阅读说明：本技术 一种用于扩增变异型靶基因片段的非淬灭型寡核苷酸探针及其应用 (Non-quenching oligonucleotide probe for amplifying variant target gene fragment and application thereof ) 是由 唐东江 赵计昌 黄雅菁 齐盼盼 乔一恺 李雁茭 于 2020-05-15 设计创作，主要内容包括：本发明涉及一种用于扩增变异型靶基因片段的非淬灭型寡核苷酸探针及其应用,非淬灭型寡核苷酸满足以下条件：①与野生型靶基因片段完全匹配,与变异型靶基因片段局部错配；②调整寡核苷酸探针长度；③在变异位点附近的特定位置进行修饰；④探针不标记荧光基团和淬灭基团；⑤探针3’末端进行修饰；⑥变异为单个或者多个碱基变异；⑦探针可单独或者联合使用。该探针在抑制野生型基因扩增与不影响变异型基因扩增上取得平衡,最大限定抑制野生型基因的扩增同时避免影响变异型基因的扩增,对变异型基因具有良好的富集效果。同时,还具有成本低,富集后的检测方式灵活性大以及效果稳定的优点。(The invention relates to a non-quenching oligonucleotide probe for amplifying a variant target gene fragment and application thereof, wherein the non-quenching oligonucleotide meets the following conditions: completely matching with a wild target gene fragment and locally mismatching with a variant target gene fragment; adjusting the length of the oligonucleotide probe; modifying at a specific position near the mutation site; fourthly, the probe does not mark a fluorescent group and a quenching group; modifying the 3' tail end of the probe; sixthly, the variation is single or multiple base variation; the probes can be used alone or in combination. The probe balances the inhibition of wild gene amplification and the no influence on the amplification of the variant gene, inhibits the amplification of the wild gene to the maximum limit, avoids influencing the amplification of the variant gene at the same time, and has good enrichment effect on the variant gene. Meanwhile, the method has the advantages of low cost, high flexibility of the enriched detection mode and stable effect.)

1. A non-quenched oligonucleotide probe for amplifying a variant target gene fragment, comprising:

(1) the nucleotide sequence of the non-quenching oligonucleotide probe is completely matched with the wild target gene fragment and is mismatched with the variant target gene fragment at the variant position;

(2) the length of the non-quenching oligonucleotide probe is 24-50 bp, wherein at least 1-6 nucleotides are modified within the range of 1-5 bp at the variation position and two sides of the variation position to enhance the thermal stability of the probe and a complementary chain, and preferably, the modification is locked nucleic acid modification or peptide nucleic acid modification; the 3' end of the non-quenched oligonucleotide probe is modified to block extension of the probe during amplification, preferably the modification is a dideoxy modification, an amino modification or a phosphorylation modification;

(3) the non-quenched oligonucleotide probe can bind to the wild-type target gene fragment and the variant target gene fragment when annealed, and only bind to the wild-type target gene fragment when extended.

2. The oligonucleotide probe of claim 1, wherein the variation comprises a single base variation or a plurality of base variations;

preferably, the single base variation comprises EGFR-T790M, EGFR-L858R, K-ras, ESR1-D538G, PIK3CA-E542K, PIK3CA-E545K, PIK3CA-H1047R, PIK3CA-H1047L or Braf-V600E;

preferably, the plurality of base variations comprises EGFR-19 Del;

preferably, the non-quenched oligonucleotide probe is probe 1, probe 2 or probe 3 for amplifying EGFR-T790M variation; the nucleotide sequence of the probe 1 is TCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCC (SEQ ID NO: 1), and the nucleotide sequence of the probe 2 is GTGCAGCTCATCACGCAGCTCATGCCCT (SEQ ID NO: 2), and the nucleotide sequence of the probe 3 is GTGCAGCTCATCACGCAGCTCATGCCCT (SEQ ID NO: 3), wherein the underlined nucleotide in probe 1, probe 2 or probe 3 is the nucleotide modified to enhance the thermal stability of the probe to the complementary strand;

or the non-quenched oligonucleotide probe is probe 4 or probe 5 for amplifying K-ras variation; the nucleotide sequence of the probe 4 is GGTAGTTGGAGCTGGTGGCGTAGGCAAGAG (SEQ ID NO: 4), nucleotides of said probe 5Sequence TGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTG (SEQ ID NO: 5), wherein the underlined nucleotide in probe 4 or probe 5 is the nucleotide modified to enhance the thermostability of the probe to the complementary strand;

