CN110541033A

CN110541033A - composition for detecting EGFR gene mutation and detection method

Info

Publication number: CN110541033A
Application number: CN201910925527.8A
Authority: CN
Inventors: 赵雨航; 王书芳; 葛志琪; 何辉煌; 李锦�
Original assignee: Mike Biological Ltd By Share Ltd
Current assignee: Mike Biological Ltd By Share Ltd
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-06
Anticipated expiration: 2039-09-27
Also published as: CN110541033B

Abstract

The invention discloses a high-sensitivity primer composition for detecting EGFR gene mutation and a detection method thereof. The primer composition comprises two upstream primers, a probe and a downstream primer, wherein the first upstream primer is only partially complementary and paired with a target sequence, and the sequences of the second upstream primer and the probe are respectively identical with partial sequences, which are not complementary and paired with the target sequence, on the first upstream primer. The detection method adopts a digital PCR method, and carries out pretreatment of melting a sample into a single strand before carrying out conventional digital PCR reaction. By using the primer composition and the detection method, the detection sensitivity and specificity of EGFR gene mutation can be improved, and the detection cost can be reduced; the primer composition and the detection method have wide application range and extremely low requirement on the DNA content in the sample.

Description

composition for detecting EGFR gene mutation and detection method

Technical Field

the invention relates to the field of molecular biology, in particular to a digital PCR primer for EGFR gene mutation detection and a detection method thereof. More specifically, the present invention relates to a digital PCR primer for improving detection sensitivity in the detection of nucleic acid sequence variation.

Background

The Polymerase Chain Reaction (PCR) is a molecular biology technique for the enzymatic replication of DNA without using living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as gene cloning, phenotypic identification of laboratory animals, transcriptome studies, detection of genetic diseases, identification of gene fingerprints, diagnosis of infectious diseases, paternity testing, and the like. Due to its incomparable replication and precision capabilities, PCR is considered by molecular biologists to be the first method of nucleic acid detection. In the later 90 s of the last century, Real Time Quantitative PCR (qPCR) technology and related products, which were introduced by ABI of America, developed PCR into a nucleic acid sequence analysis technology with high sensitivity, high specificity and accurate quantification.

However, there are many factors affecting the amplification efficiency during the PCR amplification process, and it cannot be guaranteed that the amplification efficiency remains the same during the reaction process and the amplification efficiency is the same between the actual sample and the standard sample as well as between different samples, thereby causing the basis on which the quantitative analysis depends-the cycle threshold (Ct) is not constant. Therefore, qPCR is only "relative quantitative", and the accuracy and reproducibility thereof still cannot meet the requirements of molecular biological quantitative analysis.

Histopathological diagnosis has long been the basis of gold standards for tumor diagnosis and clinical treatment. However, patients with tumors of the same histological type and stage are treated with the same treatment regimen, and often only a fraction of patients with tumors respond. According to statistics, the drug taking inefficiency of the traditional medical treatment in tumor treatment reaches 75 percent. The poor efficacy of malignant tumors is due to the difficulty in determining their malignant and pharmacodynamic characteristics at the tissue diagnosis level. Research shows that the molecular characteristics of tumor pathological changes determine the malignancy characteristics, metastasis characteristics, recurrence characteristics and drug resistance characteristics of the tumor pathological changes, and the molecular characteristics are the basic basis for judging the tumor after healing and reflecting the tumor to chemotherapeutic drugs. Therefore, individualized treatment based on molecular differences is the direction of precise treatment of tumors, and molecular typing is the basis for realizing individualized precise treatment.

Epidermal Growth Factor receptor egfr (epidermal Growth Factor receptor), a transmembrane tyrosine kinase receptor, has been implicated in the activation of multiple signaling pathways, such as cancer cell proliferation, metastasis and apoptosis. The EGFR gene is located in the short arm 7p12-14 region of chromosome 7 and consists of 28 exons. Studies have shown that there is high or abnormal expression of EGFR in many solid tumors. EGFR is involved in the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis. The high expression of EGFR plays an important role in the evolution of malignant tumors, and the high expression of EGFR is found in tissues such as glial cells, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer and the like.

Currently, EGFR gene mutation detection is mainly divided into detection of tumor tissue samples and detection of Circulating tumor DNA (ctDNA). At present, EGFR gene mutation is mainly detected clinically through tumor tissue samples obtained through tissue biopsy or operation and the like, however, since invasive means are needed for obtaining the tumor tissue samples, the pain of patients is increased, additional operation risks are generated, and tumors have heterogeneity, so that for cancer patients who have metastasized, only a certain part of the cancer tissue is taken through puncture or operation, and the overall situation of the patients cannot be reflected. Secondly, some patients decide themselves that they are not suitable for biopsy, while some tumours risk accelerated metastasis after being disturbed by puncture or surgery. Finally, tissue biopsy suffers from the problems of high cost, long waiting time, and the lag time is also disadvantageous for patient treatment. Therefore, in recent years, the concept of "liquid biopsy" has been rising, and it is a trend to acquire information on tumor gene mutation by detecting circulating DNA of tumor in a body fluid sample (mainly blood) of a patient by using a body fluid sample such as blood instead of a tumor tissue sample for pathology and molecular biology detection. Early screening, medication guidance, prognosis and relapse monitoring of tumor patients can be achieved by detecting circulating tumor dna (ctdna) in the peripheral blood of patients. However, because the background of the peripheral blood sample is complex, the ctDNA content is rare, and the detection of low abundance and rare sequences, the fluorescent quantitative PCR method, the molecular hybridization method, the capillary electrophoresis and the second-generation sequencing are all easily interfered by the background DNA, so that the detection sensitivity and the accuracy can not meet the requirements of accurate quantification.

The digital PCR (digital PCR) technique is an absolute nucleic acid molecule quantification technique, which uses the principle of limiting dilution to distribute a fluorescent quantitative PCR reaction system into thousands of individual nanoliter microreactors, so that each microreactor may or may not contain 1 or more copies of a target nucleic acid molecule (DNA target), and then simultaneously perform single-molecule template PCR amplification. 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. Especially, rare mutations are detected in complex backgrounds, and are often found in tumor liquid biopsy, noninvasive prenatal detection, organ transplantation monitoring, accurate quantification of viral load, component detection of transgenic crops and the like, for example, rare mutation markers are detected in peripheral blood of tumor patients, or gene expression difference research and the like.

Disclosure of Invention

in order to solve the above problems, the present invention provides a composition for detecting mutations in an EGFR gene and a detection method. The invention solves the problem that the gene mutation can not be detected because the content of the target sequence in the sample DNA is lower than the minimum detection limit in the detection process. The invention is particularly suitable for detecting samples with low target sequence content, such as plasma, FFPE samples and the like.

specifically, in a first aspect, the present invention provides a primer composition for detecting mutations in an EGFR gene, the primer composition comprising a mutant primer composition and/or a wild-type primer composition;

The mutant primer composition comprises an upstream primer F1, an upstream primer F1-1, a hydrolysis probe P1 and a downstream primer,

Wherein,

The upstream primer F1 sequentially comprises an upstream detection region and a target sequence binding region from the 5 'end to the 3' end:

(1) The upstream detection zone comprises from 5 'end to 3' end: part (a) having the same sequence as F1-1, and part (b) having the same sequence as hydrolysis probe P1, 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;

The wild type primer composition comprises an upstream primer F2, an upstream primer F2-1, a hydrolysis probe P2 and a downstream primer;

Wherein, the upstream primer F2 comprises an upstream detection area and a target sequence binding area from 5 'end to 3' end in sequence:

(1) The upstream detection zone comprises from 5 'end to 3' end: part (a) having the same sequence as F2-1, and part (b) having the same sequence as hydrolysis probe P2, and

(2) The 3' end of the target sequence binding region has an amplification decision site that is complementary to a mutation detection site on the wild-type target sequence, and the upstream of the amplification decision site has a mismatch region consisting of one or more bases that is not complementary to the target sequence.

