutant sample (p.E746_ A750del deletion mutation) detected in comparative example.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. 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 application.

Definition of

The term "conserved region" refers to a nucleotide segment in a DNA molecule that remains substantially unchanged in sequence, structure or function.

The term "sample" refers to a biological sample from any subject that is used to detect the presence or absence of one or more diseases, such as influenza virus or other respiratory symptoms or diseases. Such samples may include tissue samples from skin or any organ, blood, plasma, mucus, saliva, etc.

The term "probe" refers to an oligonucleotide that can selectively hybridize to amplified target nucleic acid under suitable conditions. The probe sequence may be a sense (e.g., complementary) sequence (+) or an antisense (e.g., reverse complementary) sequence (-) to the coding/sense strand. In a kinetic PCR format, the detection probe may consist of an oligonucleotide with a 5 'reporter dye (R) and a 3' quencher dye (Q). Fluorescent reporter dyes (i.e., FAM (6-carboxyfluorescein), etc.) are typically located at the 3' end. The detection probe serves as a TAQMAN probe during amplification and detection.

The term "nucleic acid" as used herein refers to a polynucleotide, such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include equivalents, analogs of RNA or DNA consisting of nucleotide analogs, as well as single-stranded (sense or antisense) and double-stranded polynucleotides applicable in the context of the instant claims.

The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, which can be attached to a nucleic acid or protein by covalent or non-covalent interactions (e.g., by ionic or hydrogen bonding, or by immobilization, adsorption, etc.). Labels typically provide a signal for detection by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzyme activity, and the like. Specific examples of labels include fluorophores, chromophores, radioactive atoms (particularly 32p and 125I), electron-dense reagents, enzymes, and ligands with specific binders.

The epidermal Growth Factor receptor EGFR (epidermal Growth Factor receptor) is a transmembrane tyrosine kinase receptor, the activation of the kinase domain of the receptor is related to a plurality of signal transduction pathways such as cancer cell proliferation, metastasis and apoptosis, and the high expression or abnormal expression of the EGFR gene exists in a plurality of solid tumors. The tyrosine kinase active site of EGFR is mainly positioned between No. 18-21 exons. Therefore, activation mutations and drug resistance mutations of EGFR have also been focused on. The most common activating mutations are a deletion mutation of exon 19 (approximately 45%) and a mutation of exon 21 at point L858R (approximately 40%), both of which result in activation of the tyrosine kinase domain. After tumor patients carrying EGFR mutation are treated by EGFR tyrosine kinase inhibitor drugs (EGFR-TKIs), the Progression Free Survival (PFS) of the patients can be remarkably prolonged and the drug response rate can be improved. Therefore, the detection and monitoring of the EGFR gene mutation state play an important role in the formulation of a clinical first-line medication scheme, the curative effect of the medicament, the prognosis and the monitoring of tumor outcome.

The mutation frequency of the EGFR gene is as high as 20% to 40% in patients of asian small cell lung cancer (NSCLC), especially in patients of asian NSCLC who are Non-smoking adenocarcinoma women. The most common EGFR gene activating mutation-19 exon deletion mutation can be up to 30-40 types, and more than 50% of deletion mutation types are accompanied by insertion mutation. The Wu-Longzhong professor of Lung cancer institute in Guangdong province classifies the mutant subtypes of 440 NSCLC patients with EGFR gene 19 exon deletion mutation, and the most common 19 # exonic mutant subtypes are p.E746_ A750del (64.6%), followed by p.L747_ P753> S (8.4%), p.L747_ T751 (4.3%), p.L747_ A750> P (3.4%), p.E746_ S752> V (2) (3.2%), p.E746_ S752> V (1.6%), p.L747_ S752del (1.4%), and the results of the research are basically consistent with the reports of other researchers (Su J, Zhong W, Zhang X, et al. Molecular characteristics and clinical outlying of EGFR 19 subunit type C [ TK J., TK J. 7 ] S7). When NSCLC patients of different subtypes are treated by EGFR-TKIs medicaments, the clinical effect has no significant difference, and therefore, typing detection is not needed.

