CN112824535B - Primer composition for gene mutation multiplex detection and kit thereof - Google Patents

Abstract

The invention discloses a primer composition for gene mutation multiplex detection and a kit thereof; the primer composition includes 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 region and a target sequence binding region: the upstream detection zone 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 determining site which is complementary to a mutation detection site on the mutant target sequence and has a mismatch region consisting of one or more bases which is not complementary to the target sequence upstream thereof; the first, different upstream primer is different for the target sequence binding region. The primer composition of the present invention further comprises a wild-type blocker. The primer composition or the kit is used for detecting multiple gene mutations, so that the detection sensitivity and the specificity can be improved, and the detection cost can be reduced; and the readiness for detection 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 detecting multiple mutation of genes and a kit thereof.

Background

The digital PCR (dPCR) technique is an absolute quantitative technique of nucleic acid molecules, and a fluorescent quantitative PCR reaction system is distributed into thousands of individual nanoliter microreactors by using the principle of limiting dilution, so that each microreactor contains or does not contain 1 or more copies of a target nucleic acid molecule (DNA target), and single-molecule template PCR amplification is performed simultaneously. Different from the method for collecting fluorescence when each amplification cycle is carried out by fluorescence quantitative PCR, the digital PCR independently collects the fluorescence signal of each reaction unit after the amplification is finished, and finally the original copy number or concentration of the target molecule is obtained by the poisson distribution principle and the proportion of the positive/negative reaction units.

Compared with fluorescent quantitative PCR, the digital PCR can perform accurate absolute quantitative detection without depending on Ct value and standard curve, and has the advantages of high sensitivity and high accuracy. Because the digital PCR only judges the 'existence/nonexistence' two amplification states when the result is interpreted, the intersection point of a fluorescent signal and a set threshold line is not required to be detected, and the identification of a Ct value is completely not relied on, so that the influence of the amplification efficiency on the digital PCR reaction and the result interpretation is greatly reduced, and the tolerance capability to PCR reaction inhibitors is greatly improved. In addition, the process of partitioning the reaction system in digital PCR experiments can greatly reduce locally the concentration of background sequences that compete with the target sequences. Thus, since digital PCR has higher sensitivity and accuracy, it represents a significant advantage over conventional fluorescent quantitative PCR when it is desired to quantify and detect differential nucleic acid molecules with low copy number with high sensitivity. Especially detection of rare mutations in complex settings, such as by detection of circulating tumor DNA (ctDNA) in the peripheral blood of tumor patients, shows great advantages, often in early screening, drug instruction, prognostic judgment and relapse monitoring applications for tumor patients.

The existing gene mutation detection reagent mostly adopts (1) TaqMan probe method, (2) primer specificity differentiating method and (3) blocking wild type amplification method. The TaqMan probe method (1) generally adopts competitive probes respectively aiming at mutant type and wild type, the two probes are competitive probes similar to point mutation detection, or a specific probe is designed on a wild type template of a gene, a universal probe capable of simultaneously indicating the wild type and the mutant type template is designed in other conserved regions of the gene, the concentration difference value measured by the two probes is utilized to quantify the concentration of the mutant type template, and the two methods both need to design amplification primers at the upstream and downstream of the probes, so that the amplicon is longer, and because ctDNA is in highly random fragmentation distribution, shorter ctDNA fragments can be missed to detect, the detection sensitivity is reduced, and meanwhile, when a certain gene mutation type is numerous, a plurality of different mutation type probes are designed aiming at different mutation types, the cost is higher, and the large-scale popularization and the use are not facilitated. (2) 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, design a universal probe capable of simultaneously indicating wild type and mutant templates and upstream and downstream primers in other conserved regions of genes, and the method still has the problem of longer amplicon, is unfavorable for detection of shorter ctDNA, has extremely high requirement on the specificity of the primers and is easy to generate cross reaction. (3) The blocking wild type amplification method usually adopts peptide nucleic acid (Peptide Nucleic Acid, PNA), and lock nucleic acid (Locked Nucleic Acid, LNA) modification to block the amplification of wild type template, and at present, the modification is expensive and is unfavorable for clinical application. Therefore, a primer probe design method with high sensitivity, good specificity and low cost is needed to meet the requirements of clinical detection by using a digital PCR technology.

