CN111349691B

CN111349691B - Composition, kit and detection method for EGFR gene deletion mutation detection

Info

Publication number: CN111349691B
Application number: CN201811571640.2A
Authority: CN
Inventors: 赵雨航; 葛志琪
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2023-10-20
Anticipated expiration: 2038-12-21
Also published as: CN111349691A

Abstract

The invention discloses a composition, a kit and a detection method for EGFR gene deletion mutation detection. The composition comprises a mutant type detection composition and/or a wild type detection composition, wherein the mutant type detection composition comprises an upstream primer F1, an upstream primer F1-1, a hydrolysis probe P1 and a downstream primer; the wild type detection composition comprises an upstream primer F2, an upstream primer F2-1, a hydrolysis probe P2 and a downstream primer; wherein the upstream primers F1 and F2 are composed of a non-matching region and a matching region, and the matching regions of the upstream primers F1 and F2 specifically bind to the region of the sequence to be detected. The mismatch region of the upstream primer F1 and F2 has the same sequence as that of the upstream primer for detection and the hydrolysis probe. Compared with the prior art, the invention has the advantages of obviously improved sensitivity and high specificity, greatly reduced primer and test cost and extremely high application value.

Description

Composition, kit and detection method for EGFR gene deletion mutation detection

Technical Field

The invention relates to the technical field of biomedical nucleic acid detection, in particular to a composition, a kit and a detection method for EGFR gene deletion mutation detection.

Background

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.

The EGFR is a transmembrane tyrosine kinase receptor, and the activation of the receptor kinase domain is related to the proliferation, metastasis, apoptosis and other various signaling pathways of cancer cells. The EGFR gene is located in the 7 th chromosome short arm 7p12-14 region and consists of 28 exons. The EGFR gene is mutated in a wide variety of ways, mainly at the tyrosine kinase active site between exons 18-21. The most common sensitive mutations are the 19 exon deletion mutation (about 45%) and the L858R point mutation of exon 21 (about 40%), both of which can result in tyrosine kinase domain activation. Currently, the deletion mutant species of 19 exons can be up to 30-40, with more than 50% of the deletion mutations being accompanied by insertion mutations. The COSIC database statistics show that the most common subset of exon 19 mutations is delE746-A750 (68.9%), followed by delL747-P753insS (6.0%), and delL747-T751 (4.1%). Patients with non-small cell lung cancer (Non small cell lung cancer, NSCLC) having 19 exon mutations, after receiving EGFR tyrosine kinase inhibitor drugs (EGFR-TKIs) treatment, can significantly prolong the progression-free survival (Progression Free Survival, PFS) of the patients and increase the drug response rate without significant differences between patients of different subtypes. Thus, detection of EGFR mutation status in NSCLC patients has become the basis for the determination of their clinical treatment regimen.

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 distributing the reaction system in the digital PCR experiment can greatly reduce the concentration of the background sequence with the competitive effect with the target sequence locally, so that the digital PCR is particularly suitable for detecting rare mutation in a complex background, and is currently applied to liquid biopsy to detect rare mutation markers in peripheral blood of tumor patients.

The existing detection reagent for gene deletion mutation adopts a TaqMan probe method, a primer specificity distinguishing method or a wild-type blocking amplification method. The basic principle of the TaqMan probe is that the 5' exonuclease activity of Taq enzyme is utilized in the amplification process to cut an oligonucleotide probe combined with a target nucleic acid sequence, the 5' end of the probe is marked with a fluorescent reporter group, the 3' end of the probe is marked with a fluorescent quenching group and is phosphorylated to prevent the probe from extending, and when a primer extends to the combining position of the oligonucleotide probe, the Taq enzyme can cut the oligonucleotide probe into small fragments so that the fluorescent reporter group is separated from the quenching group, and fluorescence is emitted. For use in tumor gene deletion mutation detection, competitive probes for mutant and wild-type, respectively, are generally used. Because EGFR 19 exon deletion mutation types are numerous, a plurality of different mutation type probes are designed aiming at different mutation types, the cost is high, and the large-scale popularization and the use are not facilitated. Another more common TaqMan probe method is to design a probe aiming at a wild type in a common deletion region of EGFR 19 exons, and design a universal probe capable of simultaneously indicating a wild type and a mutant template in other conserved regions, wherein the two probes share an upstream primer and a downstream primer. On one hand, because ctDNA is in highly random fragmentation distribution, the method needs longer target nucleic acid sequence to be detected, so shorter DNA fragments can be missed, and the detection sensitivity is reduced; on the other hand, when the method is applied to digital PCR, because the digital PCR needs to carry out partition treatment on a nucleic acid amplification system, when a micro reaction chamber contains mutant type and wild type templates at the same time, mutant type fluorescent signals can be submerged, so that the mutation abundance ration is low. The primer specificity distinguishing method is to design one or more specific primers aiming at mutant type in a common deletion area, design a hydrolysis probe and a downstream primer in a conserved area at the downstream of the specific primers, and design a universal probe capable of simultaneously indicating wild type and mutant type templates and upstream and downstream primers in conserved areas of other exons of EGFR genes. However, when the method is applied to digital PCR, the possibility of flooding the wild-type fluorescent signal exists, so that the mutation abundance ration is high. The blocking wild type amplification method usually adopts peptide nucleic acid (Peptide Nucleic Acid, PNA) and locked nucleic acid (Locked Nucleic Acid, LNA) modification to block the amplification of wild type templates, on one hand, when the method is applied to digital PCR, the possibility that wild type fluorescent signals are submerged exists, and on the other hand, the modification is high in cost and is unfavorable for clinical application.

