CN110541033B

CN110541033B - Composition for EGFR gene mutation detection and detection method

Info

Publication number: CN110541033B
Application number: CN201910925527.8A
Authority: CN
Inventors: 赵雨航; 王书芳; 葛志琪; 何辉煌; 李锦�
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2023-11-14
Anticipated expiration: 2039-09-27
Also published as: CN110541033A

Abstract

The invention discloses a primer composition for detecting EGFR gene mutation with high sensitivity and a detection method thereof. The primer composition comprises two upstream primers, a probe and a downstream primer, wherein the first upstream primer only partially complements and pairs with a target sequence, and the sequences of the second upstream primer and the probe are respectively identical to partial sequences, which are not complementarily matched with the target sequence, on the first upstream primer. The detection method adopts a digital PCR method, and the sample is subjected to pretreatment of melting into a single chain before the conventional digital PCR reaction. The primer composition and the detection method can improve the detection sensitivity and specificity of EGFR gene mutation and reduce the detection cost; 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 EGFR gene mutation detection and detection method

Technical Field

The invention relates to the field of molecular biology, and discloses 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 that improves detection sensitivity in nucleic acid sequence variation detection.

Background

Polymerase Chain Reaction (PCR) is a molecular biological technique that performs 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, laboratory animal phenotype identification, transcriptome studies, detection of genetic disease, identification of gene fingerprints, diagnosis of infectious disease, paternity test, and the like. PCR is considered by molecular biologists as the method of choice for nucleic acid detection due to its incomparable replication and precision capabilities. In the late 90 s of the last century, the real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) technology and related products proposed by the American ABI company have developed PCR into a highly sensitive, highly specific and precisely quantitative nucleic acid sequence analysis technology.

However, there are many factors affecting the amplification efficiency during PCR amplification, and it cannot be ensured that the amplification efficiency remains unchanged during the reaction and the amplification efficiency is the same between the actual sample and the standard sample and between the samples, thereby resulting in that the cycle threshold (Ct) on which quantitative analysis depends is not constant. Therefore, qPCR is only "relative quantitative", and the accuracy and reproducibility still cannot meet the requirements of quantitative analysis of molecular biology.

Histopathological diagnosis has long been the basis of gold standards for tumor diagnosis and clinical treatment. However, the same treatment regimen is adopted for patients with tumors of the same histological type and stage, and only a part of patients with tumors often respond. It is counted that the traditional medicine has no efficiency of up to 75% in tumor treatment. The curative effect of malignant tumor is bad in that it is difficult to judge its malignant characteristic and pharmacodynamic characteristic from the tissue diagnosis level. Research shows that the molecular characteristics of tumor lesions determine the malignant characteristics, metastasis characteristics, recurrence characteristics and drug resistance characteristics of the tumor lesions, and are the basic basis for judging after the tumor is healed and reflecting chemotherapeutic drugs. Therefore, the individuation treatment based on the molecular difference is the direction of the accurate treatment of the tumor, and the molecular typing is the basis for realizing the individuation accurate treatment.

Epidermal growth factor receptor EGFR (Epidermal Growth Factor Receptor) is a transmembrane tyrosine kinase receptor, and the activation of the receptor kinase domain is related to the proliferation, metastasis and apoptosis of cancer cells. The EGFR gene is located in the 7 th chromosome short arm 7p12-14 region 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 proliferation, angiogenesis, tumor invasion, metastasis and apoptosis of tumor cells. The high expression of EGFR plays an important role in the evolution of malignant tumors, and EGFR is high expressed 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 tumor circulating DNA (Circulating tumor DNA, ctDNA). At present, EGFR gene mutation is detected mainly by a tumor tissue sample obtained by means of tissue biopsy or surgery and the like clinically, but because invasive means are needed for obtaining the tumor tissue sample, the process often increases pain of a patient, extra surgery risks are generated, tumors have heterogeneity, and for a cancer patient who has already metastasized, only cancer tissue at a certain part is taken by means of puncture or surgery, and the situation of the whole patient cannot be reflected. Second, some patients themselves determine that they are not suitable for tissue biopsies, while some tumors risk accelerating metastasis after being disturbed by puncture or surgery. Finally, tissue biopsies suffer from the problems of high cost and long waiting times, and their hysteresis is also detrimental to the treatment of patients. Therefore, in recent years, the concept of "liquid biopsy" is rising, and the basic idea is to use a body fluid sample such as blood instead of a tumor tissue sample for pathological and molecular biological detection, and it has become a trend to acquire tumor gene mutation information by detecting tumor circulating DNA in a body fluid sample (mainly blood) of a patient. Early screening, medication guidance, prognosis and recurrence monitoring of tumor patients can be achieved by detecting circulating tumor DNA (ctDNA) in the patient's peripheral blood. However, because the background of the peripheral blood sample is complex, the ctDNA content is rare, and for 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 easy to be interfered by the background DNA, so that the detection sensitivity and the accuracy cannot meet the requirement of accurate quantification.

Digital PCR (dPCR) technology is an absolute quantitative technique of nucleic acid molecules that utilizes the principle of limiting dilution to distribute a fluorescent quantitative PCR reaction system into thousands of individual nanoliter microreactors, such 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. In particular, detection of rare mutations in complex settings is commonly found in tumor fluid biopsies, noninvasive prenatal detection, organ transplantation monitoring, accurate quantification of viral load, component detection of transgenic crops, etc., such as detection of rare mutation markers in peripheral blood of tumor patients, or gene expression differential studies, etc.