The invention improves on the basis of the common detection method, simultaneously detects a plurality of mutation genes with common clinic and higher mutation frequency in one reaction tube, and does not carry out parting treatment on mutation types. The method has the advantages that one tube simultaneously detects multiple mutation types, so that the method has certain challenges, on one hand, the digital PCR has extremely high sensitivity, and false positive is very easy to occur; on the other hand, there are a plurality of primer probes in one tube, and the interaction between them is not negligible, so the specificity requirement for the primer probes is extremely high, and cross reactions need to be avoided as much as possible. Therefore, when a multiple gene mutation detection system is constructed by utilizing a digital PCR technology, it is particularly important how to improve the specificity of the system, reduce the mutual interference between primers and protect the detection performance of the most common mutation subtype in clinic.

Disclosure of Invention

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

In one aspect, a primer composition for multiplex detection of gene mutations 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 region and a target sequence binding region from a 5 'end to a 3' end in sequence:

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

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

the first, different upstream primer, the target sequence binding region being different, the upstream detection region (a) being identical or different and the upstream detection region (b) being identical;

in some embodiments, the first, different upstream primer has a base spacing or no base spacing between the target sequence binding region and the portion (a) of the upstream detection region that is different from the portion (a), the portion (b), and the target sequence binding region of the upstream detection region.

The sequence of the second upstream primer is identical to part (a) of the upstream detection zone and is not complementary to any of the target nucleic acid sequence, the first upstream primer, and the downstream primer, and the sequence thereof can be freely exchanged.

The sequence of the probe is identical to part (b) of the upstream detection zone and is not complementary to any of the target nucleic acid sequence, the first upstream primer, and the downstream primer, and the sequence thereof can be freely exchanged.

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 Tm of the wild-type blocker is greater than or equal to the Tm of the first upstream primer and greater than the Tm of the second upstream primer.

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

In the digital PCR amplification, the wild-type blocker preferentially binds to the wild-type template, avoiding non-specific binding of the first upstream primer to the wild-type template; then, the first upstream primer specifically binds to the mutant template, and the mutant template is pre-amplified; then, the second upstream primer amplifies the downstream complementarily bound probe of the enriched mutant template, thereby detecting the corresponding target nucleic acid sequence.

The second upstream primer and probe pair bind to the pre-amplified product only after the first upstream primer 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 sequence of a common mutation region; the length of the oligonucleotide is 13-30 bp, the Tm value is 50-80 ℃, and the GC content is 40-80%; the 3' end of the blocker is modified with non-hydroxyl groups including, but not limited to, dideoxy base modifications, C6 spacer, C3 spacer, phosphorylation modifications, amino, halo or other modifications; in further embodiments, the 3' end of the blocker is dideoxybase modified.

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

In some embodiments, the second upstream primer has a length of 13 to 30bp, a Tm of 50 to 80℃and a GC content of 40 to 80%.

In some embodiments, the downstream primer has a length of 15 to 30bp, a Tm of 55 to 75℃and a GC content of 40 to 80%.

In the present invention, the first upstream primer and the second upstream primer are preferably the same downstream 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 upstream primer is 5 ℃ to 20 ℃ higher than the Tm value of the second upstream 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, the first, different upstream primer is the same or different in part (a) of the upstream detection zone and the same or different in part (b) of the upstream detection zone.

When the portions (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 position of the complementary pairing of the downstream primer and the target sequence is set at 1-150 bp downstream of the mutation detection site.