In the application of the digital PCR technology, since the result is interpreted only by the end point fluorescence signal intensity without depending on the Ct value and the standard curve, and the mutant type and the wild type cannot be distinguished by the delta Ct, the specificity requirement on the primer probe is extremely high, and the cross reaction should be avoided as much as possible. Therefore, a primer probe design method with high sensitivity, good specificity and low cost is needed to meet the requirements of clinical detection by using a digital PCR technology.

Disclosure of Invention

In order to solve the problems, the invention provides a composition for digital PCR detection of human EGFR gene mutation and a method thereof.

A composition for detecting deletion mutation of EGFR gene, which comprises a composition for detecting mutation type and/or a composition for detecting wild type;

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

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

wherein, the liquid crystal display device comprises a liquid crystal display device,

the upstream primer F1 sequentially comprises the following two parts from the 5 'end to the 3' end:

(1) Mismatch area: complementary pairing does not occur with both mutant and wild-type sequences; the sequence of the unmatched region is a sequence identical to the upstream primer F1-1 and a sequence identical to the hydrolysis probe P1 from the 5 'end to the 3' end in sequence;

(2) Matching area: only with the mutation site upstream and downstream sequences of the mutant type;

the upstream primer F2 sequentially comprises the following two parts from the 5 'end to the 3' end:

(1) Mismatch area: complementary pairing does not occur with the mutant and wild type sequences, and the sequence of the mismatch region is a sequence identical to the upstream primer F2-1 and a sequence identical to the hydrolysis probe P2 in sequence from the 5 'end to the 3' end;

(2) Matching area: only with the wild-type mutation site and its upstream and downstream sequences.

In a preferred embodiment, the downstream primers in the mutant-type detection composition and the wild-type detection composition 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 the invention, the upstream primer F1-1 is completely identical with the 5' end sequence of the unmatched region on the upstream primer F1, and is not complementarily paired with the target sequence before and after the occurrence of the deletion mutation and the sequence of the upstream primer F2; the upstream primer F2-1 is completely identical with the 5' -end sequence of the unmatched region on the upstream primer F2, and is not complementarily paired with the target sequence before and after the occurrence of the deletion mutation and the sequence of the upstream primer F1;

The hydrolysis probe P1 is completely identical with the downstream sequence of the same sequence of the upstream primer F1-1 on the mismatch region of the upstream primer F1, and is not complementarily paired with the target sequence before and after the occurrence of the deletion mutation and the sequence of the upstream primer F2; the hydrolysis probe P2 is identical to the sequence downstream of the same sequence as the upstream primer F2-1 in the mismatch region of the upstream primer F2 and is not complementarily paired with the target sequence before and after the occurrence of the deletion mutation and the sequence of the upstream primer F1.

In another preferred embodiment, the upstream primer F1 has or has not a base interval between the same sequence as the upstream primer F1-1 and the same sequence as the hydrolysis probe P1; the upstream primer F2 has a sequence identical to the upstream primer F2-1 and a sequence identical to the hydrolysis probe P2 with or without a base interval therebetween.

In another preferred embodiment, the sequence of the mismatch region on both the upstream primer F1 and the upstream primer F2 is freely exchangeable.

In another preferred embodiment, the hydrolysis probe P1 is modified at the 5 'and 3' ends with a reporter group and a quencher group, respectively; the 5 'end and the 3' end of the hydrolysis probe P2 are respectively modified with a reporter group and a quenching group; preferably, the reporter groups on hydrolysis probe P1 and hydrolysis probe P2 release different detection signals, respectively.

When the probe is hydrolyzed by the DNA polymerase 5'-3' exonuclease activity, the reporter group separates from the quencher group, thereby releasing a detectable signal.