In the present invention, the upstream detection region of the upstream primer F1 is not complementary to the target sequence, the upstream primer F1-1 and the hydrolysis probe P1, and the upstream detection region of the upstream primer F1 is also not complementary to the sequences of the upstream primer F2, the upstream primer F2-1 and the hydrolysis probe P2; the upstream detection region on the upstream primer F2 is not complementarily paired with the sequences of the target sequence, the upstream primer F2-1 and the hydrolysis probe P2, and the upstream detection region on the upstream primer F2 is not complementarily paired with the sequences of the upstream primer F1, the upstream primer F1-1 and the hydrolysis probe P1. That is, the upstream primer F1-1 and the probe P1 can be bound to the preamplified product only after the upstream primer F1 specifically preamplifies the target nucleic acid sequence. Only after specific pre-amplification of the target nucleic acid sequence by the upstream primer F2, the upstream primer F2-1 and the probe P2 can be paired and combined with the pre-amplified product. The upstream detection region on the upstream primer F1 does not pair identically or complementarily with the target sequence or hybridize under high stringency conditions; the upstream detection region on the upstream primer F2 does not pair identically or complementarily to the target sequence or hybridize under high stringency conditions.

The upstream detection region on the upstream primer F1 and the upstream detection region on the upstream primer F2 can be freely replaced. With or without a base separation between the upstream detection region on the upstream primer F1 and the target sequence binding region; there is no base separation between the upstream detection region on the upstream primer F2 and the target sequence binding region.

In some embodiments, the downstream primer in the mutant detection composition and the wild-type detection composition are the same or different; preferably, the downstream primer in the mutant-type detection composition and the downstream primer in the wild-type detection composition are the same.

in some more preferred embodiments, the downstream primer is positioned 1-150 bp downstream of the mutation detection site, and further 50-100 bp downstream of the complementary pair on the target sequence.

In some embodiments, the upstream primer F1 has a different Tm than the upstream primer F1-1; the difference between the Tm value of the upstream primer F1 and the Tm value of the upstream primer F1-1 is 0-20 ℃. Preferably, the Tm value of the F2 is higher than that of the upstream primer F2-1; preferably, the Tm value of the upstream primer F1 is higher than that of the upstream primer F1-1; more preferably, the Tm of the upstream primer F1 is 5 ℃ to 20 ℃ higher than the Tm of the upstream primer F1-1, and most preferably, the Tm is 10 ℃ to 15 ℃ higher. The upstream primer F2 and the upstream primer F2-1 have different Tm values; preferably, the Tm value of the upstream primer F2 is higher than that of the upstream primer F2-1; more preferably, the Tm of the upstream primer F2 is 5 ℃ to 20 ℃ higher than the Tm of the upstream primer F2-1, and most preferably, the Tm is 10 ℃ to 15 ℃ higher.

In some embodiments, the length of the mismatch region in the target sequence binding region on the forward primer F1 or the forward primer F2 is 1-20 bases. Preferably, the mismatch region is 1-15 bases in length. For example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 bases.

In some embodiments, the amplification decision site on the forward primer F1 or forward primer F2 is 1-15 bases from the upstream mismatch region. Preferably, the amplification determinant site on the forward primer F1 or the forward primer F2 is 3 to 7 bases away from the upstream mismatch region. In some embodiments of the present invention, the 3 ' end of the amplification determining site is the 3 ' end of the forward primer F1 or the 3 ' end of the forward primer F2, and no other base is downstream of the site. In other embodiments of the invention, the amplification-determining site is 1-10 bases downstream.

In some embodiments, the upstream primer F1 is separated by one or more bases between part (a) and part (b) in the upstream detection region.

In some embodiments, the reporter groups of hydrolysis probe P1 and hydrolysis probe P2 are different. The reporter group is detectable only after the hydrolysis probe is hydrolyzed. In a further embodiment, 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 another aspect, the present invention provides a method for detecting mutations in the EGFR gene, comprising the steps of:

(i) Providing a sample to be tested comprising a target nucleic acid, and pretreating the sample;

(ii) Carrying out limit dilution on the pretreated sample, randomly distributing the sample to 770-10000000 reaction units, and then carrying out uniform thermal cycle amplification on all the reaction units;

(iii) pre-amplifying the target nucleic acid with the upstream primer F1 and/or the upstream primer F2 as a primer pair at a first annealing temperature;

(iv) (iii) continuing to amplify the pre-amplification product obtained in step (ii) with a first primer composition and/or a second primer composition at a second annealing temperature, wherein the first primer composition comprises an upstream primer F1-1, a hydrolysis probe P1 and a downstream primer, the second primer composition comprises an upstream primer F2-1, a hydrolysis probe P2 and a downstream primer, and the hydrolysis probe P1 and the hydrolysis probe P2 carry a reporter group; and

(v) (iii) detecting a signal emitted by the reporter group in the reaction system after step (iv) and quantifying the target nucleic acid in the sample based on the signal;

wherein,

The upstream primer F1 comprises an upstream detection area and a target sequence binding area from the 5 'end to the 3' end in sequence:

the upstream primer F2 sequentially comprises an upstream detection region and a target sequence binding region from the 5 'end to the 3' end:

the quantification is an absolute quantification, which is intended to determine the number of molecules of the gene of interest in the sample, known as the copy number.

In some embodiments, the pretreatment in step (i) is a step of treating the target nucleic acid double strand to obtain a single strand.

the purpose of the above-mentioned "denaturation" is to break the hydrogen bonds between pairs of complementary bases on double-stranded DNA, thereby allowing the double strand to separate into two single strands.

In some embodiments, single strands can be formed by heating a mixture containing double stranded DNA, for example, heating the mixture to 90 ℃, 92 ℃, 95 ℃, or 98 ℃ to dissociate double stranded DNA. Typically, upon double-stranded dissociation, the mixture needs to be maintained at the elevated temperature for at least 10 seconds or more, e.g., 30 seconds, 1 minute, 2 minutes, 5 minutes, or even more, to achieve 90% or more dissociation of the double-stranded DNA. In order to maintain the double-stranded DNA in a single-stranded state after dissociation, the solution containing the single-stranded DNA needs to be cooled to a temperature lower than room temperature, for example, lower than 25 ℃,20 ℃ or 15 ℃.

In some embodiments, the above "denaturation" can also be accomplished by altering the solution ionic strength (e.g., adding acids, bases, salts, etc.) to break hydrogen bonds between double-stranded DNA, and an enzyme (e.g., helicase) can also be employed to effect dissociation of double-stranded DNA into single-stranded DNA.

In some embodiments, the pretreatment is any denaturation step that makes a target nucleic acid double strand single-stranded, preferably by formaldehyde heating, alkaline treatment.

In some embodiments, the Tm of the forward primer F1 is different from the Tm of the forward primer F1-1 by 0-20 ℃. Preferably, the Tm value of the upstream primer F1 is higher than that of the upstream primer F1-1; the Tm value of the upstream primer F2 is higher than that of the upstream primer F2-1.

in some embodiments, the Tm of the upstream primer F1 is 5 ℃ to 20 ℃ higher, more preferably 10 ℃ to 15 ℃ higher, than the Tm of the upstream primer F1-1.