In some embodiments, the length of the mismatched zone in the binding region of the target sequence on the first upstream primer is between 1 and 15 bases; 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 has a reporter group and a quencher group thereon. In still further embodiments, the reporter group may be a fluorescent group selected from the group consisting of: : FAM, HEX, VIC, ROX, cy5, cy3, etc.; the quenching group may be selected from the group consisting of: TAMRA, BHQ1, BHQ2, BHQ3, DABCYL, QXL, DDQI, and the like. In some embodiments, the probe does not carry any other modifications, such as MGB, LNA, PNA, BNA, superBase, etc., in addition to the reporter and quencher groups. In a preferred embodiment, the probe of the invention is a Taqman probe. In a preferred embodiment, the reporter group is located at the 5 'end of the probe and the quencher group is located at the 3' end of the probe.

In a second aspect, a kit for multiplex detection of gene mutations, the kit comprising the primer composition described above.

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

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

In some embodiments, the rF, rR primers have a length of 5 to 30bp, a Tm of 55 to 75℃and a GC content of 40 to 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 mutation of exon 19 of EGFR gene, the primer composition comprising:

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

a second upstream primer SEQ ID NO. 9;

the probe SEQ ID NO. 10;

the downstream primer SEQ ID NO. 11.

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

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

a second upstream primer SEQ ID NO. 8 and SEQ ID NO. 9;

the probe SEQ ID NO. 10;

the downstream primer SEQ ID NO. 11.

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

In some embodiments, the primer composition for detecting an exon 19 mutation of 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' end of the blocker is modified with non-hydroxyl groups including, but not limited to, dideoxy base modifications, C6 spacer, C3 spacer, phosphorylation modifications, amino, halo or other modifications; in further embodiments, the 3' end of the blocker is dideoxybase modified.

In a fourth aspect, the use of the above primer composition for the preparation of an EGFR gene 19 exon mutation detection kit.

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

(1) Fewer false positives: the blocker oligonucleotide introduced in the system is completely complementary with the antisense strand of the specific region (common mutation deletion region) of the wild type template, preferentially tightly binds with the wild type template in the PCR reaction process, and competitively inhibits the nonspecific binding of the mutant first upstream primer F1 and the wild type template, thereby reducing the generation of false positive fluorescent signals, and being particularly suitable for mutation detection by using a digital PCR method.

(2) The specificity is high: the phenomenon that mutual interference can exist among 6 mutant F1 primers in the system can be caused, in order to weaken cross reaction, detection of the most common clinical mutant subtype p.E746_A750del (64.6%) is protected from interference of other mutant first upstream primers F1, a second upstream primer F2 aiming at the mutant type is additionally added, namely 2 different F2 primers are added in the system, and other 5F 1 primers share the F2 primers except that one F2 primer is independently used for the F1 primer of the mutant subtype p.E746_A750del (64.6%). The scheme can not only improve the amplification efficiency when detecting mutant subtype p.E746_A750del (64.6%), but also ensure that the detection of other mutant subtypes is not affected, the threshold is clear, and the detection result is accurate and reliable.

(3) The cost is low: according to the invention, 6 mutant F1 primers are used for detecting 19 mutant subtypes, one probe P is used for different mutant subtypes, simultaneous detection of multiple mutant subtypes can be completed without adding multiple probes P, and in addition, the primer probes are not required to be modified by expensive PNA or LNA, so that the use cost of the primer probes is greatly reduced, and the method 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 as the probe P can be matched and combined with the 5' -end complementary sequence of the first upstream primer F1 after the pre-amplification is completed, the part which is actually matched and combined with the target nucleic acid sequence is only two parts: f1, and a sequence of a 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 at least three parts of the two methods are 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 is shorter. In the highly fragmented free DNA detection, since the fragmentation of DNA is random, a shorter detection fragment can detect more DNA targets, thereby greatly improving the sensitivity of detection.