In another preferred embodiment, the reporter group is a fluorescent group.

In another preferred embodiment, 1 to 20 mismatched bases are added to the 3' -end of the upstream primer F1 at the 2 nd to 9 th bases; 1-20 mismatched bases are added at the 2 nd-9 th base of the 3' end of the upstream primer F2.

In another preferred embodiment, 1 to 15 mismatched bases are added at the 3' end of the upstream primer F1 at the 3 rd to 7 th bases; 1-15 mismatched bases are added at the 3-7 th base of the 3' -end of the upstream primer F2.

In another preferred embodiment, the Tm value of the upstream primer F1 is higher than the Tm value 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 another preferred embodiment, the Tm value of the upstream primer F1 is 5 to 20℃higher than the Tm value of the upstream primer F1-1, and the Tm value of the upstream primer F2 is 5 to 20℃higher than the Tm value of the upstream primer F2-1; more preferably, the Tm value of the upstream primer F1 is 10 to 15℃higher than the Tm value of the upstream primer F1-1, and the Tm value of the upstream primer F2 is 10 to 15℃higher than the Tm value of the upstream primer F2-1.

In another preferred embodiment, the length of the upstream primer F1 and the upstream primer F2 is 50-90 bp, the Tm value is 60-80 ℃, and the GC content is 40% -80%.

In another preferred embodiment, the length of the upstream primer F1-1 and the upstream primer F2-1 is 13 to 30bp, the Tm value is 50 to 70℃and the GC content is 40 to 80%.

In another preferred embodiment, the hydrolysis probes P1 and P2 have a length of 10 to 30bp, a Tm value of 55 to 75℃and a GC content of 40 to 80%.

In another preferred embodiment, the downstream primer R in the mutant-type detecting composition and the wild-type detecting composition has a length of 15 to 30bp, a Tm value of 55 to 75℃and a GC content of 40 to 80%.

In another preferred embodiment, the position of complementary pairing of the downstream primer with the target nucleic acid sequence is located 20 to 100bp downstream of the region to be detected.

The region to be detected is the region of complementary pairing of the upstream primer F1 and the mutant sequence or the region of complementary pairing of the upstream primer F2 and the wild sequence.

In another aspect, a kit for detecting deletion mutation of EGFR gene, comprising the above composition for detecting mutation and/or the composition for detecting wild type.

In another aspect, a method for detecting deletion mutations in EGFR gene using the above composition, the method comprising the steps of:

firstly, carrying out limiting dilution on a sample to be detected and randomly distributing the sample to be detected into 10000-20000 reaction units;

then, carrying out unified thermal cycle amplification on all the reaction units; in the first 1-10 cycles of thermal cycle amplification, pre-amplification of the target nucleic acid sequence is performed with the upstream primer F1 or the upstream primer F2 and the downstream primer as primer pairs; then, in the last 10-50 cycles of thermal cycle amplification, the amplification of the pre-amplification product is carried out by taking the upstream primer F1-1 or the upstream primer F2-1 and the downstream primer as primer pairs; in the last 10-50 cycles of thermocycling amplification, the hydrolysis probes bound to the pre-amplified product are hydrolyzed and release the signal on the reporter group;

and finally, detecting the signal of the reporter group on the hydrolysis probe, and judging the corresponding target nucleic acid sequence according to the detection result.

Preferably, the reporter group is detected as a fluorescent signal on the hydrolysis probe.

In another embodiment, the first annealing temperature is higher than the second annealing temperature.

In another aspect, the present invention provides a reaction system for detecting deletion mutation of EGFR gene, comprising:

An upstream primer F1 with a concentration of 15-150 nM, an upstream primer F1-1 with a concentration of 150-1500 nM, and a hydrolysis probe P1 with a concentration of 50-800 nM;

and, a step of, in the first embodiment,

a downstream primer R with a concentration of 150-1800 nM;

and/or the number of the groups of groups,

an upstream primer F2 with a concentration of 15-150 nM, an upstream primer F2-1 with a concentration of 150-1500 nM, and a hydrolysis probe P2 with a concentration of 50-800 nM.

In another preferred embodiment, the concentration of the upstream primers F1 and F2 is 30 to 60nM, the concentration of the upstream primers F1-1 and F2-1 is 300 to 600nM, the concentration of the downstream primer R is 300 to 900nM, and the concentration of the hydrolysis probes P1 and P2 is 150 to 400nM.

In another aspect, the present invention provides a reaction system for detecting deletion mutation of EGFR gene, the reaction conditions of the reaction system are as follows:

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

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

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

(4) inactivating for 5-15 min at 94-98 ℃;

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

In a preferred embodiment, said step (2) is carried out for 5 to 8 cycles; step (3) is carried out for 40-45 cycles.