The present invention provides a primer composition for detecting a 19 exon deletion mutation in the EGFR gene (hereinafter referred to as "the primer composition of the present invention"):

the first upstream primer F1 is a primer aiming at common mutation deletion of an exon of an EGFR gene 19, the sequences are respectively SEQ ID NO. 1-6, the blocker oligonucleotide sequence is SEQ ID NO. 7, the sequence of the second upstream primer F2 is SEQ ID NO. 8-9, wherein the SEQ ID NO. 8 is used for detecting the most common mutation subtype p.E746_ A750del, the sequence of the mutant probe P is SEQ ID NO. 10, and the sequence of the downstream primer R is SEQ ID NO. 11; the sequence of the reference primer rF is SEQ ID NO. 12, the sequence of the reference primer rR is SEQ ID NO. 13, and the sequence of the reference probe rP is SEQ ID NO. 14. The mutant probe shown by SEQ ID NO. 10 was labeled with FAM at the 5 'end and BHQ1 at the 3' end. The reference probe shown by SEQ ID NO. 14 was labeled with HEX at the 5 'end and BHQ1 at the 3' end.

Table 1:

in the primer probe, the total length of the mutant F1 primer (SEQ ID NO: 1-6) is 60-70 bp, and the total length of the wild type blocker oligonucleotide (SEQ ID NO:7) is 33 bp. The mutant F1 primer (SEQ ID NO: 1-6) is aimed at the mutation deletion type of the EGFR gene 19 exon, and 20-25 bases of the 3' end of the primer are paired with a target nucleic acid sequence. Wild type blocker oligonucleotides were paired to wild type templates. The 20 bases at the 5' -end of the mutant F1 primer are identical to the base sequence of the corresponding F2 primer (SEQ ID NO:3 or SEQ ID NO: 4). The 20 th to 23 th bases to the 40 th to 43 th bases of the 5' -end of the mutant F1 primer are the same as the base sequence of the corresponding mutant probe P (SEQ ID NO: 10). Therefore, after the mutant F1 primer specifically amplifies the target nucleic acid, the generated amplification product will be added with a base sequence from the 5' end of the F1 primer and its complementary sequence, and then the F2 primer and the probe P can be coupled with the corresponding target nucleic acid template and hydrolyzed to emit a fluorescent signal. Since the total percentage of the mutant subtype p.E746_ A750del accounts for up to 64.6% of all EGFR gene 19 deletion mutations, in order to improve the accuracy of detection of this mutation type, one F2 primer (SEQ ID NO:8) was used alone for the F1 primer of this mutation type, and one F2 primer (SEQ ID NO:9) was used in common for the other 5F 1 primers.

The invention also provides a reaction system for detecting the 19 exon deletion mutation of the EGFR gene, which comprises the primer composition, sample DNA, DNA polymerase and buffer solution, and the specific enrichment of a target nucleic acid sequence, the specific amplification of a template after the enrichment and the hydrolysis of a fluorescence labeling probe are sequentially carried out. The reaction system comprises the following steps:

(1) firstly, in the first few cycles of PCR reaction, an F1 primer and a downstream primer R can specifically amplify a target nucleic acid sequence, and an F2 sequence, a P sequence and a complementary sequence thereof are introduced into the 5' end of an amplified product, a wild type blocker oligonucleotide is combined with a wild type template, and the non-specific combination of a mutant type F1 primer with the wild type template is competitively inhibited;

(2) after the reaction is completed, the F2 primer and the downstream primer R can be matched with the corresponding enriched template and amplified, so that the probe P combined with the enriched template is cut by utilizing the 5' exonuclease activity of DNA polymerase;

(3) throughout the PCR reaction, the reference primers rF, rR amplify the corresponding target sequence and cleave the template-bound probe rP using the 5' exonuclease activity of the DNA polymerase.

The reaction conditions described in the present invention are:

(1) pre-denaturation at 92-96 ℃ for 5-15 minutes;

(2) denaturation at 92-95 ℃ for 10-60 seconds,

annealing at 55-75 ℃ and extending for 30-90 seconds,

reacting for 35-50 cycles in the step (2);

(3) inactivating at 94-98 ℃ for 5-15 minutes;

(4) the reaction is terminated at 4-15 ℃.

The concentrations of the primer probes in the reaction system are respectively as follows:

the concentration of the F1 primer is 15 nM-150 nM;

the concentration of the F2 primer is 150 nM-1500 nM;

the concentration of the probe P and the reference probe rP is 50 nM-800 nM;

the concentration of the downstream primer R and the reference primer rF is 150 nM-1800 nM;

the concentration of the blocker oligonucleotide is 150nM to 1500 nM.