Disclosure of Invention

In order to solve the problems, the invention provides a composition for EGFR gene mutation detection and a detection method. The invention solves the problem that the gene mutation can not be detected due to the fact that 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 EGFR gene mutation, 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 the 5 'end to the 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 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 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 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 the 5 'end to the 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 determining site that is complementary to a variant detection site on the wild-type target sequence, and upstream of the amplification determining site there is a mismatch region consisting of one or more bases that is not complementary to the target sequence.

In the invention, the upstream detection region on the upstream primer F1 is not complementarily paired with the sequence of the target sequence, the upstream primer F1-1 and the hydrolysis probe P1, and the upstream detection region on the upstream primer F1 is also not complementarily paired with the sequence 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 sequence of the target sequence, the upstream primer F2-1, the hydrolysis probe P2, and the upstream detection region on the upstream primer F2 is not complementarily paired with the sequence of the upstream primer F1, the upstream primer F1-1, the hydrolysis probe P1. That is, only after the specific pre-amplification of the target nucleic acid sequence by the upstream primer F1, the upstream primer F1-1 and the probe P1 can be paired with the pre-amplified product. Only after the 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 with the pre-amplified product. The upstream detection region on the upstream primer F1 is not identical or complementary to the target sequence or hybridizes under high stringency conditions; the upstream detection region on the upstream primer F2 is not identical or complementary to the target sequence or hybridizes under high stringency conditions.

The upstream detection region on the upstream primer F1 and the upstream detection region on the upstream primer F2 are freely exchangeable. The upstream detection region on the upstream primer F1 has or has not a base interval with the target sequence binding region; there is or is not a base separation between the upstream detection region on the upstream primer F2 and the target sequence binding region.

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

In some more preferred embodiments, the position of the complementary pairing of the downstream primer and the target sequence is set at 1 to 150bp downstream of the mutation detection site, and may be further 50 to 100bp.

In some embodiments, the upstream primer F1 has a Tm value that is different from the Tm value of the upstream primer F1-1; the Tm value of the upstream primer F1 is different from that of the upstream primer F1-1 by 0-20 ℃. Preferably, the Tm value of 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, most preferably, the Tm is 10℃to 15 ℃. The Tm value of the upstream primer F2 is different from that of the upstream primer F2-1; 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, most preferably, the Tm is 10℃to 15℃higher.

In some embodiments, the length of the mismatched zone in the target sequence binding region on the upstream primer F1 or the upstream primer F2 is between 1 and 20 bases. Preferably, the mismatch region is 1 to 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 determining site on the upstream primer F1 or the upstream primer F2 is 1 to 15 bases from the upstream mismatch region. Preferably, the amplification determining site on the upstream primer F1 or the upstream primer F2 is 3 to 7 bases away from the upstream mismatch region. In some embodiments of the invention, the 3' end of the amplification determining site is the 3' end of the upstream primer F1 or the 3' end of the upstream primer F2, and there is no other base downstream of the site. In other embodiments of the invention, the amplification determining site is further 1 to 10 bases downstream.

In some embodiments, one or more bases are spaced between part (a) and part (b) in the upstream detection region on the upstream primer F1.

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 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 another aspect, the present invention provides a method for detecting EGFR gene mutation, comprising the steps of:

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

(ii) Limiting dilution is carried out on the pretreated sample, the pretreated sample is randomly distributed into 770-10000000 reaction units, and then uniform thermal cycle amplification is carried out on all the reaction units;

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

(iv) Continuing to amplify the pre-amplified product obtained in step (ii) with a first primer composition comprising an upstream primer F1-1, a hydrolysis probe P1 and a downstream primer and/or a second primer composition comprising an upstream primer F2-1, a hydrolysis probe P2 and a downstream primer, the hydrolysis probe P1 and hydrolysis probe P2 bearing a reporter group, at a second annealing temperature; and

(v) Detecting a signal emitted by a reporter group in the reaction system after the step (iv), and quantifying the target nucleic acid in the sample according to the signal;

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:

The quantification is absolute and is aimed at determining the number of molecules of the target gene in the sample, namely the copy number.

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

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

In some embodiments, single strands may be formed by heating a mixture containing double stranded DNA, for example, by heating the mixture to 90 ℃, 92 ℃, 95 ℃, or 98 ℃ to dissociate the double stranded DNA. Typically, upon double strand dissociation, the mixture needs to be maintained at an 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 strand DNA. In order to maintain the double-stranded DNA after dissociation in a single-stranded state, it is necessary to cool the solution containing the single-stranded DNA to a temperature below room temperature, for example, below 25℃and 20℃and 15 ℃.

In some embodiments, the above described "denaturation" can also be accomplished by altering the ionic strength of the solution (e.g., addition of acids, bases, salts, etc.) to break hydrogen bonds between double stranded DNA, and enzymes (e.g., helicases) 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 treats the target nucleic acid duplex to single strands, preferably formaldehyde heating, alkali treatment.

In some embodiments, the Tm of the upstream primer F1 differs from the Tm of the upstream primer F1-1 by 0℃to 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 than the Tm of the upstream primer F1-1, more preferably 10℃to 15 ℃.