In another preferred embodiment, the annealing temperature Tm value of step (2) is 5 to 20 ℃ higher than that of step (3), more preferably, the annealing temperature Tm value of step (2) is 10 to 15 ℃ higher than that of step (3).

In another preferred embodiment, a reaction system for detecting deletion mutation of EGFR gene, the reaction conditions of the reaction system are as follows:

(2) pre-denaturation at 95℃for 10min;

(2) denaturation at 94℃for 30s, annealing at 65℃for 60s, and extension for 5 cycles;

(3) denaturation at 94℃for 30s, annealing at 52℃for 60s, and extension for 40 cycles;

(4) inactivating at 98 ℃ for 10min;

(5) the reaction was stopped at 10 ℃.

The present invention will be explained in detail below.

In the invention, an upstream primer F1, an upstream primer F2 and corresponding downstream primers are used for pre-amplifying mutant templates and wild templates respectively, namely, enriching corresponding templates; and then amplifying the enriched mutant and wild type templates by using the upstream primer F1-1, the upstream primer F2-1 and the corresponding downstream primer respectively, and hydrolyzing the probes bound with the enriched mutant and wild type DNA templates to hydrolyze P1 and P2, thereby detecting the corresponding nucleotide sequences.

The upstream primer F1 and the upstream primer F2 share a downstream primer; the upstream primer F1-1 and the upstream primer F2-1 share a downstream primer; more preferably, the upstream primer F1, the upstream primer F2, the upstream primer F1-1 and the upstream primer F2-1 share one downstream primer R.

When deletion mutation detection is performed using the composition for EGFR gene deletion mutation detection, if only one of the mutant type or the wild type is required to be detected, only the composition for mutation detection or the composition for wild type detection may be used, and if both the wild type and the mutant type are required to be detected, the composition for mutation detection and the composition for wild type detection may be used, and at this time, the mutation abundance can be further calculated by detecting the content of both the wild type and the mutant type, and the mutation abundance is clinically more useful for guiding drug administration.

In order to improve the detection accuracy, none of the upstream primer F1-1, the upstream primer F2-1, the hydrolysis probe P1 and the hydrolysis probe P2 are complementarily paired with the target nucleic acid sequence; the upstream primer F1-1 and the hydrolysis probe P1 are also not complementarily paired with the sequence of the upstream primer F2, and the upstream primer F2-1 and the hydrolysis probe P2 are also not complementarily paired with the upstream primer F1. The sample nucleotide sequence is specifically pre-amplified (enriched) only under the action of the upstream primer F1 (upstream primer F2) and the downstream primer R, after the concentration of the target nucleotide sequence is increased, the upstream primer F1-1 (upstream primer F2-1) and the hydrolysis probe P1 (hydrolysis probe P2) can be paired with the enriched product, then the enriched product is amplified by the upstream primer F1-1 (upstream primer F2-1) and the downstream primer R, and the hydrolysis probe bound on the enriched product is hydrolyzed by the DNA polymerase 5'-3' exonuclease activity, so that the reporter group is separated from the quenching group, and a detectable signal is released.

In order to avoid binding of the upstream primer F1 (upstream primer F2) to the wild-type (mutant) nucleotide sequence and amplification, i.e., to improve the specificity of the upstream primer, the present invention introduces 1 to 20 mismatched bases at the 2 nd to 9 th bases of the 3' end of the upstream primer F1 (upstream primer F2), which mismatched bases do not form a pair with the target nucleic acid sequence; further, 1 to 15 mismatched bases are introduced at 3 to 7 bases of the 3' -end of the upstream primer F1 (upstream primer F2).

The annealing temperature of the upstream primer F1 (upstream primer F2) is set to be 5-20 ℃ higher than that of the upstream primer F1-1 (upstream primer F2-1) so as to realize the amplification of different purposes (enrichment and amplification of a pre-amplification product) on sample nucleotides by adjusting the annealing temperature. 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 hydrolysis probe P1 (P2) can be matched and combined with the 5' -end complementary sequence of the upstream primer F1 (F2) after the pre-amplification is finished, the part which is actually matched and combined with the target nucleic acid sequence is only two parts: the sequence of the 3' -end of the upstream primer F1 (F2) complementary to 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 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 portion actually coupled with the target nucleic acid sequence because the hydrolysis probe P1 (P2) can be coupled with the 5' -end complementary sequence of the upstream primer F1 (F2) after the pre-amplification (enrichment) is completed: the matching region of the upstream primer F1 (F2) and the sequence of the downstream primer R. Thus, for complex target nucleic acid sequence detection, the design difficulty of primer probes is lower relative to TaqMan probe methods and ARMS.