Preferably, the concentrations of the primer probes in the reaction system of the present invention are:

the concentration of the F1 primer is 30 nM-60 nM;

the concentration of the F2 primer is 300 nM-600 nM;

the concentration of the probe P is 150 nM-400 nM;

the concentration of the downstream primer R is 300 nM-900 nM;

the concentration of the reference primer rF and rR is 300 nM-900 nM;

the concentration of the blocker oligonucleotide is 300nM to 600 nM.

In the following embodiments, the EGFR gene 19 exon deletion mutation detection reaction system described in the present invention comprises the following components:

TABLE 2

The reaction conditions used in the present invention are:

(1) pre-denaturation at 95 ℃ for 10 min;

(2) the mixture is denatured at 94 ℃ for 30 seconds,

annealing at 65 ℃ and extending for 60 seconds,

reacting for 45 cycles in the step (2);

(3) inactivating at 98 ℃ for 10 minutes;

(4) the reaction was terminated at 10 ℃.

In the embodiment of the invention, the DNA sample is an isolated DNA sample extracted from peripheral plasma of a cancer patient, or a fragmented cell line DNA sample, or an artificially synthesized plasmid DNA sample. The length between the upstream primer F1 and the downstream primer R for specifically enriching the target nucleic acid in the reaction system is not more than 100bp, so the method is particularly suitable for detecting the DNA sample of short fragments.

In the specific embodiment of the present invention, the reaction buffer and the primer probe premix are mixed according to the reaction system described in table 2, DNA is extracted from the sample to be tested by a suitable method and added to the prepared reaction system, and then the partitioning of the digital PCR microreactor (microdroplet), PCR amplification and fluorescence signal detection are performed. And judging the proportion of the negative/positive microdroplets according to the existence of the fluorescent signal to obtain the concentration of the target nucleic acid mutant sample and the wild sample, and further calculating the mutation abundance of the target nucleic acid sequence in the sample.

[ (mutant concentration)/(mutant concentration + wild type concentration) ]. 100%

For example, if the concentration of the mutant target nucleic acid of the exon 19 of the EGFR gene detected by the mutant probe (FAM fluorescent signal) in the sample to be detected is 50 copies/. mu.l, and the total amount of DNA (mutant + wild type) detected by the reference probe (HEX fluorescent signal) in the system is 10000 copies/. mu.l, the abundance of the exon 19 mutation of the EGFR gene in the sample to be detected is:

[ (50 copies/. mu.L)/(10000 copies/. mu.L) ]. 100%. 0.5%

The following embodiments of the present invention are combined with QX200 droplet digital PCR system and reagent consumables of Bio-Rad for detection. Wherein 2X ddPCR Supermix for Probe sddPCR can be selected from Bio-Rad company products, Cat 1863010; the droplet generating oil is selected from Bio-Rad company, Cat 1863005; the ddPCR minidroplet generation card can be selected from Bio-Rad company, cat # 1864008; the sealing strip used for generating the droplets can be selected from Bio-Rad company products, product number 1863009; the microdroplet analysis oil can be selected from Bio-Rad company, product number 1863004; the semi-skirt 96-well plate may be selected from Bio-Rad, Inc., cat # 12001925. The detection result of the kit can be subjected to data analysis by using QuantaSoft digital PCR analysis software of Bio-Rad company, and the concentration and the mutation abundance of the target nucleic acid in the sample to be detected can be calculated.

Example 1:

the performance of this embodiment was evaluated using a mock clinical sample (p.E746_ A750del deletion mutation)

First, preparation of simulated clinical samples

DNA of HEK-293T cells and NCI-H1650 cells (p.E746_ A750del deletion mutation) was extracted using QIAamp DNA Mini and Blood Mini Kit (QIAGEN Co., Ltd.) according to the instructions of the Kit to obtain a wild-type template (HEK-293T cell line DNA) and a mutant template (NCI-H1650 cell line DNA), respectively.

And (3) respectively performing ultrasonic breaking treatment on the two templates, screening and purifying the two templates by magnetic beads, quantifying by using digital PCR, and preparing a mixed sample with mutation abundance of 10% according to a quantification result to be used as a simulated clinical sample (a d1 sample). A negative control (negative control) was also prepared using the same concentration of fragmented wild-type DNA.

Preparation of reaction system

After the preparation of the sample, the primer composition of the present invention is configured according to the ratio shown in table 2, and the experimental principle of this embodiment, i.e. the scheme of the present invention, is shown in fig. 1.

Before the generation of the microdroplets, 20 mu L of the prepared reaction solution containing the template to be detected is placed in a metal bath, denatured at 95 ℃ for 1min and immediately placed in a refrigerator at 2-8 ℃ for 2-3 min.