alternatively, the non-quenched oligonucleotide probe is probe 6 or probe 7 for amplifying EGFR-L858R variation; the nucleotide sequence of the probe 6 is CACAGATTTTGGGCTGGCCAAACTGCTGGGTG (SEQ ID NO: 6), and the nucleotide sequence of the probe 7 is ATTTTGGGCTGGCCAAACTGCTGG (SEQ ID NO: 7), wherein the underlined nucleotide in probe 6 or probe 7 is the nucleotide modified to enhance the thermostability of the probe to the complementary strand;

or the non-quenched oligonucleotide probe is a probe 8 or a probe 9 for amplifying EGFR-19Del deletion variation; the nucleotide sequence of the probe 8 is GCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACA (SEQ ID NO: 8), the nucleotide sequence of the probe 9 is GTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGG (SEQ ID NO: 9), wherein the underlined nucleotide in probe 8 or probe 9 is the nucleotide modified to enhance the thermostability of the probe to the complementary strand;

alternatively, the non-quenched oligonucleotide probe is probe 18 for amplifying variations of ESR 1-D538G; the nucleotide sequence of the probe 18 is GTGCCCCTCTATGACCTGCTGCTGGAGATG (SEQ ID NO:26), wherein the underlined nucleotides in the probe 18 are the nucleotides that have been modified to enhance the thermal stability of the probe to the complementary strand; the modified is preferably LNA modified;

alternatively, the non-quenched oligonucleotide probe is probe 19 for amplifying PIK3CA-E542K variants and PIK3CA-E545K variants; the nucleotide sequence of the probe 19 is CTTTCTCCTGCTCAGTGATTTCAGAGAGAGG (SEQ ID NO.27), wherein the underlined nucleotides in the probe 19 are the nucleotides that have been modified to enhance the thermal stability of the probe to the complementary strand; the modified is preferably LNA modified;

alternatively, the non-quenched oligoThe nucleotide probe is a probe 20 for amplifying PIK3CA-H1047R variation and PIK3CA-H1047L variation; the nucleotide sequence of the probe 20 is AACAAATGAATGATGCACATCATGGTGGCTGGACAACAA (SEQ ID NO.28), wherein the underlined nucleotide in the probe 20 is the nucleotide that has been modified to enhance the thermal stability of the probe to the complementary strand; the modified is preferably LNA modified;

alternatively, the non-quenched oligonucleotide probe is probe 21 for amplifying a Braf-V600E variation; the nucleotide sequence of the probe 21 is GGTCTAGCTACAGTGAAATCTCGATGG (SEQ ID NO.29), wherein the underlined nucleotide in the probe 21 is the nucleotide that has been modified to enhance the thermal stability of the probe to the complementary strand; the modification is preferably LNA modification.

3. A reagent and/or kit for amplifying a mutant target gene fragment, wherein the reagent and/or kit comprises the non-quenched oligonucleotide probe of any one of claims 1-2, other amplification reagents and/or amplification consumables;

optionally, the non-quenched oligonucleotide probe is one or more, preferably, a plurality of oligonucleotide probes are directed against the same variant target gene, or different variant target genes;

optionally, the probe and the other amplification reagents are provided in a separate package, or the probe and the other amplification reagents are provided in a mixed single reagent;

optionally, the other amplification reagents comprise one or more of primer pairs, DNA polymerase, buffers, dNTPs, sterile water, and double stranded DNA dyes; further optionally, the additional amplification reagents, when multiple, are provided in separate packages, or at least two of the additional amplification reagents are provided as a mixed single reagent;

optionally, the primer pair consists of an upstream primer and a downstream primer for amplifying a mutation site comprising the mutant target gene fragment, the primer pair and the non-quenched oligonucleotide probe do not overlap or partially overlap at a binding site to the target gene fragment; further optionally, the molar ratio of the upstream primer to the downstream primer is 1: 0.75-1.25, preferably 1: 1.

4. The reagent and/or kit according to claim 3, wherein the reagent and/or kit is used for amplifying EGFR-T790M variation, and the nucleotide sequences of the primer pairs are respectively as follows: CATGCGAAGCCACACTGAC (SEQ ID NO: 10) and GTCTTTGTGTTCCCGGACATAGTCCAGG (SEQ ID NO: 11);

alternatively, the reagent and/or the kit is used for amplifying K-ras variation, and the nucleotide sequences of the primer pairs are respectively shown as follows: AAGCGTCGATGGAGGAGTTTGTAAAT (SEQ ID NO: 12) and GTTGGATCATATTCGTCCACAA (SEQ ID NO: 13);

alternatively, the reagent and/or the kit is used for amplifying EGFR-L858R variation, and the nucleotide sequences of the primer pairs are respectively shown as follows: TACTTGGAGGACCGTCGCTT (SEQ ID NO: 14) and GCTGACCTAAAGCCACCTCCTTA (SEQ ID NO: 15);