(3) 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.

(4) 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.

(5) 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.

(6) Sample consumption is low: the reaction system and the kit can detect mutant target nucleic acid sequences and wild target nucleic acid sequences simultaneously 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 free tumor DNA samples.

(7) The cost is low: the primer probe design method and the reaction system do not need expensive PNA modification or LNA modification, greatly reduce the use cost of the primer probe, have better detection performance, and meet the clinical use requirement.

Drawings

FIG. 1 is a schematic diagram showing one embodiment of a composition for detecting EGFR gene deletion mutation according to the present invention; wherein, fig. 1A is a structural diagram of an upstream primer F1, and sequentially comprises a non-matching region and a matching region from a 5 'end to a 3' end, wherein the matching region is complementarily paired with a partial region sequence crossing a mutation site after the occurrence of a deletion mutation, and the non-matching region sequentially comprises a sequence identical to the upstream primer F1-1 and a sequence identical to a hydrolysis probe P1 from the 5 'end to the 3' end; FIG. 1B shows the reaction principle of a kit using a mutant type detection composition as an example, wherein the upstream primer F1 and the downstream primer R are used for specifically enriching a target nucleic acid sequence to be detected, and a sequence complementary to a mismatch region on the upstream primer F1 is newly added for pairing and identifying the subsequent upstream primer F1-1 and hydrolysis probe P1; after enrichment of the target nucleic acid sequence, the upstream primer F1-1 and the hydrolysis probe P1 are used for identifying an enrichment product, then a primer pair is formed with the downstream primer and template amplification is carried out, and the signal of the reporter group is released through the TaqMan hydrolysis probe principle.

FIG. 2 is a graph showing the results of digital PCR assays on NCI-H1650 cell line samples using the kits and comparative kits of the present invention (Bio-Rad Corp., cat# 10041170); FIG. 2A is a diagram of the digital PCR detection result of a comparative kit, and FIG. 2B is a diagram of the digital PCR detection result of the kit according to the present invention; in the digital PCR detection result diagram, the upper left area of the vertical line is a mutant signal (FAM fluorescent signal), the upper right area of the vertical line is a mutant+wild signal (fluorescent signals of FAM and HEX), the lower left area of the vertical line is a blank signal, and the lower right area of the vertical line is a wild signal (fluorescent signal of HEX);

FIG. 3 is a graph of concentration data analysis of NCI-H1650 cell line samples quantified using the kit of the invention and a comparative kit (Bio-Rad Corp., cat. No. 10041170); wherein, system 1 represents a comparative kit and system 2 represents a kit of the invention;

FIG. 4 is a graph showing the results of digital PCR detection of the negative control of example 4 of the present invention; in the digital PCR detection result diagram, the upper left area of the vertical line is a mutant signal (FAM fluorescent signal), the upper right area of the vertical line is a mutant+wild signal (fluorescent signals of FAM and HEX), the lower left area of the vertical line is a blank signal, and the lower right area of the vertical line is a wild signal (fluorescent signal of HEX);

FIG. 5 is a diagram showing the results of the detection of a simulated clinical sample using the kit and the comparative kit (Bio-Rad, cat# 10041170); FIG. 5A shows the detection result of the comparative kit, and FIG. 5B shows the detection result of the kit according to the present invention; in the digital PCR detection result graph, the upper left region of the vertical line is a mutant signal (FAM fluorescent signal), the upper right region of the vertical line is a mutant+wild type signal (fluorescent signals of FAM and HEX), the lower left region of the vertical line is a blank signal, and the lower right region of the vertical line is a wild type signal (fluorescent signal of HEX).