(5) The requirements for the target nucleic acid sequence are lower: similar to the above advantages, the primer probe design method of the present invention has only two parts of the part actually coupled with the target nucleic acid sequence because the probe P can be coupled with the 5' -end complementary sequence of the first upstream primer F1 after the pre-amplification is completed: f1 and the sequence of the downstream primer R. Thus, for complex target nucleic acid sequence detection, the design difficulty of primer probes is lower relative to TaqMan probe methods and ARMS.

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

(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 is a diagram of a primer probe design method according to the present invention; wherein, FIG. 1A is a structural diagram of a first upstream primer F1, comprising an upstream detection region and a target sequence binding region in sequence from a 5' end to a 3' end, wherein a mismatch can be added to the 3' end near the target sequence binding region; the upstream detection zone comprises, from 5 'to 3': the same sequence as the second upstream primer F2 and the probe P. FIG. 1B shows the principle of detection according to the present invention, wherein the blocker oligonucleotide is specifically bound to the wild template, the mutant first upstream primer F1 and the reverse primer R are specifically enriched in the mutant target nucleic acid sequence to be detected, and a sequence complementary to the "upstream detection region" of the first upstream primer is newly added for pairing and identifying the subsequent second upstream primer F2 and the probe P. After enrichment of the target nucleic acid template, the second upstream primer F2 and the probe P are enabled to identify a sequence complementary to the upstream detection region of the first upstream primer on the pre-amplified product, then a primer pair is formed with the reverse primer R, template amplification is carried out, and a fluorescent signal is released through the TaqMan probe principle. During the whole PCR reaction process, the reference primer rF and the reference primer rR form a primer pair amplification template (mutant type and wild type), and fluorescent signals are released through the TaqMan probe rP;

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

FIG. 3 is a two-dimensional diagram of a sample of mutations detected in example 1 (p.E746_A750del deletion mutation);

FIG. 4 is a graph showing the concentration and mutation abundance of the mutant samples (p.E746_A750del deletion mutation) and negative control samples detected in example 1;

FIG. 5 is a one-dimensional plot of the detection of different signal channels of a simulated clinical sample (clinically common subtype) in example 2; 5A is a 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 the negative control sample detected in example 3;

FIG. 7 is a two-dimensional diagram of a sample of mutations detected in example 3 (p.E746_A750del deletion mutation);

FIG. 8 is a method of designing a primer probe according to example 4; first, the first upstream primer F1 and the reverse primer R are utilized to specifically enrich the target nucleic acid sequence to be detected (mutant/wild type), and a sequence complementary to the upstream detection region of the first upstream primer is newly added for pairing and identification of the second upstream primer F2 and the probe P. After enriching the target nucleic acid template, using the change of annealing temperature to make the second upstream primer F2 and the probe P recognize the sequence complementary to the upstream detection zone of the first upstream primer on the pre-amplified product, then forming a primer pair with the reverse primer R and amplifying the template, and releasing a fluorescent signal by the TaqMan probe principle;

FIG. 9 is a two-dimensional diagram of the detection of a negative control sample of example 4;

FIG. 10 is a two-dimensional diagram of a sample of mutations detected in example 4 (p.E746_A750del deletion mutation);

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

FIG. 12 is a two-dimensional plot of comparative example detection mutation samples (p.E746_A750del deletion mutation).

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to the following examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

Definition of the definition

The term "conserved region" refers to a region of a DNA molecule in which a nucleotide fragment 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 an 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 (-) of the coding strand/sense strand. In the kinetic PCR format, the detection probes may consist of oligonucleotides with 5 'reporter dyes (R) and 3' quencher dyes (Q). Fluorescent reporter dyes (i.e., FAM (6-carboxyfluoranthene), etc.) are typically located at the 3' end. The detection probe was used as TAQMAN probe during the amplification and detection.

As used herein, the term "nucleic acid" 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 that may be used in the described embodiments.

The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal that 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 the detected signal by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic 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 conjugates.