In some embodiments, the upstream primer F1 and the upstream primer F2 have a length of 50 to 90bp, a Tm of 50 to 80℃and a GC content of 40 to 80%.

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

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

In some embodiments, the mutant and wild-type primer compositions together use a single downstream primer R having a length of 15 to 30bp, a Tm 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 group and a quencher group, and 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 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 some embodiments, only the mutant or wild-type detection primer composition is used in the same reaction system. In other embodiments, multiple mutant and/or wild-type detection primer compositions are used in the same reaction system. Preferably, for example, when a plurality of primer compositions for detecting a mutant form are used, the amplification determining sites of the upstream primer F1 in the plurality of primer compositions for detecting a mutant form are different, and the part (b) of the upstream detection region is also different; the plurality of probes have mutually different sequences and reporter groups; optionally, the upstream detection zone part (a) is also different. The method can be used to determine both wild-type and mutant target sequences simultaneously, or to determine multiple mutant target sequences simultaneously. In some embodiments using multiple F1 primers and probes, different upstream primers F1 may share the same downstream primer.

In some embodiments, the number of cycles for pre-amplification of the upstream primer F1 and the downstream primer R is 3 to 10 cycles; further, the number of cycles for the pre-amplification of the upstream primer F1 is 5 to 8 cycles. In some embodiments, the number of cycles for amplification and measurement of the upstream primer F1-1 and hydrolysis probe P1 of the present invention is 35 to 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 to 45 cycles.

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

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

(2) denaturation at 92-95 ℃ for 10-60 seconds, annealing at 55-75 ℃ and extension for 30-90 seconds, and carrying out 3-10 cycles altogether;

(3) denaturation at 92-95 ℃ for 10-60 seconds, annealing at 45-65 ℃ and extension for 30-90 seconds, and carrying out 35-50 cycles in total;

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

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

The signal detection method adopts the principle of TaqMan probe method, namely, the 5'-3' exonuclease activity of DNA polymerase is utilized to hydrolyze the probe so as to generate fluorescent signals. Because the probe is specifically combined with the template, in the digital PCR reaction, the number of microdroplets emitting fluorescent signals represents the number of the template in the reaction system, and finally the concentration of the template can be obtained through poisson correction. For TaqMan probes as a fluorescent signal generation mode, the design method of the probes is the same as that of the conventional PCR method.

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

The digital PCR used in the invention is different from the fluorescent PCR, and is used for collecting fluorescent signals in real time, detecting signals after the whole thermal cycle reaction is finished, and preferably detecting the fluorescent signals. Thus, in the first 5 to 10 cycles of thermocycling amplification, the hydrolysis probe P1 (P2) can bind to the pre-amplified product, but depending on the amplified hydrolysis of its upstream primer F1-1 (upstream primer F2-1), the hydrolysis probe P1 (hydrolysis probe P2) will not be hydrolyzed and fluoresce when the temperature is unsuitable for the binding of the upstream primer F1-1 (upstream primer F2-1) to the template. Therefore, in order to avoid hydrolysis of hydrolysis probe P1 (hydrolysis probe P2) by upstream primer F1-1 (upstream primer F2-1) at the time of pre-amplification, the annealing temperature of upstream primer F1 (upstream primer F2) of the present invention is higher than that of upstream primer F1-1 (upstream primer F2-1).

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

Compared with the prior art, the technical scheme provided by 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 as the probe P can be matched and combined with the 5' -end complementary sequence of the upstream primer F1 after the pre-amplification is completed, the part which is actually matched and combined with the target nucleic acid sequence has 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.

(2) 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 paired with the target nucleic acid sequence because the probe P can be paired with the 5' -end complementary sequence of the upstream primer F1 after the pre-amplification is completed: f1 and the sequence of the downstream primer R. Therefore, for complex target nucleic acid sequence detection, the design method of the primer probe can avoid the GC imbalance region, and particularly has lower design difficulty compared with the TaqMan probe method and ARMS.

(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 amount, the invention can be used for effectively increasing the amount of target sequences in the samples and improving the detection sensitivity, thereby reducing errors caused by the too small amount of samples. Such samples typically include plasma (cf DNA samples), biopsy puncture, or FFPE samples. This method typically involves melting double-stranded DNA into its constituent strands, such as single-stranded DNA (ssDNA), prior to formation of the microdroplet, and then separating each strand prior to amplification and counting.

(4) High sensitivity: the minimum sensitivity of the kit for detecting the target nucleic acid sequence in the complex background can reach 0.01%, and more preferably, the sensitivity of the kit for stably detecting the target nucleic acid sequence in the complex background is 0.05%, namely, the kit can ensure that 10 copies of the target nucleic acid sequence can be stably detected in 20,000 copies of the total nucleic acid background in >95% detection, or 15 copies of the target nucleic acid sequence can be stably detected in 30,000 copies of the total nucleic acid background. The invention can realize stable detection of low-concentration samples and low-mutation abundance samples so as to meet the clinical monitoring of peripheral blood circulation free DNA samples of tumor patients, reflect the current state of tumor of the patients, guide 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, no wild type or other similar or homologous target nucleic acid cross reaction exists when the mutant target nucleic acid sequence is detected. Particularly, when the wild type and the mutant are detected simultaneously, the cross reaction of the wild type and the mutant is small, which is more favorable for the detection of rare mutation.