In some embodiments, the length of the upstream primer F1 and the upstream primer F2 is 50-90 bp, the Tm value is 50-80 ℃, and the GC content is 40-80%.

In some embodiments, the upstream primer F1-1 and the upstream primer F2-1 have a length of 13-30 bp, a Tm value of 50-70 ℃ and a GC content of 40-80%.

In some embodiments, the hydrolysis probes P1 and P2 have a length of 15-30 bp, a Tm of 55-75 ℃ and a GC content of 40-80%.

in some embodiments, the primer composition for mutant detection and the primer composition for wild type detection use a downstream primer R, which has a length of 15 to 30bp, a Tm value of 55 to 75 ℃, and a GC content of 40 to 80%.

In some embodiments, the hydrolysis probe P1 and hydrolysis probe P2 carry a reporter and a quencher, and the reporter of the hydrolysis probe P1 and hydrolysis probe P2 are different. The reporter group is detectable only after the hydrolysis probe is hydrolyzed. In a further embodiment, 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 some embodiments, only the mutant-type detection primer composition or the wild-type detection primer composition is used in the same reaction system. In another embodiment, a plurality of mutant type detection primer compositions and/or wild type detection primer compositions are used in the same reaction system. Preferably, for example, when a plurality of primer compositions for mutant type detection are used, the amplification determining site of the upstream primer F1 differs among the plurality of primer compositions for mutant type detection, and the portion (b) of the cleavage upstream determining region also differs; the plurality of probes have mutually different sequences and reporter groups; optionally, part (a) of the upstream detection zone is also different. The method can be used to simultaneously determine both wild-type and mutant target sequences, or to simultaneously determine multiple mutant target sequences. In some embodiments using multiple F1 primers and probes, different forward primers, F1, may share the same reverse primer.

In some embodiments, the number of cycles for pre-amplification of the forward primer F1 and the reverse primer R is 3-10 cycles; furthermore, the number of cycles of pre-amplification by the upstream primer F1 is 5-8 cycles. In some embodiments, the number of cycles for amplification and determination of the forward primer F1-1 and the hydrolysis probe P1 is 35-50 cycles; more preferably, the number of cycles for amplification and measurement of the upstream primer F1-1 and the hydrolysis probe P1 is 40-45 cycles.

procedures and common reaction conditions (e.g., denaturation temperature, time, etc.) for digital PCR amplification are well known in the art. For example, in some exemplary embodiments, the specific amplification reaction conditions in steps (iii) and (iv) may be:

Firstly, pre-denaturation is carried out for 5-15 minutes at the temperature of 92-96 ℃;

Secondly, performing denaturation at the temperature of 92-95 ℃ for 10-60 seconds, annealing at the temperature of 55-75 ℃ and extending for 30-90 seconds for 3-10 cycles;

thirdly, denaturation is carried out for 10-60 seconds at the temperature of 92-95 ℃, annealing and extension are carried out for 30-90 seconds at the temperature of 45-65 ℃, and 35-50 cycles are carried out;

Inactivating at 94-98 ℃ for 5-15 minutes; the reaction is terminated at 4-15 ℃.

the concentration of the primers and probes in the reaction system described in the present invention can be determined by routine experiments in the art. In some exemplary embodiments, the concentration of F1 primer in the reaction system is 15nM to 150nM, the concentration of F1-1 primer is 150nM to 1500nM, the concentration of hydrolysis probe P1 is 50nM to 800nM, and the concentration of R primer is 150nM to 1800 nM. In some more preferred embodiments, the concentration of the F1 primer in the reaction system is 30nM to 60nM, the concentration of the F1-1 primer is 300nM to 600nM, the concentration of the hydrolysis probe P1 is 150nM to 400nM, and the concentration of the primer R is 300nM to 900 nM.

the signal detection method disclosed by the invention adopts the principle of a TaqMan probe method, namely, the 5 '-3' exonuclease activity of DNA polymerase is utilized to hydrolyze the probe so as to generate a fluorescent signal. Due to the specific binding of the probe and the template, in the digital PCR reaction, the number of droplets emitting fluorescence signals represents the number of templates in the reaction system, and finally the concentration of the templates can be obtained through Poisson correction. For the TaqMan probe as a fluorescent signal generation means, the probe is designed in the same manner as the conventional PCR method.

In some embodiments, the sample providing the target sequence may be a biological sample, such as a biological fluid, a living tissue, a frozen tissue, a paraffin section, and the like. In some preferred embodiments, the sample is, e.g., peripheral blood, urine, lavage, cerebrospinal fluid, stool, saliva, and the like.

The digital PCR used in the present invention is different from the fluorescence PCR, and does not collect the fluorescence signal in real time, but detects the signal at the end point, preferably the fluorescence signal, after the whole thermal cycling reaction is finished. Therefore, in the first 5-10 cycles of thermal cycle amplification, the hydrolysis probe P1(P2) can be combined with the pre-amplification product, but the hydrolysis is dependent on the amplification hydrolysis of the upstream primer F1-1 (upstream primer F2-1), and when the temperature is not suitable for the combination of the upstream primer F1-1 (upstream primer F2-1) and the template, the hydrolysis probe P1 (hydrolysis probe P2) is not hydrolyzed and fluorescence is emitted. Therefore, in order to avoid hydrolysis of the hydrolysis probe P1 (hydrolysis probe P2) by the forward primer F1-1 (forward primer F2-1) during preamplification, the annealing temperature of the forward primer F1 (forward primer F2) of the present invention is higher than that of the forward primer F1-1 (forward primer F2-1).

in the reaction system of the present invention, the length between the upstream primers F1, F2 and the downstream primer R for specifically enriching the target nucleic acid sequence does not exceed 100bp, and thus, the present invention is particularly suitable for detecting a DNA sample with a short fragment.

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

(1) 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 extremely short, and the probe P can be matched and combined with the complementary sequence at the 5' end of the upstream primer F1 after the pre-amplification is finished, so that the part actually matched and combined with the target nucleic acid sequence is only 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.

(2) 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 upstream primer F1 after the pre-amplification is completed, the portion actually coupled to the target nucleic acid sequence is only two: the 3' end sequence of F1 and the sequence of the downstream primer R. Therefore, when a complex target nucleic acid sequence is detected, the design method of the primer probe can avoid a GC unbalanced area, and is particularly lower than a TaqMan probe method and ARMS in the design difficulty of the probe.

(3) The requirement on the content of target DNA in a sample is low: when analyzing samples with the target DNA content lower than the optimal quantity, the invention can be used for effectively increasing the quantity of target sequences in the samples and improving the detection sensitivity, thereby reducing errors caused by too small quantity of samples. Such samples typically include plasma (cf DNA samples), biopsy punctures, or FFPE samples. This method typically involves melting double-stranded DNA into its constituent strands, such as single-stranded DNA (ssdna), prior to droplet formation, and then separating each strand prior to amplification and counting.

(4) High sensitivity: the lowest sensitivity of the kit of the invention for detecting the target nucleic acid sequence in the complex background can reach 0.01%, and more preferably, the kit of the invention can stably detect the target nucleic acid sequence in the complex background by 0.05%, namely, the kit can ensure that 10 copies of the target nucleic acid sequence can be stably detected in a total nucleic acid background of 20,000copies in the detection of more than 95%, or 15 copies of the target nucleic acid sequence can be stably detected in a total nucleic acid background of 30,000 copies. The invention can realize the stable detection of the low-concentration sample and the low-mutation abundance sample, so as to meet the requirement of monitoring the free DNA sample in the peripheral blood circulation of a tumor patient in clinic, reflect the current state of the tumor of the patient, guide the targeted medication and be used for prognosis monitoring.