Epidermal growth factor receptor EGFR (Epidermal Growth Factor Receptor) is a transmembrane tyrosine kinase receptor, the activation of which is related to the proliferation, metastasis, apoptosis and other signal transduction pathways of cancer cells, and the high expression or abnormal expression of EGFR genes exists in many solid tumors. The tyrosine kinase active site of EGFR is located mainly between exons 18-21. Therefore, activating mutations and drug-resistant mutations of EGFR are concentrated. The most common activating mutations are the 19 exon deletion mutation (about 45%) and the L858R point mutation of exon 21 (about 40%), both of which can result in tyrosine kinase domain activation. Tumor patients carrying EGFR mutation can remarkably prolong the progression-free survival time (Progression Free Survival, PFS) of the patients and improve the drug response rate after being treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs). Therefore, detection and monitoring of EGFR gene mutation status plays a vital role in the establishment of clinical first-line medication schemes, drug efficacy and monitoring of tumor prognosis and prognosis.

Mutation frequencies of the EGFR gene are as high as 20% to 40% in patients with Asian-African small cell lung carcinoma (Non small cell lung cancer, NSCLC), especially in non-smoking adenocarcinoma female Asian NSCLC patients. The most common EGFR gene activating mutation, exon 19 deletion mutation, can be up to 30-40 types, and more than 50% of deletion mutation types are accompanied by insertion mutation. The classification of the mutant subtype of 440 NSCLC patients harboring an EGFR gene 19 exon deletion mutation was taught by the cantonese lung cancer institute Wu Yilong, the most common 19 exon mutant subtype was p.e746_a753 del (64.6%), followed by p.l747_p753> S (8.4%), p.l747_t751del (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%), which was substantially consistent with the reports of other scholars (Su J, zhong W, zhang X, et al Molecular characteristics and clinical outcomes of EGFR exon 19indel subtypes to EGFR TKIs in NSCLC patients[J ]. Oncotargent, 2017,8 (67)). When NSCLC patients of different subtypes are treated by EGFR-TKIs drugs, clinical effects are not significantly different, so that typing detection is not needed.

The invention provides a primer composition for detecting EGFR gene 19 exon deletion mutation (the following "primer composition of the invention"):

the sequence of the first upstream primer F1 is 1-6 of SEQ ID NO, the sequence of the blocker hanging nucleotide sequence is 7 of SEQ ID NO, the sequence of the second upstream primer F2 is 8-9 of SEQ ID NO, wherein the sequence of the SEQ ID NO 8 is 10 of SEQ ID NO and the sequence of the downstream primer R is 11 of SEQ ID NO; 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 is labeled with FAM at the 5 'end and BHQ1 at the 3' end. The reference probe shown by SEQ ID NO. 14 is labeled HEX at the 5 'end and BHQ1 at the 3' end.

Table 1:

in the primer probe, the full length of the mutant F1 primer (SEQ ID NO: 1-6) is 60-70 bp, and the full length of the wild type blocker oligonucleotide (SEQ ID NO: 7) is 33bp. Mutant F1 primer (SEQ ID NO: 1-6) was directed against the mutation deletion type of exon 19 of EGFR gene, and 20-25 bases at the 3' end of the primer was paired with the target nucleic acid sequence. The wild type blocker oligonucleotide is paired with a wild type template. The 20 bases at the 5' -end of the mutant F1 primer were identical to the base sequence of the corresponding F2 primer (SEQ ID NO:3 or SEQ ID NO: 4). The base sequences from 20 to 23 th bases to 40 to 43 th bases on the 5' end of the mutant F1 primer are identical to the base sequences of the corresponding mutant probes P (SEQ ID NO: 10). Therefore, after the mutant F1 primer specifically amplifies the target nucleic acid, the generated amplified product is added with a section of base sequence from the 5' end of the F1 primer and the complementary sequence thereof, and then the F2 primer and the probe P can be paired with the corresponding target nucleic acid template and hydrolyzed to emit fluorescent signals. Because the mutant subtype p.E746_A750del generally accounts for up to 64.6% of all EGFR gene 19 deletion mutations, one F2 primer (SEQ ID NO: 8) is used alone for the mutant type F1 primer and one F2 primer (SEQ ID NO: 9) is used for the other 5F 1 primers in order to improve the accuracy of detection of this mutant type.