(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) Sample consumption is low: the reaction system and the kit can detect mutant target nucleic acid sequences and wild target nucleic acid sequences in one reaction tube, absolute quantification and mutation abundance statistics are carried out on the mutant target nucleic acid sequences and the wild target nucleic acid sequences, and the kit is 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 to be modified by expensive MGB or LNA, so that the use cost of the primer probe is greatly reduced, and the primer probe has better detection performance and meets the clinical use requirement. In addition, after the target DNA double strand is unwound into a single strand, the unbalanced free DNA is not required to be repaired by using the tail end 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 end repair enzyme is not required to be added into the reaction liquid for repair, so that the experimental steps are reduced, and the experimental time is saved.

Drawings

FIG. 1 schematically shows the design of primers for the kit of the invention

Taking the upstream primer F1 as an example, the upstream primer F comprises an upstream detection region and a target sequence binding region from the 5' end to the 3' end, wherein the 3' end of the target sequence binding region is provided with an amplification decision site, the amplification decision site is complementary to a mutation detection site on the target sequence, and a mismatch region which consists of one or more bases and is not complementary to the target sequence is arranged upstream of the amplification decision site; the upstream detection zone comprises, from 5 'to 3': part (a) having the same sequence as the upstream primer F1-1, and part (b) having the same sequence as the probe;

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

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

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

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

Detailed Description

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

Definition of the definition

The term "Gene" (Gene, mendelian factor) in the present application is also referred to as a genetic factor. 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, so that the character expression of the biological individual is controlled.

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

At the molecular level, a gene mutation refers to a change in the base pair composition or arrangement order of a gene in structure.

Gene mutation refers to a change in the structure of a gene caused by 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 longer fragment of DNA.

The length of a nucleic acid can be expressed as a base, base pair (abbreviated "bp"), nucleotide/nucleotide residue (abbreviated "nt"), or kilobase ("kb") according to conventions used in the art. The terms "base", "nucleotide residue" may describe polynucleotides that are single-stranded or double-stranded, where the context permits. When this term is applied to a double stranded molecule, it is used to refer to the entire length and is understood to correspond to the term "base pair".

The term "primer" refers to a macromolecule of a particular nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, covalently attached to a reactant, such a molecule being referred to as a primer. Primer pairs are typically two oligonucleotide sequences that are synthesized artificially, one primer being complementary to one DNA template strand at one end of the target region and the other primer being complementary to the other DNA template strand at the other end of the target region, and function as a starting point for nucleotide polymerization, from the 3' end of which a nucleic acid polymerase can begin to synthesize a new nucleic acid strand.

The term "upstream primer", also called 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 sense strand, also called the coding strand, is generally located at the upper end of the double-stranded DNA, and the direction is 5'-3' from left to right, and the base sequence is basically the same as that of the mRNA of the gene; the primer bound to the strand is a reverse primer; the negative strand, i.e., the nonsense strand, also known as the non-coding strand, is complementary to the positive strand, and the primer that binds to this strand is the forward primer. It will be appreciated that when designations of sense and antisense strands are interchanged, the corresponding forward and reverse primer designations may also be interchanged therewith.

The terms "upstream", "upstream/on … …", "upstream with … …", etc., as used herein, refer to a portion of the same nucleic acid sequence that is closer to the 5' end than the reference region, e.g., either immediately adjacent to the reference region or at one or more bases from the reference region, in the context of describing the nucleic acid sequence. The terms "downstream", "downstream/downstream of … …", "downstream with … …", etc., as used herein, in the context of describing nucleic acid sequences, refer to portions of the same nucleic acid sequence that are closer to the 3' end than the indicated region, e.g., may be immediately adjacent to the indicated region or may be spaced one or more bases from the indicated region. It will be appreciated that where the nucleic acid is described as a double stranded nucleic acid, the expression "upstream" and "downstream" will generally be based on the 5 'and 3' ends of the sense strand, unless otherwise indicated.

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 kinetic PCR format, the detection probe may consist of an oligonucleotide with a 5 'reporter group (R) and a 3' quencher group (Q). Fluorescent reporter groups (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.

The term "label" 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. Examples of labels include fluorophores, chromophores, radioactive atoms (particularly 32p and 125I), electron dense reagents, enzymes, and ligands with specific conjugates.

The terms "target sequence binding region", "upstream detection region", "amplification determining site", "mismatch region", "(a) part" and "(b) part" as used herein are different segments located on the F1 primer, wherein the amplification determining site and the mismatch region are located within the target sequence binding region and the (a) part and the (b) part are located within the upstream detection region. It will be appreciated that the target sequence binding region means that the region is intended to hybridize or anneal to a target sequence, and does not mean that the region must be capable of 100% base complementary pairing with the target sequence, and that there may be a mismatch region within the target sequence binding region. The term "mutation detection site" is located on a target sequence, and herein refers to a segment that differs between different target sequences to be detected, e.g., a segment that differs in sequence between a wild-type and a mutant. The mutation detection site may be one or more base pairs in length. Typically, the mutation may be, for example, a point mutation (as compared to the wild type), a deletion mutation, an insertion mutation, a base inversion mutation, or the like.