(5) High specificity: the primer probe design method and the reaction system can well avoid cross reaction, namely, when detecting mutant target nucleic acid sequences, no wild type or other similar or homologous target nucleic acid cross reaction exists. Particularly, when the wild type and the mutant are detected simultaneously, the cross reaction between the wild type and the mutant is small, and the detection of rare mutation is more facilitated.

(6) The application range is wide: the reaction system can detect short-fragment DNA smaller than 200bp, 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.

(7) The sample consumption is less: the reaction system and the kit can simultaneously detect the mutant type and the wild type target nucleic acid sequence in one reaction tube, and carry out absolute quantification and mutation abundance statistics on the mutant type and the wild type target nucleic acid sequence, and are particularly suitable for detecting rare samples, such as peripheral blood circulation tumor DNA samples.

(8) The cost is low: the primer probe design method and the reaction system do not need expensive MGB modification or LNA modification, greatly reduce the use cost of the primer probe, have better detection performance and meet the requirement of clinical use. In addition, after the target DNA double strand is uncoiled into the single strand, the unbalanced free DNA does not need to be repaired by using a terminal repair enzyme, so that the detection cost is reduced.

(9) The experimental steps are simple and convenient: according to the invention, after the reaction system containing the template is subjected to the melting step, the terminal repair enzyme is not required to be added into the reaction liquid for repair, so that the experiment steps are reduced, and the experiment time is saved.

Drawings

FIG. 1 is a schematic diagram showing the design of primers for use in the kit of the present invention

Taking the upstream primer F1 as an example, the upstream primer comprises an upstream detection region and a target sequence binding region in sequence from the 5 ' end to the 3 ' end, wherein the 3 ' end of the target sequence binding region has an amplification decision site, the amplification decision site is complementary with a variation detection site on the target sequence, and the upstream of the amplification decision site has a mismatch region consisting of one or more bases and not complementary with the target sequence; the upstream detection zone comprises from the 5 'to the 3' end: a portion (a) having the same sequence as that of the forward primer F1-1, and a portion (b) having the same sequence as that of the probe;

FIG. 2 shows the results of the test of the kit of example 1 of the present invention on a sample with a theoretical dilution of 10%;

FIG. 3 shows the result of the test of the kit of example 2 of the present invention on a sample with a theoretical dilution of 10%;

FIG. 4 is a schematic diagram showing the comparison of the detection concentrations of the kit of the present invention and the kit of a comparative manufacturer for the same sample;

FIG. 5 is a schematic diagram showing the comparison of the detected concentrations of the same sample for kits of different target sequence lengths.

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 "Gene" (Gene, Mendelian factor) in the present invention is also called a genetic element. Refers to the basic genetic unit of DNA or RNA sequence carrying genetic information and controlling biological traits. The gene expresses the genetic information carried by the gene by guiding protein synthesis, thereby controlling the character expression of the organism individual.

The term "wild-type gene" as used herein refers to the allele that is most abundant in nature and is often used as a standard control gene in biological experiments. The concept corresponding to this is a mutant gene. The latter is often mutated from the wild-type gene. In the present invention, the wild type is an EGFR gene sequence which is not subjected to deletion mutation, and the mutant type is an EGFR gene sequence which is subjected to deletion mutation.

from a molecular level, a gene mutation is a change in the base pair composition or arrangement order of a gene in its structure.

Gene mutation refers to the alteration of the structure of a gene by the addition, deletion and substitution of base pairs in a DNA molecule. Deletion mutation refers to a mutation of a gene due to deletion of a long segment of DNA.

According to the customary terminology in the art, the length of a nucleic acid can be expressed as bases, base pairs (abbreviated "bp"), nucleotides/nucleotide residues (abbreviated "nt"), or kilobases ("kb"). The terms "base", "nucleotide residue" may describe a polynucleotide, either single-stranded or double-stranded, where the context permits. When the term is applied to double-stranded molecules, it is used to refer to the entire length and should be understood as equivalent to the term "base pair".

The term "primer" refers to a molecule having a specific nucleotide sequence, which is covalently linked to a reactant, at the initiation of nucleotide polymerization, and stimulates synthesis of the molecule, and such a molecule is referred to as a primer. The primer pair is usually two oligonucleotide sequences synthesized artificially, one primer is complementary to one DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region, and functions as a starting point for nucleotide polymerization, and the nucleic acid polymerase can synthesize a new nucleic acid strand from its 3' end.

The term "forward primer", also referred to as forward primer, as used herein, is an oligonucleotide that extends uninterrupted along the negative strand; the term "downstream primer", also called reverse primer, as used herein, is an oligonucleotide that extends uninterrupted along the forward strand. The positive strand, i.e., the sense strand, also called the coding strand, is generally located at the upper end of the double-stranded DNA in the direction from left to right 5 '-3', and the base sequence is substantially identical to the mRNA of the gene; the primer binding to the strand is a reverse primer; the negative strand, i.e., the nonsense strand, is also called the noncoding strand, is complementary to the positive strand, and the primer that binds to this strand is the forward primer. It is understood that when the designations of sense and antisense strands are interchanged, the corresponding forward and reverse primer designations may be interchanged accordingly.

as used herein, the terms "upstream," "at/upstream of … …," "… …" and the like, in the context of describing nucleic acid sequences, refer to a portion of the same nucleic acid sequence that is closer to the 5' end than a reference region, e.g., can be immediately adjacent to the reference region or can be separated from the reference region by one or more bases. As used herein, the terms "downstream," "at/downstream of … …," "having … …" and the like, in the context of describing nucleic acid sequences, refer to portions of the same nucleic acid sequence that are 3' of the referenced region, e.g., can be immediately adjacent to the referenced region or can be separated from the referenced region by one or more bases. It will be appreciated that, unless otherwise indicated, where the nucleic acid being described is a double-stranded nucleic acid, the designations "upstream" and "downstream" are generally based on the 5 'and 3' ends of the sense strand.

The term "probe" refers to an oligonucleotide that selectively hybridizes 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 (R) and a 3' quencher (Q). Fluorescent reporter groups (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 "label" 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. Examples of labels include fluorophores, chromophores, radioactive atoms (especially 32p and 125I), electron-dense reagents, enzymes and ligands with specific binders.

as used herein, the terms "target sequence binding region", "upstream detection region", "amplification decision region", "mismatch region", "part (a)" and part (b) "are different segments located on the F1 primer, wherein the amplification decision region and the mismatch region are located within the target sequence binding region and part (a) and part (b) are located within the upstream detection region. It will be understood that the target sequence binding region indicates that the region is for hybridisation or annealing to the target sequence and does not imply that the region necessarily enables 100% base complementary pairing with the target sequence and that there may be regions of mismatch within the target sequence binding region. The term "mutation detection site" is located on a target sequence and refers herein to a segment that differs between different target sequences to be detected, e.g., a segment that differs in sequence between wild type and mutant types. The mutation detection site may be one or more base pairs in length. Commonly, the variation may be, for example, a point mutation (compared to the wild type), a deletion mutation, an insertion mutation, a base inversion mutation, or the like.