alternatively, the reagent and/or the kit is used for amplifying EGFR-19Del deletion variation, and the nucleotide sequences of the primer pairs are respectively shown as follows: ACGTCTTCCTTCTCTCTCTGTCATA (SEQ ID NO: 16) and GCCAGACATGAGAAAAGGT (SEQ ID NO: 17);

alternatively, the reagent and/or the kit is used for amplifying ESR1-D538G variation, and the nucleotide sequences of the primer pair are respectively shown as follows: FP-538: CAGTAACAAAGGCATGGAGCAT (SEQ ID NO.30) and RP-538: CCCTCCACGGCTAGTGGG (SEQ ID NO: 31);

alternatively, the reagents and/or kits are used to amplify PIK3CA-E542K variants and PIK3CA-E545K variants, the nucleotide sequences of the primer pairs are shown below: FP-542: AATGACAAAGAACAGCTCAAAGCAA (SEQ ID NO.32) and RP-542: TTAGCACTTACCTGTGACTCCAT (SEQ ID NO. 33);

alternatively, the reagent and/or the kit is used for amplifying the PIK3CA-H1047R variation and the PIK3CA-H1047L variation, and the nucleotide sequences of the primer pairs are respectively shown as follows: FP-1047: TCGAAAGACCCTAGCCTTAGAT (SEQ ID NO.34) and RP-1047: TTGTGTGGAAGATCCAATCCAT (SEQ ID NO. 35);

alternatively, the reagents and/or kits are used to amplify Braf-V600E variants, and the nucleotide sequences of the primer pairs are shown below: FP-600: TGAAGACCTCACAGTAAAAATAGGT (SEQ ID NO.36) and RP-600: AGCCTCAATTCTTACCATCCACA (SEQ ID NO. 37).

5. A mixed reaction system for amplifying a mutant target gene fragment, comprising the reagent according to claim 3 or 4 and a sample to be tested;

optionally, the sample to be tested is a low copy number sample or a low variation frequency sample, preferably, the low copy number is 800 to 20000 copies, for example, 1000 copies, 4000 copies, 8000 copies or 16000 copies, and the low variation frequency is 0.03% to 5% variation rate, for example, 0.05% variation rate, 0.075% variation rate, 0.1% variation rate, 0.15% variation rate, 0.25% variation rate, 0.3% variation rate, 0.5% variation rate, 1% variation rate, 1.2% variation rate or 4% variation rate;

optionally, the sample to be detected is a low copy number and low variation rate sample; preferably, the low copy number is 800 to 20000 copies, for example, 1000 copies, 4000 copies, 8000 copies or 16000 copies, and the low variation frequency is 0.03% to 5% variation rate, for example, 0.05% variation rate, 0.075% variation rate, 0.1% variation rate, 0.15% variation rate, 0.25% variation rate, 0.3% variation rate, 0.5% variation rate, 1% variation rate, 1.2% variation rate or 4% variation rate; more preferably, the low copy number is 800-1200 copies (e.g., 1000 copies), and the low variation frequency is 0.3% -5% (e.g., 0.4%, 1.2%, or 4%); alternatively, the low copy number is 3000-5000 copies (e.g., 4000 copies), and the low variation frequency is 0.075-1.25% (e.g., 0.1%, 0.3%, or 1%); alternatively, the low copy number is 7000-9000 copy numbers (e.g., 8000 copy numbers), and the low variation frequency is 0.03% -1%, e.g., (0.05%, 0.15%, or 0.5%); alternatively, the low copy number is 15000-17000 copies (e.g., 6000 copies) 1, and the low variation frequency is 0.05% to 0.3% (e.g., 0.075% or 0.25%).

6. A method for amplifying a variant target gene fragment, comprising the steps of: (1) preparing the mixed reaction system of claim 5; (2) performing a PCR reaction, wherein the PCR reaction comprises denaturation, annealing and extension; wherein, upon annealing, the non-quenched oligonucleotide probe binds to the wild-type target gene fragment and the variant target gene fragment; upon extension, the non-quenched oligonucleotide probe binds only to the wild-type target gene fragment.