And adding the cooled 20 mu L of PCR reaction solution into a sample hole of the microdroplet generation card, then adding 70 mu L of microdroplet generation oil into an oil hole of the microdroplet generation card, and finally sealing the microdroplet generation card by using a sealing strip.

The prepared droplet generation card is placed into a droplet generator and droplet generation is initiated. After about 2 minutes, the droplet preparation is complete, the card slot is removed, and approximately 40 μ Ι _ of droplets are carefully transferred from the uppermost row of wells to a 96-well PCR plate.

Third, amplification reading

After the 96-well plate was subjected to a membrane sealing treatment, it was placed in a PCR thermal cycler and PCR amplification was performed using the primer composition shown in Table 1.

The reaction procedure for amplification was:

(1) pre-denaturation at 95 ℃ for 10 min;

(2) the mixture is denatured at 94 ℃ for 30 seconds,

annealing at 65 ℃ and extending for 60 seconds,

reacting for 45 cycles in the step (2);

(3) inactivating at 98 ℃ for 10 minutes;

(4) the reaction was terminated at 10 ℃.

After the PCR amplification is finished, the 96-well plate is placed in a microdroplet analyzer to select FAM/HEX channel for signal reading. As a result, as shown in FIGS. 2 and 3, it can be seen that no false positive fluorescence signal is generated in the reaction test of the negative control; when the reaction detection of a simulated clinical sample (p.E746_ A750del deletion mutation) is used, the threshold division is clear, the data statistics difficulty is low, and the accuracy is high.

The intensity and number of fluorescence signals were analyzed by QuantaSoft analysis software to obtain copy numbers and concentrations of EGFR gene 19 exon mutant and wild type, and the mutation abundance was calculated, as shown in fig. 4, the mutation abundance of p.e746_ a750del deletion mutation (d1 sample) was 10.1%.

In order to solve the generation of false positive fluorescent signals in the system, the method introduces a blocker oligonucleotide (SEQ ID NO:7), and the blocker oligonucleotide is preferentially combined with a wild-type template during PCR amplification, so that the non-specific combination of a mutant F1 primer and the wild-type template is avoided, and the generation of the false positive fluorescent signals is eliminated. Furthermore, aiming at the detection of the most common clinical mutant subtype (p.E746_ A750del, 64.6%), in order to reduce the interference of other 5 mutant F1 primers on the detection template, two mutant F2 primers are added into the system, namely, one F2 primer (namely, mutant F2-1, SEQ ID NO:8) aiming at the mutant F1-1 primer (SEQ ID NO:1) is added separately; the threshold division difficulty after detection is further reduced; the accuracy of the analysis is further improved.

Example 2

Experimental protocol performance was assessed using simulated clinical specimens (common mutant subtypes).

Preparation of simulated clinical samples

DNA of HEK-293T cells, DNA of NCI-H1650 cells (p.E746_ A750del mutation (1)) and DNA of HCC-827 cells (p.E746_ A750del mutation (2)) were each extracted using QIAamp DNA Mini and Blood Mini Kit (QIAGEN Co., Ltd.) according to the instructions of the Kit, and wild-type templates (DNA of HEK-293T cell line) and 2 mutant templates (DNA of NCI-H1650 cell line and DNA of HCC-827 cells) were obtained, respectively.

The wild-type template and the mutant template are respectively subjected to ultrasonic disruption treatment, and after being screened and purified by magnetic beads, 2 mixed samples of the mutation and the wild template, namely a D1 sample (p.E746_ A750del mutation (1)) and a D2 sample (p.E746_ A750del mutation (2)) are respectively prepared and used as a simulated clinical sample.

EGFR gene 19 exon deletion mutant plasmids (p.L747_ P753> S mutation, p.E746_ S752> V mutation, p.L747_ T751del mutation, p.L747_ A750> P mutation and p.L747_ T751> P mutation) are respectively synthesized artificially, and are used as corresponding mutant templates, the plasmids are subjected to ultrasonic disruption and magnetic bead screening and purification and then respectively mixed with fragmented HEK-293T cell line DNA (wild-type template), and D3 samples (p.L747_ P753> S mutation), D8 samples (p.E746_ S752V mutation), D13 samples (p.L747_ T751del mutation), D15 samples (p.L747_ A750> P mutation) and D19 samples (p.L747_ T751> P) are respectively prepared. A negative control (negative control) was also prepared using the fragmented wild-type DNA, and TE Buffer was used as a no-template control (NTC).