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 present application without making any inventive effort, shall fall within the scope of the present 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 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 "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 composition for detecting EGFR gene 19 exon deletion mutation, and a primer probe design method of the composition is shown in figure 1. Taking a mutant-type detection composition as an example, FIG. 1A is a block diagram of an upstream primer F1, the upstream primer F1 comprising a mismatch region and a matching region in this order from the 5 'end to the 3' end; the unmatched area is a detection area, and the sequence from the 5 'end to the 3' end is the same as the upstream primer F1-1 and the sequence same as the hydrolysis probe P1 in sequence; the matching region is a region that binds to the target nucleic acid sequence, or a region of the target nucleic acid sequence that is to be detected. Specifically, the region to be detected is a continuous sequence upstream and downstream of the deletion mutation site on the mutant target sequence, or is a continuous sequence upstream and downstream of the deletion mutation site on the wild-type target sequence. Wherein, the black triangle is a deletion mutation region, and 1-20 mismatched bases can be added at the 3' -end of the matching region. FIG. 1B is a schematic diagram of the reaction of the kit of the present invention, wherein the upstream primer F1 (F2) and the reverse primer R are used to specifically enrich the target nucleic acid sequence in the sample to be detected, and a sequence complementary to the unmatched region of the upstream primer F1 (F2) is newly added for pairing and identifying the subsequent upstream primer F1-1 (F2-1) and hydrolysis probe P1 (P2). After enrichment of the target nucleic acid sequence, the upstream primer F1-1 (F2-1) and the hydrolysis probe P1 (P2) are enabled to identify the sequence complementary to the unmatched area of the upstream primer F1 (F2) on the pre-amplified product by utilizing the change of annealing temperature, then a primer pair is formed with the reverse primer R, amplification of the target nucleic acid is carried out, and a fluorescent signal is released by the TaqMan hydrolysis probe principle. The upstream primer F2 matching region and the upstream primer F1 matching region in the wild-type detection composition differ by one more mutation deletion region from the upstream primer F1 in the mutant-type detection composition.

The invention provides a kit for detecting EGFR gene 19 exon deletion mutation, which comprises the following components:

table 1 shows the components and main components of the kit of the present invention

In the kit of the present invention, a reaction buffer from Bio-Rad company, cat# 1863010 was used. The blank is Tris-EDTA buffer without any DNA. The negative control is a fragmented healthy human genome DNA sample, the deletion mutation of the exon 19 of the EGFR gene is confirmed to be absent by sequencing, and the negative control is prepared by quantitative digital PCR and is prepared into 20000 copies/mu L by using a Tris-EDTA buffer solution. The positive control is a fragmented NCI-H1650 mutant cell line DNA sample comprising an EGFR gene 19 exon deletion mutation, specifically the mutant subtype delE746-a750; specifically, a deletion mutation at the base level of c.2235-2249 del15 (Cosmic ID: 6223); the preparation was quantified by digital PCR and diluted with negative control to prepare a positive control with a mutation abundance of 0.5% (20000 copies/. Mu.L total DNA concentration background). The NCI-H1650 mutant cell line was purchased from a Kobura organism.

In the embodiment of the invention, a microdroplet digital PCR system (ddPCR) is mainly adopted, and microdroplet processing is carried out on a sample before the 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. Wherein ddPCR droplet generation oil is selected from Bio-Rad company product, cat# 1863005; the ddPCR droplet generation card is selected from Bio-Rad company product, cat# 1864008; the sealing strip used when the microdroplet occurs can be selected from Bio-Rad company product, product number 1863009; the microdroplet analysis oil is selected from Bio-Rad company product, cat# 1863004; the half-skirt 96-well plate can be manufactured by Bio-Rad, cat# 64088955.

Table 2 shows the reaction system of the digital PCR amplification of the present invention

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:

a composition for detecting deletion mutation of exon 19 of EGFR gene comprising:

composition for detecting mutation:

an upstream primer F1 having a nucleotide sequence shown as SEQ ID NO. 1;

the upstream primer F1-1 has a nucleotide sequence shown as SEQ ID NO. 3;

hydrolysis probe P1 with a nucleotide sequence shown as SEQ ID NO. 5;

a downstream primer R having a nucleotide sequence shown as SEQ ID NO. 7; '

Wild-type detection composition:

an upstream primer F2 having a nucleotide sequence shown as SEQ ID NO. 2;

the upstream primer F2-1 has a nucleotide sequence shown as SEQ ID NO. 4;

hydrolysis probe P2 with the nucleotide sequence shown as SEQ ID NO. 6;

The downstream primer R has a nucleotide sequence shown as SEQ ID NO. 7.

The 5' end of the hydrolysis probe P1 is marked with FAM fluorescent group, the 5' end of the hydrolysis probe P2 is marked with HEX fluorescent group, and the 3' ends of the hydrolysis probes P1 and P2 are marked with BHQ1 quenching group.

In the primer probe, the total length of the upstream primer F1 (SEQ ID NO: 1) is 62bp, and the total length of the upstream primer F2 (SEQ ID NO: 2) is 69bp. Wherein the wild type sequence of EGFR gene corresponding to the upstream primer F2 is NC_000007.14 sequence in the International public nucleic acid sequence database (GeneBank).

The upstream primer F1 (SEQ ID NO: 1) has 21 bases at its 3 'end paired with the target nucleic acid sequence, and 1 mismatched base was introduced at the 6 th base position at the 3' end to improve the specificity.

The 3' -end 25 bases of the upstream primer F2 (SEQ ID NO: 2) was paired with the target nucleic acid sequence. The 18/20 bases at the 5' -end of the upstream primer F1/F2 are identical to the base sequences of the corresponding upstream primer F1-1 (SEQ ID NO: 3)/upstream primer F2-1 (SEQ ID NO: 4), respectively.