The term "Taqman probe" is used interchangeably herein with "hydrolysis probe". The Taqman probe is a fluorescence detection technology developed on a Real-time PCR technology platform, wherein the 5 'end of the probe contains a fluorescence report group, and the 3' end contains a fluorescence quenching group. When the probe is complete, fluorescent signals emitted by the reporter group are 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 is used for carrying out enzyme digestion degradation on the probe, so that the reporter group and the quenching group are separated, fluorescent signals are emitted, and the accumulation of the fluorescent signals and the formation of PCR products are completely synchronous. Specifically, the reporter group may use FAM, HEX, VIC, ROX, cy, cy3, etc., and the quencher group may use 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 a minor groove binding molecule (minor groove binder, MGB) at the 3' -end, so that the Tm value of the probe is improved, the length of the probe is shortened, and the simultaneous detection of multiple mutation sites is facilitated.

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

The terms "target sequence," "target nucleic acid," or "target" are used interchangeably herein and refer to a portion of a nucleic acid sequence to be amplified, detected, or amplified and detected, which can be annealed or hybridized 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. Hybridization does not require that 2 nucleic acids have 100% complementarity over their entire length.

The term "base" refers to derivatives of purines and pyrimidines, and is a component of nucleic acids, nucleosides, and nucleotides. There are 5 bases: cytosine (abbreviated as C), guanine (G), adenine (a), thymine (T, DNA-specific) and uracil (U, RNA-specific), the bases of DNA being cytosine (abbreviated as C), guanine (G), adenine (a), thymine (T).

The term "mismatched base" refers to the principle that the pairing between bases in a DNA duplex is not random, always adenine (a) pairs with thymine (T), guanine (G) pairs with cytosine (C). If a pairs with C or G, or T pairs with G or C, then a base mismatch occurs.

The term "stringent conditions" as used herein may be any of low stringency conditions, medium stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide at 32 ℃; furthermore, "medium stringent conditions" such as conditions of 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide at 42 ℃; "high stringency conditions" are, for example, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 50% formamide at 50 ℃. Under these conditions, it is expected that higher temperatures are more effective in obtaining polynucleotides, such as DNA, having high homology. Although there are various factors affecting the stringency of hybridization, such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., those skilled in the art can obtain similar stringency by appropriately selecting these factors.

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

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

The invention provides a primer composition for detecting EGFR gene mutation. EGFR protein tyrosine kinase functional region is encoded by EGFR gene exons 18-24, wherein exons 18-20 encode N-Lobe and exons 21-24 encode C-Lobe. EGFR mutations found to date are predominantly located in exons 19 to 21; more than 90% of non-small cell lung cancer EGFR mutations occur as deletion mutations of exon19 and L858R point mutations of exon 21. The base deletion of exon19 is mainly the deletion mutation of codons 746-752, resulting in the loss of amino acid sequence in EGFR protein, thus changing the sensitivity of the cell to TKIs; the occurrence of T-M transition mutation at codon 790 of exon 20 (nucleotide position 2669 base substitution) is the main cause of drug resistance; the point mutation of exon21 is mainly T-G conversion at 858 codon, so that leucine at the position is converted into arginine, which is called L858R for short.

A schematic design of the primer composition of the present invention is shown in FIG. 1. Taking the upstream primer F1 as an example, the upstream primer F comprises an upstream detection region and a target sequence binding region from the 5' end to the 3' end, wherein the 3' end of the target sequence binding region is provided with an amplification decision site, the amplification decision site is complementary to a mutation detection site on the target sequence, and a mismatch region which consists of one or more bases and is not complementary to the target sequence is arranged upstream of the amplification decision site; the upstream detection zone comprises, from 5 'to 3': part (a) having the same sequence as the upstream primer F1-1, and part (b) having the same sequence as the probe. After the target nucleic acid template enrichment is carried out by using the primer pair, the upstream primer F1-1 and the hydrolysis probe P1 recognize the sequence complementary to the upstream detection region of the upstream primer F1 on the pre-amplified product by using the change of annealing temperature, then form the primer pair with the reverse primer R and carry out template amplification, and fluorescent signals are released by using 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 according to the base-pairing principle by conventional means known in the art.

Experimental equipment and materials

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

in the embodiment of the invention, a microdroplet digital PCR system (ddPCR) is mainly adopted, DNA double chains in a sample are melted into single chains before microdroplet generation, and then microdroplet treatment is carried out on the sample before traditional PCR amplification, namely a reaction system containing nucleic acid molecules is divided into thousands of nano-scaled 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 droplet is detected one by one, the droplet with fluorescent signal is interpreted as 1, the droplet without fluorescent signal is interpreted as 0, and the initial copy number or concentration of the target molecule can be obtained according to the Poisson distribution principle and the number and proportion of positive droplets.

The using method of the kit comprises the following steps:

the reaction buffer and primer probe premix are mixed according to the reaction system shown in the following table, and then DNA is extracted from the 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 performed.

The kit can be matched with a Bio-Rad company QX200 microdroplet digital PCR system (ddPCR) and consumable materials to detect.

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

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 the EGFR gene 19 exon mutant target nucleic acid is detected to be 50 copies/. Mu.L and the concentration of the EGFR gene 19 exon wild type target nucleic acid is detected to be 9950 copies/. Mu.L in the sample to be tested, the abundance of EGFR gene 19 exon mutation 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, so as to calculate the concentration and mutation abundance of the target nucleic acid in the sample to be detected.