The terms "Taqman probe (TaqMan probe)" and "hydrolysis probe (hydrolysis probe)" are used interchangeably herein. The Taqman probe is a fluorescence detection technology developed on a Real-time PCR technology platform, the 5 'end of the probe contains a fluorescence reporter group, and the 3' end of the probe contains a fluorescence quenching group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group, and when PCR amplification is carried out, the exonuclease activity from the 5 'end to the 3' end of Taq DNA polymerase enzyme cuts and degrades the probe, so that the reporter group and the quenching group are separated, and the fluorescent signal is emitted, thereby achieving the complete synchronization of the accumulation of the fluorescent signal and the formation of a PCR product. Specifically, the reporter group may be FAM, HEX, VIC, ROX, Cy5, Cy3, or the like, and the quencher group may be TAMRA, BHQ1, BHQ2, BHQ3, DABCYL, QXL, DDQI, but is not limited thereto. In addition, other modification forms are derived from the Taqman probe, for example, the Taqman-MGB probe is a Taqman probe with minor groove binding Molecules (MGBs) at the 3' end, the Tm value of the probe is improved, the length of the probe is shortened, and simultaneous detection of multiple mutation sites is facilitated.

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 applicable in the context of the instant claims.

The term "target nucleic acid sequence" refers to a nucleic acid sequence that is detected, amplified, or both using the primer/probe sets provided herein. In addition, while the term target sequence refers in some instances to being single-stranded, one of ordinary skill in the art will recognize that the target sequence is actually double-stranded. Thus, in the case where the target is double stranded, the primer sequences of the invention will amplify both strands of the target sequence.

The terms "target sequence", "target nucleic acid" or "target" as used herein are used interchangeably and refer to the portion of a nucleic acid sequence to be amplified, detected or amplified and detected, which can anneal or hybridize to a probe or primer under hybridization, annealing or amplification conditions.

The term "hybridization" refers to a base pairing interaction between two nucleic acids that results in the formation of a duplex. It is not required that 2 nucleic acids have 100% complementarity over their entire length to achieve hybridization.

The term "base" refers to purine and pyrimidine derivatives, which are components of nucleic acids, nucleosides, nucleotides. The total number of bases is 5: cytosine (abbreviated as C), guanine (G), adenine (A), thymine (T, exclusive to DNA) and uracil (U, exclusive to RNA), and bases of DNA are cytosine (abbreviated as C), guanine (G), adenine (A) and thymine (T).

the term "mismatched base" refers to a base pair in the double helix structure of DNA which is not an arbitrary base, and is always the principle that adenine (A) is paired with thymine (T) and guanine (G) is paired with cytosine (C). If A is paired with C or G, or T is paired with G or C, it is a base mismatch.

The "stringent conditions" described herein may be any of low stringency conditions, medium stringency conditions and high stringency conditions. "Low stringency conditions" such as 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide, 32 ℃; further, "medium stringent conditions" include, for example, conditions of 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide, 42 ℃; "high stringency conditions" include, for example, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide, and 50 ℃ conditions. Under these conditions, it is expected that a polynucleotide having a high homology, such as DNA, can be obtained more efficiently at a higher temperature. Although there are various factors that affect the stringency of hybridization, such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., one skilled in the art can obtain similar stringency by appropriately selecting these factors.

The term "mutation abundance" as used herein refers to a relative or absolute quantitative value of the mutant target gene, and is generally defined as the ratio of the number of mutant target gene molecules to the total number of DNA molecules in the assay.

In the present invention, the DNA sample is derived from free DNA extracted from peripheral plasma of a cancer patient, or derived from a fragmented cell line DNA sample, or derived from an artificially synthesized plasmid DNA sample.

the invention provides a primer composition for detecting EGFR gene mutation. The EGFR protein tyrosine kinase functional region is coded by EGFR gene exons 18-24, wherein exons 18-20 code N-Lobe, and exons 21-24 code C-Lobe. EGFR mutations found to date are located predominantly in exons 19 to 21; more than 90% of EGFR mutations of non-small cell lung cancer are deletion mutation of exon19 and L858R point mutation of exon 21. The base deletion of the 19 th exon is mainly deletion mutation at codon 746-752 leading to the loss of amino acid sequence in EGFR protein, thereby changing the sensitivity of cells to TKIs; the T-M transition mutation of codon 790 of exon 20 (2669 bp substitution at nucleotide position) is the main cause of drug resistance; the point mutation of exon21 is mainly the T-G conversion at codon 858 to convert leucine to arginine, abbreviated as L858R.

The design scheme of the primer composition of the invention is shown in FIG. 1. Taking the upstream primer F1 as an example, the upstream primer comprises an upstream detection region and a target sequence binding region in sequence from the 5 ' end to the 3 ' end, wherein the 3 ' end of the target sequence binding region has an amplification decision site, the amplification decision site is complementary with a variation detection site on the target sequence, and the upstream of the amplification decision site has a mismatch region consisting of one or more bases and not complementary with the target sequence; the upstream detection zone comprises from the 5 'to the 3' end: a portion (a) having the same sequence as that of the forward primer F1-1, and a portion (b) having the same sequence as that of the probe. After the primer pair is used for enriching the target nucleic acid template, the change of annealing temperature is utilized to lead the upstream primer F1-1 and the hydrolysis probe P1 to identify a sequence on a pre-amplification product, which is complementary with an upstream detection area of the upstream primer F1, then the upstream primer and the reverse primer R form a primer pair for template amplification, and a fluorescent signal is released by the TaqMan hydrolysis probe principle.

The downstream primer in the present invention may be a conventional primer complementary to a sequence downstream of the target sequence variation detection site, for example, a primer designed by a conventional technique known in the art according to the base complementary pairing principle.

Experimental apparatus and materials

the kit for detecting EGFR gene mutation comprises the following components:

In the embodiment of the invention, a droplet digital PCR system (ddPCR) is mainly used, double strands of DNA in a sample are melted into single strands before microdroplets are generated, and then the microdroplets are processed before traditional PCR amplification, namely, a reaction system containing nucleic acid molecules is divided into thousands of nano-upgrade microdroplets, wherein each microdroplet contains no nucleic acid target molecules to be detected or contains one to a plurality of nucleic acid target molecules to be detected. After PCR amplification, each microdroplet is detected one by one, the microdroplet with a fluorescent signal is judged to be 1, the microdroplet without the fluorescent signal is judged to be 0, and the initial copy number or the concentration of the target molecule can be obtained according to the Poisson distribution principle and the number and the proportion of the positive microdroplets.

the use method of the kit is as follows:

the reaction buffer solution and the primer probe premix are mixed according to a reaction system shown in the following table, then DNA is extracted from a sample to be detected by a proper method and added into the prepared reaction system, and then the partitioning of a digital PCR microreactor (microdroplet), the PCR amplification and the detection of a fluorescent signal are carried out.

The kit can be matched with a QX200 micro-drop digital PCR system (ddPCR) and consumables of Bio-Rad company for detection.

The specific amplification reaction system is shown in the following table:

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 exon19 of the EGFR gene detected in the sample to be tested is 50 copies/. mu.L, and the concentration of the wild-type target nucleic acid of the exon19 of the EGFR gene detected in the sample to be tested is 9950 copies/. mu.L, the abundance of the exon19 mutation of the EGFR gene in the sample to be tested is:

[ (50 copies/. mu.L)/(50 copies/. mu.L +9950 copies/. mu.L) ]. 100% -0.5%

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 the primer composition for detection and the detection method was evaluated by simulating the L858R point mutation of the EGFR gene in a clinical specimen.