The 20 th to 39 th base sequences at the 5' end of the upstream primer F1 are identical to the base sequences of the corresponding hydrolysis probes P1 (SEQ ID NO: 5). The 23 rd to 43 rd base sequences of the 5' -end of the upstream primer F2 are identical to the base sequences of the corresponding hydrolysis probes P2 (SEQ ID NO: 6). Therefore, after the specific amplification of the target nucleic acid by the upstream primer F1 and the upstream primer F2, respectively, the generated amplified products are added with a segment of the base sequence from the 5' end on the upstream primer F1 or the upstream primer F2 and the complementary sequence thereof, and then the upstream primer F1-1, the upstream primer F2-1 hydrolysis probe P1 and the hydrolysis probe P2 can be paired with the corresponding target nucleic acid templates and can be hydrolyzed to emit fluorescent signals.

The 19 exon delE746-A750 mutation site can be detected by using the above-described composition for detecting a mutant; specifically, a deletion mutation at the base level of c.2235-2249 del15 (Cosmic ID: 6223).

Example 2

A kit for detecting EGFR gene 19 exon deletion mutation comprises primer probe premix, reaction buffer, negative control, positive control and blank control, wherein the main components are shown in table 1.

The primer probe premix in the kit is a mixture obtained by uniformly mixing the composition of the example 1 according to the proportion of the table 3;

the negative control is a fragmented healthy human genome DNA sample, the deletion mutation of the exon 19 of the EGFR gene is confirmed by sequencing, and the negative control is prepared by quantitative digital PCR and is prepared into 20000 copies/mu L by using Tris-EDTA buffer solution, and the negative control is specifically shown in figure 4.

The positive control is a fragmented NCI-H1650 mutant cell line DNA sample comprising an EGFR gene 19 exon deletion mutation, specifically the mutant subtype delE746-a750; specifically, a deletion mutation at the base level of c.2235-2249 del15 (Cosmic ID: 6223).

The amplification reaction system for detecting the deletion mutation by the kit is sample and primer probe premix liquid Reaction buffer (2 XddPCR from Bio-Rad Co.) ^TM Supermix for Probes, cargo number: 1863010 And water in the proportions shown in Table 3.

Table 3 shows the reaction system of the digital PCR amplification of the present invention

Example 3

Cell line samples are tested.

Sample preparation:

using QIAGEN CoThe DNA Mini Kit performs nucleic acid extraction on the NCI-H1650 cell line containing EGFR gene 19 exon mutation deletion according to the operation instruction of the Kit to obtain the genome DNA of the EGFR gene 19 exon mutation deletion cell line.

And (3) preparing the following reaction:

after sample preparation was completed, the reaction system was prepared and a PCR reaction system was prepared according to the ratios described in table 3.

EGFR WT & E746_A750del Assay Kit (cat# 10041170) from Bio-Rad was also used as a control reagent.

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

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

Amplification reading:

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 seconds, annealing at 65℃for 60 seconds, and a total of 5 cycles; denaturation at 94℃for 30 seconds, annealing at 52℃for 60 seconds, and a total of 40 cycles; 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.

The intensity and number of fluorescent signals are analyzed by using QuantaSoft analysis software to obtain the copy number and concentration of EGFR gene 19 exon deletion mutant type and the copy number and concentration of EGFR gene 19 exon wild type, and the mutation abundance is calculated.

Analysis and statistics:

the results of detection of NCI-H1650 cell line samples using the Bio-Rad company contrast kit are shown in FIG. 2A, and the results of detection of the same samples using the kit of the present invention are shown in FIG. 2B. The threshold value of the two is divided clearly, and the detection performance is close; and the specificity of the kit according to the invention is significantly better than that of the control kit as seen by the observation of the double cationic microdroplets.

The results of detection and quantification of the same NCI-H1650 cell line sample using the kit of the present invention and the Bio-Rad company comparison kit are shown in FIG. 3, and the means of the mutation abundance quantified by the two are 68.82% and 68.20%, respectively, without significant difference (P=0.287).

Example 4

The simulated clinical samples are tested.

Preparation of samples:

And (3) performing enzyme digestion and disruption on the extracted EGFR gene 19 exon mutation deletion cell line DNA by using a KAPA fragment Kit to obtain fragmented mutant DNA with the fragment length of about 120-130bp, and simulating the fragment size of clinical free tumor DNA.

Meanwhile, preparing genome DNA from healthy people, determining that the genome DNA does not contain EGFR gene 19 exon deletion 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.