The invention also provides a reaction system for detecting EGFR gene 19 exon deletion mutation, which comprises the primer composition, sample DNA, DNA polymerase and buffer solution, and is used for carrying out specific enrichment of target nucleic acid sequences, specific amplification of templates after enrichment and hydrolysis of fluorescent marked probes in sequence. The reaction system comprises the following steps:

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

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

(3) Throughout the PCR reaction, the reference primers rF, rR amplify the corresponding target sequences 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 deg.c and extending for 30-90 sec,

step (2) reacting for 35-50 cycles;

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

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

The primer probe concentration in the reaction system of the invention is respectively as follows:

f1 primer concentration is 15 nM-150 nM;

f2 primer concentration 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 150 nM-1500 nM.

Preferably, the primer probe concentration in the reaction system of the invention is respectively as follows:

f1 primer concentration is 30 nM-60 nM;

f2 primer concentration 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 600nM.

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) Denaturation at 94℃for 30 seconds,

annealing at 65 ℃ and extending for 60 seconds,

step (2) reacting for 45 cycles;

(3) Inactivating at 98 ℃ for 10 minutes;

(4) The reaction was stopped at 10 ℃.

In one embodiment of the invention, the DNA sample is a free DNA sample from the peripheral plasma of a cancer patient, or a fragmented cell line DNA sample, or a synthetic plasmid DNA sample. Because 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, the reaction system is particularly suitable for detecting a short-fragment DNA sample.

In the specific embodiment of the invention, the reaction buffer solution and the primer probe premix solution are mixed according to the reaction system shown in Table 2, DNA is extracted from a sample to be tested by a proper method and added into the prepared reaction system, and then digital PCR micro-reactor (microdroplet) partitioning, PCR amplification and fluorescent signal detection are carried out. 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 concentration of 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, when the concentration of mutant target nucleic acid of exon 19 of EGFR gene is 50 copies/. Mu.L detected by a mutant probe (FAM fluorescent signal) in a sample to be detected, and the total amount of DNA (mutant+wild type) in a system is 10000 copies/. Mu.L detected by a reference probe (HEX fluorescent signal), the abundance of exon mutation of EGFR gene 19in the sample to be detected is:

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

The following specific embodiments of the present invention are used in conjunction with a Bio-Rad QX200 microdroplet digital PCR system and reagent consumables for detection. Wherein 2X ddPCR Supermix for ProbesddPCR is selected from Bio-Rad company product, product number 1863010; the droplet generation oil is selected from Bio-Rad company product, cat# 1863005; the ddPCR droplet generation card is selected from Bio-Rad company product, cat# 1864008; the sealing strip used when the microdroplet occurs can be selected from Bio-Rad company product, product number 1863009; the microdroplet analysis oil is selected from Bio-Rad company product, cat# 1863004; the half-skirt 96-well plate is available from Bio-Rad company under the trade designation 12001925. The detection result of the kit can be subjected to data analysis by using the QuantaSoft digital PCR analysis software of Bio-Rad company, so as to calculate the concentration and mutation abundance of the target nucleic acid in the sample to be detected.

Example 1:

evaluation of the Performance of this embodiment Using a mock clinical sample (p.E746_A750del deletion mutation)

1. Simulated clinical sample preparation

DNA of HEK-293T cells and NCI-H1650 cells (deletion mutation of p.E746_A750del) was extracted using QIAGEN QIAamp DNA Mini and Blood Mini Kit according to the protocol of the kit, and wild-type template (HEK-293T cell line DNA) and mutant template (NCI-H1650 cell line DNA) were obtained, respectively.