7. The method of claim 6, wherein the method is used for amplifying the EGFR-T790M variant gene fragment, and the non-quenched nucleotide probe is probe 1, probe 2 or probe 3; the annealing temperature of the probes 1-3 is preferably 56-60 ℃, more preferably 56.5-57.5 ℃, and most preferably 57 ℃; the extension temperature is preferably 66-69 ℃, more preferably 67.5-68.5 ℃, and most preferably 68 ℃;

the method is used for amplifying a K-ras variant gene fragment, the non-quenched oligonucleotide probe is probe 4, the annealing temperature is preferably 61-63 ℃, more preferably 61.5-62.5 ℃, most preferably 62 ℃, the extension temperature is preferably 71-73 ℃, more preferably 71.5-72.5 ℃, and most preferably 72 ℃; or the non-quenched oligonucleotide probe is probe 5, the annealing temperature is preferably 65 ℃ to 67 ℃, more preferably 65.5 ℃ to 66.5 ℃, and most preferably 66 ℃, the extension temperature is preferably 67 ℃ to 69 ℃, more preferably 67.5 ℃ to 68.5 ℃, and most preferably 68 ℃;

or, the method is used for amplifying EGFR-L858R variation, the non-quenched oligonucleotide probe is probe 6 or probe 7, the annealing temperature of the probe 6-7 is preferably 58-61 ℃, more preferably 59.5-60.5 ℃, most preferably 60 ℃, the extension temperature is preferably 67-70 ℃, more preferably 68.5-69.5 ℃, and most preferably 69 ℃;

or, the method is used for amplifying EGFR-19-Del, the non-quenched oligonucleotide probe is probe 8 or probe 9, the annealing temperature of the probe 8-9 is preferably 59-61 ℃, more preferably 59.5-60.5 ℃, most preferably 60 ℃, the extension temperature is preferably 65-68 ℃, more preferably 65.5-66.5 ℃, and most preferably 66 ℃;

alternatively, the method is used to amplify ESR1-D538G variation, the non-quenched oligonucleotide probe is probe 18, and the annealing temperature of probe 18 is preferably 56 ℃ to 60 ℃, more preferably 56.5 ℃ to 57.5 ℃, and most preferably 57 ℃; the extension temperature is preferably 66-70 ℃, more preferably 67-69 ℃, and most preferably 68 ℃;

alternatively, the method is used to amplify a PIK3CA-E542K variant and a PIK3CA-E545K variant, the non-quenched oligonucleotide probe is probe 19, and the probe is annealed at a temperature preferably between 56 ℃ and 60 ℃, more preferably between 56.5 ℃ and 57.5 ℃, and most preferably at 57 ℃; the extension temperature is preferably 56 ℃ to 60 ℃, more preferably 56 ℃ to 58 ℃, and most preferably 56 ℃;

alternatively, the method is used to amplify a PIK3CA-H1047R variant and a PIK3CA-H1047L variant, the unquenched oligonucleotide probe is probe 20; the annealing temperature of the probe is preferably 56-60 ℃, more preferably 56.5-57.5 ℃, and most preferably 57 ℃; the extension temperature is preferably 60 ℃ to 68 ℃, more preferably 60 ℃ to 65 ℃, and most preferably 62 ℃;

alternatively, the method is used to amplify a Braf-V600E variation, and the non-quenched oligonucleotide probe is probe 21; the annealing temperature of the probe is preferably 56-60 ℃, more preferably 56-58 ℃ and most preferably 56 ℃; the elongation temperature is preferably from 58 ℃ to 62 ℃, more preferably from 59 ℃ to 61 ℃, and most preferably 60 ℃.

8. The mixed reaction system according to claim 5 or the method according to any one of claims 6 to 7, wherein the sample to be detected is blood, body fluid, tissue, circulating tumor cells, cfDNA or a sample of early fetal test.

9. The mixed reaction system of claim 8 or the method of any one of claims 6 to 7, wherein the sample of the fetal pre-detection is selected from the group consisting of maternal blood, villus punch sample, and amniotic fluid punch sample.

10. Use of the oligonucleotide probe of any one of claims 1 to 2, the reagent and/or kit of any one of claims 3 to 4, the mixed reaction system of claim 5 or the method of any one of claims 6 to 8 for enrichment prior to detection or detection of a mutated target gene fragment, preferably said enrichment is pooling prior to sequencing.

Technical Field

The invention relates to the field of gene detection, in particular to a non-quenched oligonucleotide probe for amplifying a variant target gene fragment and application thereof.