Example 1:

the performance of the primer composition for detection and the detection method were evaluated using simulated clinical samples for EGFR gene L858R point mutation.

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

A primer composition for detecting L858R point mutation of EGFR gene:

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 is labeled with FAM at the 5 'end and BHQ1 at the 3' end. The wild type probe shown by SEQ ID NO. 6 is labeled HEX at the 5 'end and BHQ1 at the 3' end. See in particular table 1.

TABLE 1 (nucleotide sequence of the following 5 '. Fwdarw.3' from left to right)

In the primer probe, the full length of a mutant F1 primer (SEQ ID NO: 1) is 65bp, the full length of a wild F2 primer (SEQ ID NO: 2) is 66bp, 23 bases at the 3 'end of the mutant F1 primer and the wild F2 primer are paired 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 th to 40 th base sequences at the 5' end of the mutant F1 primer are identical to the base sequences of the corresponding mutant probes P1 (SEQ ID NO: 5). The 22 th to 40 th base sequences at the 5' end of the wild type F2 primer are identical to the base sequences of the corresponding wild type probe P2 (SEQ ID NO: 6). Therefore, after the mutant F1 primer and the wild F2 primer respectively amplify the target nucleic acid specifically, the generated amplified product is added with a section of base sequence from the 5' ends of the F1 primer and the F2 primer and the complementary sequence thereof, and then the F1-1 primer and the probe P1 and the F2-1 primer and the probe P2 can be paired with the corresponding target nucleic acid templates and hydrolyzed to emit fluorescent signals.

Reaction system

The PCR reaction mixture (20. Mu.L system as an example) was prepared at the following concentration

TABLE 2

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

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

Sample preparation

Free DNA from healthy human plasma was prepared, and it was confirmed by second generation sequencing that the EGFR gene L858R mutation was not contained, and a wild-type free DNA sample was obtained as a negative sample.

Meanwhile, preparing NCI-H1975 cell line DNA samples quantified by digital PCR, and after fragmentation treatment, diluting the wild type DNA samples to obtain EGFR gene L858R mutation samples with mutation abundance of 10% as positive samples.

The primer composition is used for detecting the EGFR gene L858R mutation site, and the method comprises the following steps:

preparation of the reaction System

After this preparation, the reaction system was prepared, and a PCR reaction solution was prepared in accordance with the ratio shown in Table 1.

The primer probes used are 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).

The DNA double strand in 20 μl of the prepared template-containing reaction solution was melted into single strands prior to droplet generation.

The prepared PCR reaction solution was taken in an amount of 20. Mu.l and added to the sample well of the droplet-generating card, then 70. Mu.l of the droplet-generating oil was added to the oil well of the droplet-generating card, and finally the droplet-generating card was sealed with a sealing tape.

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

Amplification reads

Sealing the 96-well plate, and placing the sealed membrane in a PCR thermal cycler for PCR amplification. The procedure used was: pre-denaturation at 95 ℃ for 10 min; denaturation at 94℃for 30 sec, annealing at 54℃for 60 sec; denaturation at 94℃for 30 seconds, annealing at 54℃for 60 seconds, and 47 cycles in total; inactivating at 98 ℃ for 10 minutes; 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.

Analysis statistics

The intensity and number of fluorescent signals were analyzed using QuantaSoft analysis software to obtain copy numbers and concentrations of EGFR gene L858R mutant and wild type, and mutation abundance was calculated.

The test results of the mixed sample with 10% theoretical dilution using the kit of the present invention are shown in fig. 2.

The detection results of the negative control and the blank control by using the kit provided by the invention have the negative coincidence rate of 100%.

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

Example 2

The performance of the primer composition for detection and the detection method were evaluated 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 is labeled with FAM at the 5 'end and BHQ1 at the 3' end. The wild type probe shown by SEQ ID NO. 13 is labeled HEX at the 5 'end and BHQ1 at the 3' end. See in particular table 3.

TABLE 3 (nucleotide sequence of the following 5 '. Fwdarw.3' from left to right)

In the primer probe, the full length of the mutant F1 primer (SEQ ID NO: 8) is 65bp, the full length of the wild type F2 primer (SEQ ID NO: 9) is 67bp, 21 bases at the 3' end of the mutant F1 primer pair with a target nucleic acid sequence, 25 bases at the 3' end of the wild type F2 primer pair with a nucleotide sequence, and the vicinity of 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 th to 41 st base sequences at the 5' end of the mutant F1 primer are identical to the base sequences 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 are identical to the base sequences of the corresponding wild type probe P2 (SEQ ID NO: 6). Therefore, after the mutant F1 primer and the wild F2 primer respectively amplify the target nucleic acid specifically, the generated amplified product is added with a section of base sequence from the 5' ends of the F1 primer and the F2 primer and the complementary sequence thereof, and then the F1-1 primer and the probe P1 and the F2-1 primer and the probe P2 can be paired with the corresponding target nucleic acid templates and hydrolyzed to emit fluorescent signals. Wherein, R base is expressed as A or G base.

Reaction system

TABLE 4 Table 4

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

wherein, the negative control is a fragmented healthy human genome DNA sample, the 19Del specific mutation of EGFR gene is not existed through sequencing, and the negative control of 20,000 copies/microliter is prepared through digital PCR quantification and using Tirs-EDTA buffer solution.