Respectively carrying out ultrasonic breaking treatment on the two templates, carrying out magnetic bead screening and purification, then quantifying by utilizing 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 (d 1 sample). A negative control (negative control) was also prepared using the same concentration of fragmented wild-type DNA.

2. Preparation of the reaction System

After sample preparation, the primer composition of the present invention was subjected to the configuration of the reaction system according to the ratios shown in Table 2, and the experimental principle of this example, i.e., the scheme of the present invention, is shown in FIG. 1.

Before generating microdrops, placing 20 mu L of prepared reaction solution containing a template to be tested in a metal bath, and immediately placing in a refrigerator at 2-8 ℃ for 2-3 min after denaturation at 95 ℃ for 1 min.

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

The prepared droplet generation card is placed into a droplet generator to initiate droplet generation. After about 2 minutes, droplet preparation was complete, the card slot was removed, and about 40 μl of droplets were carefully transferred from the uppermost row of wells to a 96-well PCR plate.

3. Amplification reads

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

The amplification reaction procedure was:

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

(2) Denaturation at 94℃for 30 seconds,

annealing at 65 ℃ and extending for 60 seconds,

step (2) reacting for 45 cycles;

(3) Inactivating at 98 ℃ for 10 minutes;

(4) The reaction was stopped at 10 ℃.

After the PCR amplification is finished, the 96-well plate is placed in a microdroplet analyzer, and a FAM/HEX channel is selected for signal reading. The results are shown in fig. 2 and 3, and it can be seen that no false positive fluorescent signal is generated in the reaction detection of the negative control; when the response detection of a simulated clinical sample (p.E746_A750del deletion mutation) is used, the threshold value is clear, the data statistics difficulty is low, and the accuracy is high.

The intensity and number of fluorescent signals were analyzed using QuantaSoft analysis software to obtain copy numbers and concentrations of the exon mutant and wild type of EGFR gene 19, and the mutation abundance was calculated, as shown in fig. 4, with a mutation abundance of 10.1% for the p.e746_a750del deletion mutation (d 1 sample).

In order to solve the generation of false positive fluorescent signals in a system, the embodiment 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. Further, for detection of the most common clinical mutant subtype (p.E746_A750del, 64.6%), to reduce interference of other 5 mutant F1 primer pairs when detecting templates, two mutant F2 primers are added in the system, namely one F2 primer (i.e. mutant F2-1, SEQ ID NO: 8) for mutant F1-1 primer (SEQ ID NO: 1) is added alone; the threshold dividing difficulty after detection is further reduced; the accuracy of the analysis is further improved.

Example 2

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

1. Preparation of simulated clinical samples

DNA of HEK-293T cells and NCI-H1650 cells (p.E746_A750del mutation (1)), and DNA of HCC-827 cells (p.E746_A750del mutation (2)) were extracted using QIAGEN QIAamp DNA Mini and Blood Mini Kit according to the protocol of the kit, respectively, to obtain wild-type templates (HEK-293T cell line DNA) and 2 mutant templates (NCI-H1650 cell line DNA and HCC-827 cell DNA).

The wild type template and the mutant template are respectively subjected to ultrasonic breaking treatment, and after magnetic bead screening and purification, 2 mixed samples of mutation and wild type, namely a D1 sample (p.E746_A750del mutation (1)), and a D2 sample (p.E746_A75del mutation (2)), are respectively prepared and used as simulated clinical samples.