Background

Genetic variation (mutation) refers to the change of genetic material and the resulting phenotypic change, including point mutation, deletion, duplication, rearrangement, etc. Various techniques are available in the market for detecting and analyzing gene mutation, for example, ARMS-PCR (amplification recovery mutation system PCR), NGS (next generation mutation), dd-PCR (repeat digital-PCR) and the like can detect mutant sequences from wild type, but all have disadvantages. Wherein, the detection sensitivity of ARMS-PCR is lower; the detection sensitivity of NGS and dd-PCR is higher, but dd-PCR and NGS have the defects of high detection cost, expensive equipment, complex operation, easy pollution, difficult clinical popularization and the like. In addition, the current gene detection reagents are usually used for detecting serum and tissue samples, and a reagent or a kit which can be compatible with Circulating Tumor Cell (CTC) sample detection is lacked.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a non-quenching oligonucleotide probe, a reagent and/or a kit, a mixed reaction system, an amplification method and application of a target amplification variant target gene fragment, wherein the probe can continuously and effectively block amplification of a wild type gene fragment in an amplification reaction process, but basically has no influence on the variant gene fragment, so that the effective enrichment of the variant gene fragment is realized, the detection of gene variation, especially low-frequency gene variation, is favorable, and has the advantages of high detection sensitivity, simple operation, low cost and high cost performance.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a non-quenched oligonucleotide probe for amplifying a variant target gene fragment, wherein (1) the nucleotide sequence of the non-quenched oligonucleotide probe perfectly matches a wild-type target gene fragment and mismatched with the variant target gene fragment at a variant position;

(2) the length of the non-quenching oligonucleotide probe is 24-50 bp, wherein at least 1-6 nucleotides are modified within the range of 1-5 bp at the variation position and two sides of the variation position to enhance the thermal stability of the probe and a complementary chain, and preferably, the modification is locked nucleic acid modification or peptide nucleic acid modification; the 3' end of the non-quenched oligonucleotide probe is modified to block extension of the probe during amplification, preferably the modification is a dideoxy modification, an amino modification or a phosphorylation modification;

(3) the non-quenched oligonucleotide probe can bind to the wild-type target gene fragment and the variant target gene fragment when annealed, and only bind to the wild-type target gene fragment when extended.

The invention designs the non-quenching oligonucleotide probe, and the non-quenching oligonucleotide probe meets the following conditions: matching with wild target gene segment and mismatching with mutant target gene segment; adjusting the length of the oligonucleotide probe; and modifying at specific positions to enhance the thermal stability of the probe and the complementary link. The probe can be combined with a wild type template with high binding force and a variant template with low binding force in an annealing state, and is only combined with the wild type template in an extension state, so that the balance between the inhibition of the amplification of the wild type gene fragment and the inhibition of the amplification of the variant gene fragment is achieved, the inhibition of the amplification of the wild type gene fragment is limited to the maximum extent, and the influence on the amplification of the variant gene fragment is avoided.

The aforementioned probe of the present invention also satisfies the following conditions: fourthly, the probe is a non-quenching oligonucleotide probe; the 3' end of the probe is subjected to dideoxy modification, amino modification or phosphorylation modification and the like. Compared with the common fluorescence quenching type probe, the non-quenching type oligonucleotide probe has the advantages of low cost, large flexibility of the detection mode after enrichment and stable effect. Specifically, firstly, the probe does not need the labeling modification of a fluorescent group and a quenching group, and the cost of the probe is reduced. Secondly, the application range of the non-quenching oligonucleotide probe is wide, and the non-quenching oligonucleotide probe is suitable for various platforms. For example, the non-quenched oligonucleotide probe can be used in combination with a double-stranded DNA dye (such as SYBR Green or EvaGreen) during a PCR amplification stage to obtain a detection result of the variant gene; alternatively, the amplified and enriched PCR product can be directly used for sequencing; alternatively, other low-sensitivity genetic variation detection kits are used to detect the enriched sample. Finally, the 3 'end of the probe is subjected to dideoxy modification, amino modification or phosphorylation modification and the like, so that the extension of the 3' end of the probe in the amplification process can be avoided, and the increase of Tm of the probe due to the increase of the length can be prevented, and the inhibition effect on the amplification of the variant gene can be generated.

In some specific embodiments, the probe has a length of 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 33bp, 34bp, 35bp, 36bp, 37bp, 38bp, 39bp, 40bp, 41bp, 42bp, 43bp, 44bp, 45bp, 46bp, 47bp, 48bp, 49bp, or 50 bp; preferably, the probe is 24bp, 28bp, 30bp, 32bp, 33bp, 43bp, 48bp or 49bp in length.

In some embodiments, the variation comprises a single base variation or a plurality of base variations.

Preferably, the single base variation comprises EGFR-T790M, EGFR-L858R, K-ras, ESR1-D538G, PIK3CA-E542K, PIK3CA-E545K, PIK3CA-H1047R, PIK3CA-H1047L or Braf-V600E;

preferably, the plurality of base variations comprises EGFR-19 Del; .