Sample preparation

Ultrasound purified 293T cell line DNA was prepared and was determined to contain no EGFR gene 19del mutation by second generation sequencing as a negative sample.

Meanwhile, preparing NCI-H1650 cell line DNA sample quantified by digital PCR, and after fragmentation treatment, diluting the wild type DNA sample to obtain EGFR gene 19del mutation sample with mutation abundance of 10% as positive sample.

The primer composition is used for detecting 19del mutation sites of EGFR genes, and the method comprises the following steps:

preparation of the reaction System

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

The primer probes used are 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 reads

Analysis statistics

The intensity and number of fluorescent signals are analyzed by using QuantaSoft analysis software to obtain the copy number and concentration of 19del mutant forms of EGFR genes, and mutation abundance is calculated.

The test results of the mixed sample with 10% theoretical dilution using the kit of the present invention are shown in fig. 3.

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

The following comparative examples will take the L858R gene mutation of EGFR as an example.

Comparative example 1

The primer pair comparison experiments were performed using fragmented NCI-H1975 cell line DNA samples (EGFR L858R mutation) with similar manufacturer kits.

Sample preparation

Clinical circulating tumor DNA was simulated using a digital PCR quantified sample of fragmented NCI-H1975 cell line DNA containing the EGFR gene L858R mutation with a mutation abundance of 75%.

Meanwhile, preparing genome DNA from healthy people, determining that the genome DNA does not contain EGFR gene L858R mutation through second generation sequencing, and then performing enzyme cutting and breaking to obtain fragmented wild type DNA and simulate a clinical free DNA sample.

Mixing the prepared fragmented mutant DNA with fragmented wild DNA according to a certain proportion, quantifying the mixture by adopting digital PCR, and diluting the mutant DNA by using the fragmented wild DNA to obtain a mixed sample with the theoretical mutation abundance of 30 percent, and detecting the mixed sample with 15ng per reaction.

Preparation of the reaction System

After sample preparation is completed, preparing a reaction system, and preparing a PCR reaction solution according to the proportion shown in tables 1 and 2; namely, the reaction system 1. The specific primer probes are 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 for detecting EGFR L858R mutation developed by Aide corporation, i.e., reaction system 2, were synthesized according to patent CN105349654B of Aide corporation for comparison. Specifically, mutant F (SEQ ID NO: 15), mutant probe P (SEQ ID NO: 16), and downstream primer R (SEQ ID NO: 17).

Wherein, the primer probe used in the reaction system 2 is as follows:

TABLE 5

The target sequence length of the reaction system 1 and the target sequence length of the reaction system 2 are respectively 60bp and 121bp according to different designs of the primer and the probe.

Method for reaction system

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

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

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

For the second reaction (inventive reagent), the remaining steps were completed using reaction system 1 without melting pretreatment of the sample

For the third reaction (comparative manufacturer reagent), the sample was subjected to melting pretreatment, and the rest was completed using reaction system 2.

Amplification reads

Sealing the 96-well plate, and placing the sealed membrane in a PCR thermal cycler for PCR amplification. The procedure used was: pre-denaturation at 95 ℃ for 10 min; denaturation at 94℃for 30 sec, annealing at 52℃for 60 sec; denaturation at 94℃for 30 seconds, annealing at 52℃for 60 seconds, and 47 cycles in total; inactivating at 98 ℃ for 10 minutes; the reaction was stopped at 10 ℃.

Analysis statistics

The intensity and number of fluorescent signals were analyzed using QuantaSoft analysis software to obtain the copy number and concentration of the EGFR gene L858R mutant and the copy number and concentration of the EGFR gene L858R wild type. The test results are shown in Table 6.

TABLE 6 concentration results (units: copy/. Mu.L) of targets detected using different reaction systems and detection methods

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The results show that the concentration detected using the primer composition of the present invention (60.2 copies/. Mu.L) is significantly higher than that of the comparative manufacturer (29.6 copies/. Mu.L) when the type and concentration content of the added sample are the same. It can be seen that the primer composition of the present invention has higher detection sensitivity than that of the comparative manufacturer. This is because the primer composition of the present invention can be designed to have a length that is suitably shortened, shorter than that of a conventional primer such as TaqMan primer probe. Thus, the primer composition of the present invention has higher sensitivity in detecting a randomly fragmented nucleic acid sample.

Furthermore, after the sample is subjected to melting treatment, the detection sensitivity can be improved by about 2 times theoretically, however, mariana Fitarelli-Kiehl and the like are mentioned in the literature (Mariana Fitarelli-Kiehl, et al clinical Chemistry,64:12, 2018), and when the conventional TaqMan primer is used for detecting clinical cfDNA after melting, the number of positive droplets is only increased by 1.4-1.6 times after melting compared with that before melting; therefore, the sample is subjected to the melting pretreatment, and the detection sensitivity cannot be increased by times. In Table 6, it is shown that the sample was subjected to a pretreatment of double-strand melting to single strand, and then subjected to detection after melting only with the primer composition of the present invention; the average concentration of the detected mutant was 114.0 copies/microliter, and 60.2 copies/microliter compared to the detection using conventional digital PCR; the overall detection concentration is about 1.9 times of that of the conventional digital PCR; further, the primer composition of the present invention was demonstrated to have better sensitivity.