The simulated clinical sample is a sample obtained by mixing prepared fragmented mutant DNA (in this example, the EGFR gene with the L858R point mutation is randomly fragmented) and fragmented wild-type DNA (the EGFR gene without the mutation is randomly fragmented) according to a certain ratio.

a primer composition for detecting an EGFR gene L858R point mutation:

The sequence of the mutant F1 primer is SEQ ID NO. 1, the sequence of the wild F2 primer is SEQ ID NO. 2, the sequence of the mutant F1-1 is SEQ ID NO. 3, the sequence of the wild F2-1 is SEQ ID NO. 4, the sequence of the mutant probe P1 is SEQ ID NO. 5, the sequence of the wild probe P2 is SEQ ID NO. 6, and the sequence of the downstream primer R is SEQ ID NO. 7. The mutant probe shown by SEQ ID NO. 5 was labeled with FAM at the 5 'end and BHQ1 at the 3' end. The wild-type probe shown by SEQ ID NO. 6 was labeled with HEX at the 5 'end and BHQ1 at the 3' end. See table 1 for details.

Table 1 (nucleotide sequence from left to right below is 5 '→ 3')

In the primer probe, the total length of the mutant F1 primer (SEQ ID NO:1) is 65bp, the total length of the wild F2 primer (SEQ ID NO:2) is 66bp, 23 bases at the 3 'ends of the mutant F1 primer and the wild F2 primer are matched with a target nucleic acid sequence, and the 3' end is an EGFR gene L858R mutation site. The 19 bases at the 5 'end of the mutant primer were identical to the base sequence of the corresponding F1-1 primer (SEQ ID NO:3), and the 18 bases at the 5' end of the wild-type F2 primer were identical to the base sequence of the corresponding F2-1 primer (SEQ ID NO:4), respectively. The 22 nd to 40 th base sequences at the 5' -end of the mutant F1 primer were identical to the base sequence of the corresponding mutant probe P1(SEQ ID NO: 5). The 22 nd to 40 th base sequences at the 5' -end of the wild-type F2 primer were identical to the base sequence of the corresponding wild-type probe P2(SEQ ID NO: 6). Therefore, after the mutant F1 primer and the wild-type F2 primer specifically amplify the target nucleic acid, respectively, a base sequence and a complementary sequence thereof from the 5' end of the F1 primer and the F2 primer are added to the generated amplification product, and then the F1-1 primer, the probe P1, the F2-1 primer and the probe P2 can be coupled with the corresponding target nucleic acid template in a pairing manner and hydrolyzed to emit a fluorescent signal.

reaction system

The PCR reaction mixture was prepared at the following concentrations (taking 20. mu.L as an example)

TABLE 2

The kit for detecting EGFR gene mutation comprises the following components:

Wherein, the negative control is a fragmented healthy human genome DNA sample, the absence of EGFR gene L858R mutation is confirmed by sequencing, and the negative control is prepared into a negative control of 20,000 copies/microliter by digital PCR quantification and using Tirs-EDTA buffer solution.

Sample preparation

Free DNA extracted from healthy human plasma is prepared, and the DNA does not contain EGFR gene L858R mutation through second-generation sequencing, so that a wild type free DNA sample is obtained and used as a negative sample.

Meanwhile, NCI-H1975 cell line DNA samples quantified by digital PCR are prepared, and after fragmentation treatment, EGFR gene L858R mutation samples diluted to 10% mutation abundance by wild-type DNA samples are used as positive samples.

the method for detecting the L858R mutation site of the EGFR gene by using the primer composition comprises the following steps:

Preparation of the reaction System

After the preparation, the reaction system was prepared, and the PCR reaction solution was prepared according to the ratio shown in table 1.

The primer probes used were respectively: mutant F1(SEQ ID NO:1), wild-type F2(SEQ ID NO:2), mutant F1-1(SEQ ID NO:3), wild-type F2-1(SEQ ID NO:4), mutant probe P1(SEQ ID NO:5), wild-type probe P2(SEQ ID NO:6), and downstream primer R (SEQ ID NO: 7).

Before the generation of the droplets, the prepared reaction solution containing the template was double-stranded and single-stranded in 20. mu.l.

And taking 20 microliters of the prepared PCR reaction liquid, adding the prepared PCR reaction liquid into a sample hole of the microdroplet generation card, then adding 70 microliters 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 about 40 microliters of droplet suspension is carefully transferred from the uppermost row of wells to a 96-well PCR plate.

Amplification reading

And (3) sealing the membrane of the 96-well plate, and then placing the 96-well plate in a PCR thermal cycler for PCR amplification. The procedure used was: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds and annealing and extension at 54 ℃ for 60 seconds; denaturation at 94 ℃ for 30 seconds and annealing and extension at 54 ℃ for 60 seconds for 47 cycles; inactivating at 98 ℃ for 10 minutes; 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.

Analytical statistics

And (3) analyzing the intensity and the number of the fluorescence signals by using QuantaSoft analysis software to obtain the copy number and the concentration of the mutant and the wild type of the EGFR gene L858R, and calculating the abundance of the mutation.

the results of the test using the kit of the present invention on the mixed sample at a theoretical dilution of 10% are shown in FIG. 2.

By using the kit, the negative coincidence rate of the detection results of the negative reference substance and the blank reference substance is 100%.

By using the kit, 4 independent repeated tests are carried out on a diluted sample with the theoretical mutation abundance of 10%, so that the CV value of the quantitative result is 5.24%, less than 20%, the average value is 10.3% and the standard deviation is 0.54% in the sample with the theoretical mutation abundance of 10%.

Example 2

The performance of the primer composition for detection and the detection method was evaluated by using a simulated clinical sample EGFR gene 19Del point mutation.

A primer composition for detecting 19Del point mutation of EGFR gene:

the sequence of the mutant F1 primer is SEQ ID NO. 8, the sequence of the wild F2 primer is SEQ ID NO. 9, the sequence of the mutant F1-1 is SEQ ID NO. 10, the sequence of the wild F2-1 is SEQ ID NO. 11, the sequence of the mutant probe P1 is SEQ ID NO. 12, the sequence of the wild probe P2 is SEQ ID NO. 13, and the sequence of the downstream primer R is SEQ ID NO. 14. The mutant probe shown by SEQ ID NO. 12 was labeled with FAM at the 5 'end and BHQ1 at the 3' end. The wild-type probe shown by SEQ ID NO. 13 was labeled with HEX at the 5 'end and BHQ1 at the 3' end. See table 3 for details.

Table 3 (nucleotide sequence from left to right below 5 '→ 3')

in the primer probe, the total length of the mutant F1 primer (SEQ ID NO:8) is 65bp, the total length of the wild type F2 primer (SEQ ID NO:9) is 67bp, 21 bases at the 3 ' end of the mutant F1 primer are matched with a target nucleic acid sequence, 25 bases at the 3 ' end of the wild type F2 primer are matched with a target nucleic acid sequence, and the position near the 3 ' end of the mutant F1 primer and the wild type F2 is an EGFR gene 19Del mutation site. The 20 bases at the 5 '-end of the mutant primer were identical to the base sequence of the corresponding F1-1 primer (SEQ ID NO:10), and the 19 bases at the 5' -end of the wild-type F2 primer were identical to the base sequence of the corresponding F2-1 primer (SEQ ID NO:11), respectively. The 22 nd to 41 th base sequences at the 5' -end of the mutant F1 primer were identical to the base sequence of the corresponding mutant probe P1(SEQ ID NO: 12). The 20 th to 40 th base sequences at the 5' -end of the wild-type F2 primer were identical to the base sequence of the corresponding wild-type probe P2(SEQ ID NO: 6). Therefore, after the mutant F1 primer and the wild-type F2 primer specifically amplify the target nucleic acid, respectively, a base sequence and a complementary sequence thereof from the 5' end of the F1 primer and the F2 primer are added to the generated amplification product, and then the F1-1 primer, the probe P1, the F2-1 primer and the probe P2 can be coupled with the corresponding target nucleic acid template in a pairing manner and hydrolyzed to emit a fluorescent signal. Wherein, the R base is represented by A or G.