In some specific embodiments, the non-quenched oligonucleotide probe is probe 1, probe 2, or probe 3 for amplifying EGFR-T790M variation; the nucleotide sequence of the probe 1 is TCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCC (SEQ ID NO: 1), and the nucleotide sequence of the probe 2 is GTGCAGCTCATCACGCAGCTCATGCCCT (SEQ ID NO: 2), and the nucleotide sequence of the probe 3 is GTGCAGCTCATCACGCAGCTCATGCCCT (SEQ ID NO: 3), wherein the underlined nucleotide in probe 1, probe 2 or probe 3 is the nucleotide modified to enhance the thermal stability of the probe to the complementary strand.

In some embodiments, the non-quenched oligonucleotide probe is probe 4 or probe 5 for amplification of a K-ras mutation; the nucleotide sequence of the probe 4 is GGTAGTTGGAGCTGGTGGCGTAGGCAAGAG (SEQ ID NO: 4), and the nucleotide sequence of the probe 5 is TGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTG (SEQ ID NO: 5), wherein the underlined nucleotide in probe 4 or probe 5 is the nucleotide modified to enhance the thermostability of the probe to the complementary strand.

In some specific embodiments, the non-quenched oligonucleotide probe is probe 6 or probe 7 for amplifying EGFR-L858R variation; the nucleotide sequence of the probe 6 is CACAGATTTTGGGCTGGCCAAACTGCTGGGTG (SEQ ID NO: 6), and the nucleotide sequence of the probe 7 is ATTTTGGGCTGGCCAAACTGCTGG (SEQ ID NO: 7), wherein the underlined nucleotide in probe 6 or probe 7 is the modified so as to enhance the thermostability of the probe and the complementary strandThe nucleotide of (a).

In some specific embodiments, the non-quenched oligonucleotide probe is probe 8 or probe 9 for amplification of EGFR-19Del deletion variants; the nucleotide sequence of the probe 8 is GCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACA (SEQ ID NO: 8), the nucleotide sequence of the probe 9 is GTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGG (SEQ ID NO: 9), wherein the underlined nucleotides in probe 8 or probe 9 are the nucleotides that have been modified to enhance the thermal stability of the probe to the complementary strand.

In some specific embodiments, the non-quenched oligonucleotide probe is probe 18 for amplifying variations in ESR 1-D538G; the nucleotide sequence of the probe 18 is GTGCCCCTCTATGACCTGCTGCTGGAGATG (SEQ ID NO:26), wherein the underlined nucleotides in the probe 18 are the nucleotides that have been modified to enhance the thermal stability of the probe to the complementary strand; the modification is preferably LNA modification.

In some specific embodiments, the non-quenched oligonucleotide probe is probe 19 for amplifying PIK3CA-E542K variations and PIK3CA-E545K variations; the nucleotide sequence of the probe 19 is CTTTCTCCTGCTCAGTGATTTCAGAGAGAGG (SEQ ID NO.27), wherein the underlined nucleotide in the probe 19 is the nucleotide that has been modified to enhance the thermal stability of the probe to the complementary strand; the modification is preferably LNA modification.

In some specific embodiments, the non-quenched oligonucleotide probe is probe 20 for amplifying PIK3CA-H1047R variations and PIK3CA-H1047L variations; the nucleotide sequence of the probe 20 is AACAAATGAATGATGCACATCATGGTGGCTGGACAACAA (SEQ ID NO.28), wherein the underlined nucleotide in the probe 20 is the nucleotide that has been modified to enhance the thermal stability of the probe to the complementary strand; the modification is preferably LNA modification.

In some specific embodiments, the non-quenched oligonucleotide probe is probe 21 for amplifying a Braf-V600E variation; of said probe 21The nucleotide sequence is GGTCTAGCTACAGTGAAATCTCGATGG (SEQ ID NO.29), wherein the underlined nucleotide in the probe 21 is the nucleotide that has been modified to enhance the thermal stability of the probe to the complementary strand; the modification is preferably LNA modification.

The known resistance mechanisms of oxitinib include re-mutation of the EGFR gene itself (mutation at the Target On Target), other gene mutation (Off Target mutation Off Target), pathological type transformation, etc. BRAF mutations are associated with a variety of cancers. Cancers such as melanoma, lymphoma, thyroid cancer, and non-small cell lung cancer, with about 1-3% occurring in patients with non-small cell lung cancer (both squamous and adenocarcinoma), with about 50% of patients at V600E; BRAF causes a higher incidence of non-small cell lung cancer in the african-asian smoking population. Besides causing third-generation TKI resistance, researches show that part of KRAS gene wild patients have unsatisfactory single-drug treatment effect on panitumumab and are related to mutation of BRAF (BRAF) which is a downstream gene of the patients. Patients can regain sensitivity to cetuximab or panitumumab by the use of BRAF inhibitors (sorafenib and dojimet). Meanwhile, BRAF gene mutation is often an index of poor prognosis.