In addition, the combination of the kit and the detection method of the present invention was compared with the case where the same sample was detected by the comparative manufacturer (alder), but the concentration of the positive sample detected by the kit and the detection method of the present invention was increased 3.8 times as compared with the case where the positive sample was detected by the comparative manufacturer, as shown in fig. 4. It can be considered that the detection sensitivity is greatly increased and the detection accuracy is improved by using the kit provided by the invention in combination with the detection method; and the sensitivity is high, so that the difficulty of an analysis process can be reduced, the corresponding requirement on the complexity and fineness of equipment is low, the equipment can be effectively simplified, and the cost is reduced.

Comparative example 2

The fragmented NCI-H1975 cell line DNA samples were detected using a system of different target sequence lengths.

Sample preparation

Reaction preparation

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

The primer probes used in the reaction system 1 are respectively 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).

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

The downstream primers used in reaction systems 2 and 3 were: the downstream primer R2 (SEQ ID NO: 18) and the downstream primer R3 (SEQ ID NO: 19) were identical to the system 1 with respect to the remaining primers and probes.

Table 7:

since free DNA in plasma and 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, the higher the detection rate of the template.

Amplification reads

Analysis statistics

The intensity and number of fluorescent signals were analyzed using QuantaSoft analysis software to obtain the copy number and concentration of the EGFR gene L858R mutant and the copy number and concentration of the EGFR gene L858R wild type.

The detection results of the kit for the fragmented NCI-H1975 cell line samples are shown in Table 8, the detection results of the downstream primer designed by the patent for the same samples are shown in Table 9 by replacing the downstream primer R2 (other primers and probes are unchanged), and the detection results of the downstream primer designed by the patent for the same samples are shown in Table 10 by replacing the downstream primer designed by the patent for the downstream primer R3 (other primers and probes are unchanged).

Table 8:

numbering device	Sample of	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 device	Sample of	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 the concentration contents of the added samples are the same, the detection results of the reagent and the detection method are shown in the table 11, namely, when the length of a 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 the downstream primer R2, namely when the length of a target sequence is 82bp, the average concentration of the detected mutant is 89.0 copies/microliter; when the downstream primer R is changed into the downstream primer R3 in the patent, namely the length of the target sequence is 105bp, the average concentration of the detected mutant is 59.7 copies/microliter, and the mutual difference ratio of the mutant and the target sequence is more than 20%.

Table 11:

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

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.

Sequence listing

<110> Mike organism Co., ltd

<120> composition for EGFR gene mutation detection and detection method

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Claims

1. A method of using an EGFR gene mutation kit, and for non-diagnostic purposes, characterized by:

the using method of the kit comprises the following steps:

(iii) Pre-amplifying the target nucleic acid with the upstream primer mutant F1 and/or the upstream primer wild type F2 and the downstream primer R as primer pairs at a first annealing temperature;

(iv) Continuing to amplify 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 mutant type F1-1, a mutant type probe P1 and a downstream primer R, the second primer composition comprises an upstream primer wild type F2-1, a wild type probe P2 and a downstream primer R, and the mutant type probe P1 and the wild type probe P2 are provided with a report group; and

the pretreatment in the step (i) is a step of double-strand treatment of the target nucleic acid to obtain a single strand;

wherein, the EGFR gene mutation kit comprises the following primer probe compositions:

mutant F1 with sequence shown in SEQ ID NO. 1;

Wild F2 has a sequence shown as SEQ ID NO. 2;

mutant F1-1 with sequence shown in SEQ ID NO. 3;

wild F2-1 has a sequence shown in SEQ ID NO. 4;

the sequence of the mutant probe P1 is shown as SEQ ID NO. 5;

the sequence of the wild probe P2 is shown as SEQ ID NO. 6;

the sequence of the downstream primer R is shown as SEQ ID NO. 7.

2. The method of use according to claim 1, wherein: the mutant probe P1 and the wild-type probe P2 have a reporter group and a quencher group, and the reporter groups of the mutant probe P1 and the wild-type probe P2 are different.

3. The method of use according to claim 2, wherein: the reporter group is selected from one or more of FAM, HEX, VIC, ROX, cy and Cy 3; the quenching group is selected from one or more of TAMRA, BHQ1, BHQ2, BHQ3 and DABCYL, QXL, DDQI.

4. The method of use according to claim 1, wherein: the means for double-strand treatment of the target nucleic acid to obtain a single strand include a formaldehyde heating method, an alkali treatment method, and a helicase treatment method.

5. The method of use according to claim 1, wherein: the number of pre-amplification cycles in step (iii) is 5 to 8 cycles; the number of consecutive amplification cycles in the step (iv) is 35 to 50 cycles.

6. The digital PCR kit for detecting EGFR gene L858R point mutation is characterized by comprising the following primer probe composition:

mutant F1 with sequence shown in SEQ ID NO. 1;

wild F2 has a sequence shown as SEQ ID NO. 2;

mutant F1-1 with sequence shown in SEQ ID NO. 3;

wild F2-1 has a sequence shown in SEQ ID NO. 4;

the sequence of the mutant probe P1 is shown as SEQ ID NO. 5;

the sequence of the wild probe P2 is shown as SEQ ID NO. 6;

the sequence of the downstream primer R is shown as SEQ ID NO. 7.