Reaction system

TABLE 4

The kit for detecting EGFR gene mutation comprises the following components:

wherein, the negative control is a fragmented healthy human genome DNA sample, the absence of EGFR gene 19Del specific mutation is confirmed through sequencing, and the negative control is prepared into a negative control substance of 20,000 copies/microliter through digital PCR quantification and using Tirs-EDTA buffer solution.

Sample preparation

293T cell line DNA purified by ultrasonic is prepared, and a negative sample which does not contain the 19del mutation of the EGFR gene is determined by second-generation sequencing.

meanwhile, a DNA sample of the NCI-H1650 cell line which is quantified by digital PCR is prepared, and after fragmentation treatment, a 19del mutation sample of the EGFR gene which is diluted to 10% of mutation abundance by using a wild type DNA sample is used as a positive sample.

the primer composition is used for detecting the 19del mutation site of the EGFR gene:

Preparation of the reaction System

After the preparation of the sample was completed, the reaction system was prepared, and PCR reaction solutions were prepared according to the ratios shown in tables 3 and 4.

The primer probes used were respectively: mutant F1(SEQ ID NO:8), wild-type F2(SEQ ID NO:9), mutant F1-1(SEQ ID NO:10), wild-type F2-1(SEQ ID NO:11), mutant probe P1(SEQ ID NO:12), wild-type probe P2(SEQ ID NO:13), and downstream primer R (SEQ ID NO: 14).

Amplification reading

analytical statistics

And analyzing the intensity and the number of the fluorescence signals by using QuantaSoft analysis software to obtain the copy number and the concentration of the 19del mutation type of the EGFR gene, and calculating the abundance of the mutation.

The results of the test using the kit of the present invention on the mixed sample at a theoretical dilution of 10% are shown in FIG. 3.

by using the kit, 4 independent repeated tests are carried out on a diluted sample with the theoretical mutation abundance of 10%, so that the CV value of the quantitative result is 2.02% and less than 20%, the average value is 9.9% and the standard deviation is 0.20% in the sample with the theoretical mutation abundance of 10%.

The following comparative examples will be made by exemplifying the mutation of the L858R gene of EGFR.

Comparative example 1

the experiment was performed using fragmented NCI-H1975 cell line DNA samples (EGFR L858R mutation) in comparison to the primer probes used in the same manufacturer's kit.

Sample preparation

A fragmented NCI-H1975 cell line DNA sample which is quantified by digital PCR and contains EGFR gene L858R mutation with mutation abundance of 75% is used for simulating clinical circulating tumor DNA.

meanwhile, genome DNA from healthy people is prepared, after the fact that the genome DNA does not contain EGFR gene L858R mutation is determined through second-generation sequencing, enzyme digestion breaking is carried out similarly, fragmented wild type DNA is obtained, and clinical free DNA samples are simulated.

Mixing the prepared fragmented mutant DNA and the fragmented wild type DNA according to a certain proportion, quantifying the mixture by adopting digital PCR, diluting the mutant DNA by using the fragmented wild type DNA to obtain a mixed sample with the theoretical mutation abundance of 30%, and taking 15ng of the mixed sample for each reaction for detection.

Preparation of the reaction System

After the sample preparation is finished, configuring a reaction system, and preparing PCR reaction solution according to the proportion in the tables 1 and 2; namely, reaction system 1. The specific primer probe is as follows: mutant F1(SEQ ID NO:1), wild-type F2(SEQ ID NO:2), mutant F1-1(SEQ ID NO:3), wild-type F2-1(SEQ ID NO:4), mutant probe P1(SEQ ID NO:5), wild-type probe P2(SEQ ID NO:6), and downstream primer R (SEQ ID NO: 7).

Meanwhile, a mutant primer and a mutant probe which are developed by the company Ed and used for detecting EGFR L858R mutation, namely the reaction system 2, are synthesized according to the patent CN105349654B of the company Ed for comparison. Specifically, the mutant type F (SEQ ID NO:15), the mutant type probe P (SEQ ID NO:16) and the downstream primer R (SEQ ID NO: 17).

The primer probes used in the reaction system 2 are shown in the following table:

TABLE 5

The lengths of the target sequences of the reaction system 1 and the reaction system 2 are different due to different designs of the primers and the probes, and are respectively 60bp and 121 bp.

Reaction system related method

The following reactions used the same amount and concentration of sample.

For the first reaction (inventive reagent + melting), 20. mu.l of the prepared reaction solution containing the template was melted into single strands of DNA double strands before droplet generation.

And adding 20 microliters of the prepared PCR reaction solution and the reaction system 1 into a sample hole of the microdroplet generation card, then adding 70 microliters 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.

For the second reaction (reagent of the present invention), the sample was not subjected to the melting pretreatment, and the remaining steps were carried out using reaction system 1

For the third reaction (comparative manufacturer's reagents), the sample was subjected to a melting pretreatment and the remaining steps were completed using reaction system 2.

Amplification reading

And (3) sealing the membrane of the 96-well plate, and then placing the 96-well plate in a PCR thermal cycler for PCR amplification. The procedure used was: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30 seconds and annealing and extension at 52 ℃ for 60 seconds; denaturation at 94 ℃ for 30 seconds and annealing and extension at 52 ℃ for 60 seconds for 47 cycles; inactivating at 98 ℃ for 10 minutes; the reaction was terminated at 10 ℃.

Analytical statistics

the intensity and the number of the fluorescence signals are analyzed by QuantaSoft analysis software, and the copy number and the concentration of the EGFR gene L858R mutant and the copy number and the concentration of the EGFR gene L858R wild type are obtained. The results are shown in Table 6.

TABLE 6 results of the concentration of target detected using different reaction systems and detection methods (unit: copy/. mu.L)

the results show that the concentration measured by the primer composition of the invention (60.2 copies/. mu.L) is significantly higher than that measured by the comparative manufacturer (29.6 copies/. mu.L) when the primer composition of the invention is used compared with the primer composition of the comparative manufacturer, under the same type and concentration content of the sample added. It can be seen that the primer composition of the present invention has higher detection sensitivity than the reference manufacturer. This is because the primer composition of the present invention can suitably shorten the length of the target sequence, which is shorter than the target sequence targeted by conventional primers such as TaqMan primer probes. Therefore, the primer composition of the present invention has higher sensitivity in detecting a nucleic acid sample fragmented at random.

Furthermore, after the sample is subjected to melting treatment, the detection sensitivity can be theoretically improved by about 2 times, however, in the literature (Mariana Fitarelli-Kiehl, et al clinical Chemistry,64:12,2018), the number of positive droplets is only increased to 1.4-1.6 times after melting compared with that before melting when the clinical cfDNA is subjected to melting detection by using a conventional TaqMan primer; therefore, the sample is not subjected to the melting pretreatment, and the detection sensitivity cannot be doubled. Table 6 shows that the sample was subjected to a pre-treatment for melting double strands into single strands and then to a post-melting detection using only the primer composition of the present invention; the average concentration of the detected mutant type is 114.0 copies/microliter, compared with the detection by using the conventional digital PCR, the average concentration of the detected mutant type is 60.2 copies/microliter; the integral detection concentration is about 1.9 times of that of the conventional digital PCR; further proves that the primer composition has better sensitivity.