Mutations in PIK3CA gene are commonly found in cancer species such as liver cancer (about 36%), breast cancer (about 26%), colorectal cancer (9%), and the like, and the common mutations include mutations in E542K, E545K and E545D of the ninth exon, and mutations in H1047R, H1047L of 20 exons, and the like. When activated as an EGFR downstream signaling molecule, the PI3K gene may lead to resistance of tumor cells to EGFR-TKI drugs, for example, mutation of PIK3CA gene may lead to resistance of cetuximab, panitumumab to metastatic colorectal cancer treatment, and gefitinib, erlotinib to treatment of patients with NSCLC and advanced esophageal cancer. Meanwhile, the PIK3CA mutation is ineffective to treat the breast cancer individualized targeting drug trastuzumab, and the wild type treatment is effective.

The estrogen receptor comprises two subtypes of estrogen receptor alpha and estrogen receptor beta, wherein the estrogen receptor alpha protein is coded by ESR1 gene and is closely related to the occurrence and development of breast cancer. The activation of the ligand-independent pathway caused by the mutation of ESR1 is an important cause of AI endocrine resistance, the main mutation hotspots Y537S and D583G are very rare in untreated primary breast cancer patients, and the mutation rate is only 3%, but the ESR1 mutation rate is increased in advanced breast cancer patients, especially patients treated with aromatase inhibitor, and the ESR1 mutation rate is 25% and 29% in patients who have progressed endocrine therapy and patients who have been treated with aromatase inhibitor in phase 3 clinical study PALOMA3, respectively. In the SoFFA study, the ESR1 mutation rate was approximately 39% in patients susceptible to aromatase inhibitor treatment. For MBC patients with ER positivity and ESR1 mutation, a combination therapy of Apigliocide and fulvestrant as endocrine therapy, and single drug or combination therapy can be selected.

The detection of the mutation states of EGFR-T790M, K-ras, EGFR-L858R, EGFR-19Del, ESR1-D538G, PIK3CA-E542K, PIK3CA-E545K, PIK3CA-H1047R, PIK3CA-H1047L and Braf-V600E is of great significance for the targeted treatment of cancer, and the detection of the mutation states of EGFR-T790M, K-ras, EGFR-L58858 23 and EGFR-19Del, PIK3CA-E542K, PIK 3-E545K, PIK3CA-H1047L, PIK3CA-H1047R and Braf-V600E is of great significance particularly for the targeted treatment of patients with Tyrosine Kinase Inhibitors (TKI) for lung cancer. The invention designs, optimizes and obtains the non-quenching type oligonucleotide probe, and is used for amplifying EGFR-T790M, K-ras, EGFR-L858R, EGFR-19Del, ESR1-D538G, PIK3CA-E542K, PIK3CA-E545K, PIK3CA-H1047R, PIK3CA-H1047L and Braf-V600E variant genes. However, it is noteworthy that the present invention found that the length of the probe and the modification position of the locked nucleic acid have a significant influence on the effect of the non-quenched oligonucleotide probe in the optimization process. Taking EGFR-T790M as an example, the locked nucleic acid modification positions of probe 1 and probe 3 are the same, the lengths of the probes are different, and although the amplification of wild-type genes can be inhibited, the inhibition effect of probe 1 is obviously better than that of probe 3. Taking K-ras as an example, probe 4 and probe 5 have basically the same length and different nucleotide modification positions, and both of them show blocking effect on wild-type K-ras, but probe 4 has no amplification inhibition effect on G12V and G13D, while probe 5 inhibits the amplification of G13D.

The invention also relates to: a reagent and/or kit for amplifying a variant target gene fragment, comprising the aforementioned non-quenched oligonucleotide probe, other amplification reagents and/or amplification consumables.

In some embodiments, the non-quenched oligonucleotide probe is one or more, preferably, a plurality of oligonucleotide probes are directed against the same variant target gene, or different variant target genes.

In some embodiments, the probe and the additional amplification reagent are provided in a separate package, or the probe and the additional amplification reagent are provided in a mixed single reagent.

In some specific embodiments, the additional amplification reagents comprise one or more of primer pairs, DNA polymerases, buffers, dNTPs, sterile water, and double-stranded DNA dyes; further optionally, the additional amplification reagents are provided in separate packages when they are multiple, or at least two of the additional amplification reagents are provided as a mixed single reagent.

In some specific embodiments, the primer pair consists of an upstream primer and a downstream primer for amplifying a mutation location comprising the mutant target gene fragment, the primer pair and the non-quenched oligonucleotide probe do not overlap or partially overlap at a binding site to the target gene fragment; further optionally, the molar ratio of the upstream primer to the downstream primer is 1: 0.75-1.25, preferably 1: 1.