Second, preparation of reaction system and reading of amplification

After the preparation of the sample is completed, the primer composition of the present invention is prepared into a reaction system according to the ratio shown in table 2. The droplet generation and PCR reaction process, amplification reading process are the same as example 1. A

The amplification result is shown in fig. 5, when the 7 groups of deletion mutations of the simulated clinical samples (clinical common subtypes) are detected, the detection threshold is clearly divided, and no false positive fluorescence signal is generated; therefore, when the clinical common subtype samples are detected, the data analysis difficulty after detection is low, and the accuracy is high.

Example 3

The primer composition designed in example 3 was as follows: namely, 6 mutant F1 primers share one F2 primer (namely mutant F2-1 and SEQ ID NO:8), and the sequence of the corresponding mutant F1-1 is (SEQ ID NO: 15: ACTCGTACTCAGTCAACTCTCCTAACCGTTCCGCCTGTTCCAAACGTCGCTATCAARRCATCTCC) to observe the influence on the detection of the most common clinical mutant subtype (p.E746_ A750del) (the primer composition in example 3); the other 5 first upstream primers are respectively as follows: 2-6 of SEQ ID NO; the probe, the downstream primer, the blocker oligonucleotide and the reference primer composition are the same as those in example 1.

Example 3 primer composition the reaction system was configured according to the formulation described in table 3. Wild-type samples and mock clinical samples (p.E746_ A750del deletion mutant, i.e., d1 samples) were the same as in example 1, microdroplet generation and PCR reaction processes, and amplification reading processes were the same as in example 1.

Table 3:

as shown in FIGS. 6 and 7, no false positive fluorescence signal was generated in the negative control reaction; when d1 samples are detected, the detection result of the embodiment shows that the double-positive fluorescence signal slightly deviates from the single-positive fluorescence signal of the wild-type probe, and the threshold division difficulty is larger compared with that of embodiment 1.

Example 4

The primer composition of example 4 was designed as follows: that is, a wild-type F1 primer is designed in the common deletion mutation region, the F1 primer spans the common deletion mutation region and competes with the mutant F1 primer, and a sequence which is not matched with the template is also added at the 5' end of the F1 primer, and the F3578 primer consists of two parts: the specific principle of the wild type F2 primer and the wild type probe P for detecting the wild type template is the same as that of the mutant type F1 primer for detecting the mutant type template, and the specific principle is shown in FIG. 8. The sequences of the primers and probes in the system are shown in Table 4.

The specific primer composition is as follows:

no blocker oligonucleotide, two mutant F2 primers, namely a F2 primer (namely mutant F2-1 and SEQ ID NO:8) aiming at the mutant F1-1 primer (SEQ ID NO:1) are added into the system, and the specific primer composition is shown in Table 4. The two reaction systems are respectively corresponding to the table 5.

TABLE 4

Table 5:

the wild-type and mock clinical samples (p.E746_ A750del deletion mutant, i.e., d1 samples) used in this example 4 were the same as in example 1, the microdroplet generation and PCR reaction processes, and the amplification reading process was the same as in example 1.

As a result, as shown in FIGS. 9 to 10, it was found that only the blocker oligonucleotide was absent when the primer composition of example 4 was used.

When a negative control sample is detected, although a few false positive fluorescence signals appear, the system specificity is improved;

when the d1 sample is detected, the threshold division is clear, the data analysis is easy, and the accuracy is high.

Comparative example

The primer composition of the comparative example was designed as follows:

the 6 mutant F1 primers are non-blocker oligonucleotides and share one F2 primer (namely mutant F2-2 and SEQ ID NO:9), and the specific primer composition is shown in Table 4 and only does not contain F2-1. The two reaction systems are respectively corresponding to the table 6.

Table 6:

the wild-type sample and the mock clinical sample (p.E746_ A750del deletion mutant, i.e., the d1 sample) used in this comparative example were the same as in example 1, the microdroplet generation and PCR reaction processes, and the amplification reading process was the same as in example 1.

When the primer composition of the comparative example, no blocker oligonucleotide and no F2-1;

when a negative control sample is detected, a false positive fluorescence signal appears, and the system specificity is insufficient;

when the d1 sample is detected, the double positive fluorescence signal is slightly biased to the single positive fluorescence signal of the wild-type probe, which is not beneficial to threshold division.