The EGFR gene 19 exon deletion mutant plasmids (p.L747_P 753> S mutation, p.E746_S752> V mutation, p.L747_T751del mutation, p.L747_A750> P mutation, p.L747_T751> P mutation) were artificially synthesized as corresponding mutant templates, respectively, and after disruption of plasmid ultrasound and purification by bead screening, were mixed with fragmented HEK-293T cell line DNA (wild-type template) to prepare D3 samples (p.L747_P753 > S mutation), D8 samples (p.E746_S752 > V mutation), D13 samples (p.L747_T751 del mutation), D15 samples (p.L747_A750 > P mutation), D19 samples (p.L747_T751 > P) templates, respectively. A negative control (negative control) was also prepared using fragmented wild-type DNA, using TE Buffer as a template-free control (NTC).

2. Preparation of reaction System and amplification reading

After sample preparation was completed, the primer composition of the present invention was subjected to the configuration of a reaction system in accordance with the ratios shown in Table 2. The procedure of the droplet generation and PCR reaction, the amplification read-out procedure was the same as in example 1. A step of

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

Example 3

The primer composition designed in example 3 was as follows: that is, 6 mutant F1 primers share one F2 primer (i.e., mutant F2-1, SEQ ID NO: 8), and the corresponding mutant F1-1 sequence is (SEQ ID NO:15: ACTCGTACTCACGTACTCACTCTAACCGTTCCCTGTTCCAACGTCTAARRCTCC) observed for the effect on detection of the clinically most common mutant subtype (p.E746_A750del) (primer composition of example 3); the remaining 5 first upstream primers were: SEQ ID NO. 2-6; the probe, downstream primer, blocker oligonucleotide, and reference primer composition were the same as in example 1.

Example 3 primer compositions the reaction system was configured in the proportions described in Table 3. Wild-type samples and mock clinical samples (p.E746_A750del deletion mutation, d1 samples) were the same as in example 1, and the procedure for droplet generation and PCR reactions, and the procedure for amplification reads were the same as in example 1.

Table 3:

the detection results are shown in fig. 6 and 7, and no false positive fluorescent signal is generated in the negative control reaction; in the case of detecting the d1 sample, the detection result of this example shows that the double positive fluorescent signal is slightly biased toward the single positive fluorescent signal of the wild-type probe, and the difficulty in threshold division is greater than that of example 1.

Example 4

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

The specific primer composition is as follows:

in the absence of the blocker oligonucleotide, two mutant F2 primers were added to the system, i.e., one F2 primer (i.e., mutant F2-1, SEQ ID NO: 8) was added to the mutant F1-1 primer (SEQ ID NO: 1) alone, and the specific primer compositions are shown in Table 4. The two reaction sets correspond to Table 5, respectively.

TABLE 4 Table 4

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Table 5:

the wild type sample and the mock clinical sample (p.E746_A750del deletion mutation, i.e., d1 sample) used in this example 4 were the same as in example 1, and the droplet generation and PCR reaction procedure, and the amplification reading procedure were the same as in example 1.

As a result, as shown in FIGS. 9 to 10, it can be seen 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 fluorescent signals appear, the system specificity is improved;

when d1 samples are detected, the threshold is clearly divided, the data analysis is easy, and the accuracy is higher.

Comparative example

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

for the non-blocker oligonucleotide, 6 mutant F1 primers shared one F2 primer (i.e., mutant F2-2, SEQ ID NO: 9), and the specific primer compositions are shown in Table 4, with no F2-1 alone. The two reaction sets correspond to Table 6, respectively.

Table 6:

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the wild type sample and the mock clinical sample (p.E746_A750del deletion mutation, i.e., d1 sample) used in this comparative example were the same as in example 1, and the droplet generation and PCR reaction procedure, the amplification read procedure was the same as in example 1.

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

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

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

It is to be understood that this invention 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 also encompassed by the appended claims.

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Claims (2)

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

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

a second upstream primer SEQ ID NO. 8 and SEQ ID NO. 9;

the probe SEQ ID NO. 10;

the downstream primer SEQ ID NO. 11;

the wild-type blocker SEQ ID NO. 7.

2. Use of the primer composition of claim 1 for preparing an EGFR gene 19 exon mutation detection kit.

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