The prepared fragmented mutant DNA and fragmented wild DNA are mixed according to a certain proportion to obtain a simulated clinical sample. And negative controls were prepared using the same concentration of fragmented wild-type DNA.

Preparing a reaction system:

after sample preparation is completed, the configuration of the reaction system is performed, and specifically, a PCR reaction system is prepared according to the ratio described in Table 3.

EGFR WT & E746-A750del Assay Kit (cat# 10041170) from Bio-Rad was also used as a control reagent.

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

Amplification reading:

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 seconds, annealing at 65℃for 60 seconds, and a total of 5 cycles; denaturation at 94℃for 30 seconds and annealing at 55℃for 60 seconds, followed by 40 cycles in total; inactivating at 98 ℃ for 10 minutes; the reaction was stopped at 10 ℃.

The intensity and number of fluorescent signals are analyzed by using QuantaSoft analysis software to obtain the copy number and concentration of EGFR gene 19 exon mutant type and the copy number and concentration of GFR gene 19 exon wild type, and mutation abundance is calculated.

Analysis and statistics:

the detection result of the negative control product by using the kit is shown in figure 4.

The results of detection of simulated clinical samples using the Bio-Rad company comparison kit are shown in FIG. 5B, and the results of detection of the same sample using the kit of the present invention are shown in FIG. 5A. The threshold value of the two is divided clearly, and the detection performance is close; the specificity of the kit according to the invention is clearly better than that of the control kit as seen by the observation of the double cationic microdroplets. The cross reaction is smaller when the wild type and the mutant type are detected simultaneously, the interference on the fluorescence signal intensity of the double-positive microdroplet is smaller, and the detection of rare mutation targets is facilitated;

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

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

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Claims

1. A composition for detecting EGFR gene deletion mutation, characterized in that: the composition comprises:

composition for detecting mutation:

an upstream primer F1, a nucleotide sequence shown as SEQ ID NO. 1;

the upstream primer F1-1 has a nucleotide sequence shown as SEQ ID NO. 3;

hydrolysis probe P1, nucleotide sequence shown as SEQ ID NO. 5;

a downstream primer R, a nucleotide sequence shown as SEQ ID NO. 7;

wild-type detection composition:

an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO. 2;

an upstream primer F2-1 with a nucleotide sequence shown as SEQ ID NO. 4;

hydrolysis probe P2, nucleotide sequence shown as SEQ ID NO. 6;

the downstream primer R is shown as a nucleotide sequence shown in SEQ ID NO. 7.

2. The composition for detecting deletion mutation of EGFR gene as set forth in claim 1, wherein: the 5 'end of the hydrolysis probe P1 is marked with FAM fluorescent group, and the 5' end of the hydrolysis probe P2 is marked with HEX fluorescent group.

3. The composition for detecting deletion mutation of EGFR gene as set forth in claim 2, wherein: the 3 '-end of the hydrolysis probe P1 and the 3' -end of the hydrolysis probe P2 are marked with BHQ1 quenching groups.

4. Use of the composition of any one of claims 1 to 3 for the preparation of a kit for the detection of deletion mutations in the EGFR gene.

5. The use according to claim 4, characterized in that: the using method of the kit comprises the following steps:

And finally, detecting the signal of the reporter group, and judging the corresponding target nucleic acid sequence according to the detection result.

6. The use according to claim 4, characterized in that: the reaction procedure of the kit is as follows:

pre-denaturation at 95 ℃ for 10 min;

denaturation at 94℃for 30 seconds, annealing at 65℃for 60 seconds, and a total of 5 cycles;

denaturation at 94℃for 30 seconds, annealing at 52 ℃;

extending for 60 seconds, and performing 40 cycles in total; inactivating at 98 ℃ for 10 minutes; the reaction was stopped at 10 ℃.

7. The use according to claim 4, characterized in that: the reaction system of the kit comprises:

an upstream primer F1 with a concentration of 15-150 nM, an upstream primer F1-1 with a concentration of 150-1500 nM, and a hydrolysis probe P1 with a concentration of 50-800 nM; and, a downstream primer R having a concentration of 150 to 1800 nM; and/or,

8. The use according to claim 7, characterized in that: the reaction system of the kit comprises:

an upstream primer F1 with a concentration of 30-60 nM, an upstream primer F1-1 with a concentration of 300-600 nM, and a hydrolysis probe P1 with a concentration of 150-400 nM; and, a downstream primer R having a concentration of 300 to 900 nM; and/or,

An upstream primer F2 with a concentration of 30-60 nM, an upstream primer F2-1 with a concentration of 300-600 nM, and a hydrolysis probe P2 with a concentration of 150-400 nM.