It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence listing

<110> Mike Bio-Ltd

Primer composition for multiplex detection of <120> gene mutation and kit thereof

<130> MB2019

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 65

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

ccgtctcgca ttaacatatt cctaaccgtt ccgcctgttc caaagtcgct atcaarrcat 60

ctccg 65

<210> 2

<211> 65

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

actcgtactc agtcaactct cctaaccgtt ccgcctgttc caatcgctat caaggaatcg 60

aaagc 65

<210> 3

<211> 64

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

actcgtactc agtcaactct cctaaccgtt ccgcctgttc caagtcgcta tcaagggtcc 60

gaaa 64

<210> 4

<211> 63

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

actcgtactc agtcaactct cctaaccgtt ccgcctgttc cagtcgctat caaggaatct 60

ccg 63

<210> 5

<211> 64

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<213> Artificial sequence (artificial sequence)

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actcgtactc agtcaactct cctaaccgtt ccgcctgttc ccgctatcaa ggaaccaaca 60

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actcgtactc agtcaactct cctaaccgtt ccgcctgttc ccgctatcaa ggaaccatct 60

ccg 63

<210> 7

<211> 33

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

tcgctatcaa ggaattaaga gaagcaacat ctc 33

<210> 8

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<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

ccgtctcgca ttaacatatt 20

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<400> 9

actcgtactc agtcaactct 20

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<213> Artificial sequence (artificial sequence)

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ctaaccgttc cgcctgttcc 20

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<213> Artificial sequence (artificial sequence)

<400> 11

caaagcagaa actcacatcg 20

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

cctccagagg atgttcaa 18

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<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

gcacataggt aatttccaaa 20

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tcccaaggac cacctcacag 20

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<213> Artificial sequence (artificial sequence)

<400> 15

actcgtactc agtcaactct cctaaccgtt ccgcctgttc caaacgtcgc tatcaarrca 60

tctcc 65

<210> 16

<211> 67

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

cgcctctcct aatcatctta ctctcgtctc agcctccatc cacgctatca aggaattaag 60

agaagca 67

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 17

cgcctctcct aatcatctt 19

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Claims

1. A primer composition for multiplex detection of gene mutation, characterized in that: the primer composition comprises a second upstream primer, a probe, a downstream primer and at least two different first upstream primers;

the different first upstream primers are different in target sequence binding region; the second forward primer does not pair with the target sequence and the first forward primer.

2. The primer composition for multiplex detection of gene mutation according to claim 1, wherein: said different first upstream primers being different in said target sequence binding region, said upstream detection region being identical or different in part (a), and said upstream detection region being identical in part (b); preferably, the different first upstream primers are different in both the target sequence binding region and the portion (a) of the upstream detection region.

3. The primer composition for multiplex detection of gene mutation according to claim 2, characterized in that: the primer composition further comprises a wild-type blocker; the Tm value of the wild type blocker is more than or equal to the Tm value of the first upstream primer and more than the Tm value of the second upstream primer.

4. The primer composition for multiplex detection of gene mutation according to any one of claims 1 to 3, characterized in that: the complementary pairing position of the downstream primer and the target sequence is arranged at the position 1-150 bp downstream of the mutation detection site.

5. The primer composition for multiplex detection of gene mutation according to any one of claims 1 to 4, wherein: the length of a mismatching region in a target sequence binding region on the first upstream primer is 1-15 bases; the distance between the amplification determining site and the mismatch region on the first upstream primer is 1-15 bases.

6. A gene mutation multiplex detection kit is characterized in that: the kit comprises the primer composition according to any one of claims 1 to 5.

7. A primer composition for detecting mutation of 19 exons in EGFR gene, which is characterized in that: the primer composition comprises:

a second upstream primer SEQ ID NO. 9;

probe SEQ ID NO 10;

the downstream primer is SEQ ID NO. 11.

8. A primer composition for detecting mutation of 19 exons in EGFR gene, which is characterized in that: the primer composition comprises:

the second upstream primer SEQ ID NO. 8, SEQ ID NO. 9;

probe SEQ ID NO 10;

the downstream primer is SEQ ID NO. 11.

9. The primer composition for detecting 19 exon mutation in EGFR gene according to claim 8 or 9, further comprising a wild-type blocker; preferably, the wild-type blocker is oligonucleotide SEQ ID NO 7.

10. Use of the primer composition of claim 7 or 9 for the preparation of a kit for detecting an exon 19 mutation in the EGFR gene.