In addition, the combination of the kit and the detection method of the present invention is compared, and although the same samples were detected by the same manufacturer (alder), the detection concentration of the positive samples detected by the kit and the detection method of the present invention is 3.8 times higher than that of the manufacturer, as shown in fig. 4. The kit provided by the invention is combined with a detection method, so that the detection sensitivity is greatly improved, and the detection accuracy is improved; and because sensitivity is high, can reduce the degree of difficulty of analytic process, corresponding to the complicated meticulous degree requirement of equipment low, can effectively simplify equipment, reduce cost.

Comparative example 2

Fragmented NCI-H1975 cell line DNA samples were tested using a system of varying target sequence lengths.

sample preparation

Reaction preparation

After the preparation of the sample was completed, the reaction system was prepared, and PCR reaction solutions were prepared according to the ratios shown in tables 1 and 2.

The primer probes used in the reaction system 1 were: mutant F1(SEQ ID NO:1), wild-type F2(SEQ ID NO:2), mutant F1-1(SEQ ID NO:3), wild-type F2-1(SEQ ID NO:4), mutant probe P1(SEQ ID NO:5), wild-type probe P2(SEQ ID NO:6), and downstream primer R (SEQ ID NO: 7).

In addition to the above primers and probes, two downstream primers R2(SEQ ID NO:18) and R3(SEQ ID NO:19) were used for detecting mutation of EGFR gene L858R, and the length of the template bound when the downstream primer in this patent was changed to the downstream primer R2 was 82bp, and the length of the template bound when the downstream primer was changed to the downstream primer R3 was 105 bp.

The downstream primers used in reaction system 2 and reaction system 3 were: the reverse primer R2(SEQ ID NO:18) and the reverse primer R3(SEQ ID NO:19), the remaining primers and probe were the same as in System 1.

Table 7:

Since the free DNA in plasma and the circulating tumor DNA are highly fragmented, wherein the average length of the free DNA is about 160bp, and the average length of the circulating tumor DNA is about 130bp, the shorter the target sequence length of the detection system is, the higher the detection rate of the template is.

Amplification reading

Analytical statistics

The intensity and the number of the fluorescence signals are analyzed by QuantaSoft analysis software, and the copy number and the concentration of the EGFR gene L858R mutant and the copy number and the concentration of the EGFR gene L858R wild type are obtained.

The detection results of the fragmented NCI-H1975 cell line samples by using the kit of the invention are shown in Table 8, the detection results of the same samples by replacing the downstream primer designed in the patent with the downstream primer R2 (the other primers and the probes are not changed) are shown in Table 9, and the detection results of the same samples by replacing the downstream primer designed in the patent with the downstream primer R3 (the other primers and the probes are not changed) are shown in Table 10.

Table 8:

Numbering	Sample(s)	Target concentration (copy/microliter)
			1	Blank control	0
2	negative sample	0
			3	Positive sample	106
4	Positive sample	113
			5	Positive sample	114
6	Positive sample	109
			7	positive sample	115
8	positive sample	117

Table 9:

Numbering	Sample(s)	Target concentration (copy/microliter)
			1	Blank control	0
2	Negative sample	0
			3	Positive sample	87.9
4	Positive sample	92.6
			5	Positive sample	88.5
6	Positive sample	87.0
			7	Positive sample	96.7
8	Positive sample	81.6

Table 10:

The results show that, under the condition that the types and concentration contents of the added samples are the same, the detection results of the reagent and the detection method are shown in Table 11, namely when the length of the target sequence is 60bp, the average concentration of the detected mutant is 112.3 copies/microliter; the downstream primer R in the patent is changed into a downstream primer R2, namely when the length of a target sequence is 82bp, the average concentration of the mutant type to be detected is 89.0 copies/microliter; the downstream primer R in the patent is changed into the downstream primer R3, namely when the length of a target sequence is 105bp, the average concentration of the detected mutant is 59.7 copies/microliter, and the difference ratio of the mutant and the primer is more than 20%.

Table 11:

As can be seen from FIG. 5, different target sequence lengths have significant differences in detection results, and when a fragmented DNA template is detected, a shorter target sequence length can detect more target nucleic acid templates, resulting in higher concentration and copy number in the result, and greatly improving the detection sensitivity. Therefore, for the detection of rare mutation, especially for the detection of tumor mutation targets in peripheral blood or other body fluids, the reaction system and the kit have better detection performance.

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.

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Claims

1. A primer composition for detecting EGFR gene mutation, characterized in that: the primer composition comprises a mutant primer composition and/or a wild-type primer composition;

Wherein the upstream primer F1 sequentially comprises an upstream detection region and a target sequence binding region from the 5 'end to the 3' end:

Wherein the upstream primer F2 sequentially comprises an upstream detection region and a target sequence binding region from the 5 'end to the 3' end:

2. The primer composition for detecting mutations in the EGFR gene according to claim 1, wherein: 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.

3. The primer composition for detecting mutations in the EGFR gene according to claim 1 or 2, wherein: the difference between the Tm value of the upstream primer F1 and the Tm value of the upstream primer F1-1 is 0-20 ℃; preferably, the Tm value of the F1 is higher than that of the upstream primer F1-1; the difference between the Tm value of the upstream primer F2 and the Tm value of the upstream primer F2-1 is 0-20 ℃; preferably, the Tm value of the F2 is higher than that of the upstream primer F2-1.

4. The primer composition for detecting mutations in EGFR genes according to any one of claims 1 to 3, wherein: the length of the mismatch region in the target sequence binding region on the upstream primer F1 or the upstream primer F2 is 1-15 bases.

5. the primer composition for detecting mutations in EGFR genes according to any one of claims 1 to 4, wherein: the distance between the amplification decision site on the upstream primer F1 or the upstream primer F2 and the upstream mismatch region is 1-15 bases.

6. The primer composition for detecting mutations in EGFR genes according to any one of claims 1 to 5, wherein: the hydrolysis probe P1 and the hydrolysis probe P2 differ in reporter group.

7. a method for detecting EGFR gene mutation, which is characterized in that: the detection method comprises the following steps:

(iv) (iv) continuing amplification of the pre-amplification product obtained in step (iii) with the first primer composition and/or the second primer composition at a second annealing temperature; wherein the first primer composition comprises an upstream primer F1-1, a hydrolysis probe P1 and a downstream primer, the second primer composition comprises an upstream primer F2-1, a hydrolysis probe P2 and a downstream primer, and the hydrolysis probe P1 and the hydrolysis probe P2 carry reporter groups; and

Wherein,

8. The method for detecting mutations in the EGFR gene of claim 7, wherein: the pretreatment in the step (i) is a step of treating a target nucleic acid double strand to obtain a single strand.

9. The method for detecting mutations in the EGFR gene according to claim 7 or 8, wherein: the difference between the Tm value of the upstream primer F1 and the Tm value of the upstream primer F1-1 is 0-20 ℃; preferably, the Tm value of the F1 is higher than that of the upstream primer F1-1; the difference between the Tm value of the upstream primer F2 and the Tm value of the upstream primer F2-1 is 0-20 ℃; preferably, the Tm value of the F2 is higher than that of the upstream primer F2-1.

10. The method for detecting mutations in the EGFR gene according to any one of claims 7 to 9, wherein: the hydrolysis probe P1 and the hydrolysis probe P2 are provided with a reporter group and a quenching group, and the reporter groups of the hydrolysis probe P1 and the hydrolysis probe P2 are different.