In some specific embodiments, the reagents and/or kits are used to amplify EGFR-T790M variation, and the nucleotide sequences of the primer pairs are shown below: CATGCGAAGCCACACTGAC (SEQ ID NO: 10) and GTCTTTGTGTTCCCGGACATAGTCCAGG (SEQ ID NO: 11).

In some specific embodiments, the reagents and/or kits are used for amplifying K-ras variants, and the nucleotide sequences of the primer pairs are respectively as follows: AAGCGTCGATGGAGGAGTTTGTAAAT (SEQ ID NO: 12) and GTTGGATCATATTCGTCCACAA (SEQ ID NO: 13).

In some specific embodiments, the reagents and/or kits are used to amplify EGFR-L858R variation, and the nucleotide sequences of the primer pairs are shown below: TACTTGGAGGACCGTCGCTT (SEQ ID NO: 14) and GCTGACCTAAAGCCACCTCCTTA (SEQ ID NO: 15).

In some specific embodiments, the reagents and/or kits are used for amplifying EGFR-19Del deletion variants, and the nucleotide sequences of the primer pairs are respectively as follows: ACGTCTTCCTTCTCTCTCTGTCATA (SEQ ID NO: 16) and GCCAGACATGAGAAAAGGT (SEQ ID NO: 17).

In some specific embodiments, the reagents and/or kits are used to amplify ESR1-D538G variants, and the nucleotide sequences of the primer pairs are respectively as follows: FP-538: CAGTAACAAAGGCATGGAGCAT (SEQ ID NO.30) and RP-538: CCCTCCACGGCTAGTGGG (SEQ ID NO: 31).

In some embodiments, the reagents and/or kits are used to amplify PIK3CA-E542K and PIK3CA-E545K variants, the nucleotide sequences of the primer pairs are shown below: FP-542: AATGACAAAGAACAGCTCAAAGCAA (SEQ ID NO.32) and RP-542: TTAGCACTTACCTGTGACTCCAT (SEQ ID NO. 33).

In some specific embodiments, the reagents and/or kits are used to amplify PIK3CA-H1047R and PIK3CA-H1047L variants, the nucleotide sequences of the primer pairs are shown below, respectively: FP-1047: TCGAAAGACCCTAGCCTTAGAT (SEQ ID NO.34) and RP-1047: TTGTGTGGAAGATCCAATCCAT (SEQ ID NO. 35).

In some specific embodiments, the reagents and/or kits are used to amplify Braf-V600E variants, and the nucleotide sequences of the primer pairs are shown below: FP-600: TGAAGACCTCACAGTAAAAATAGGT (SEQ ID NO.36) and RP-600: AGCCTCAATTCTTACCATCCACA (SEQ ID NO. 37).

The method further defines a primer pair matched with the oligonucleotide probe for use in amplification of EGFR-T790M, K-ras, EGFR-L858R, EGFR-19Del, ESR1-D538G, PIK3CA-E542K, PIK3CA-E545K, PIK3CA-H1047R, PIK3CA-H1047L and Braf-V600E mutant genes, wherein an amplification fragment of the primer pair covers the mutation position of a target gene, and the method has the advantages of good specificity, no non-specific amplification, no primer dimer and the like.

The invention also relates to: a mixed reaction system for amplifying the mutant target gene fragment, wherein the mixed reaction system comprises the reagent and a sample to be detected; optionally, the sample to be tested is a low copy number sample or a low variation frequency sample, preferably, the low copy number is 800 to 20000 copies, for example, 1000 copies, 4000 copies, 8000 copies or 16000 copies, and the low variation frequency is 0.03% to 5% variation rate, for example, 0.05% variation rate, 0.075% variation rate, 0.1% variation rate, 0.15% variation rate, 0.25% variation rate, 0.3% variation rate, 0.5% variation rate, 1% variation rate, 1.2% variation rate or 4% variation rate;

optionally, the sample to be detected is a low copy number and low variation rate sample; preferably, the low copy number is 800 to 20000 copies, for example, 1000 copies, 4000 copies, 8000 copies or 16000 copies, and the low variation frequency is 0.03% to 5% variation rate, for example, 0.05% variation rate, 0.075% variation rate, 0.1% variation rate, 0.15% variation rate, 0.25% variation rate, 0.3% variation rate, 0.5% variation rate, 1% variation rate, 1.2% variation rate or 4% variation rate; more preferably, the low copy number is 800-1200 copies (e.g., 1000 copies), and the low variation frequency is 0.3% -5% (e.g., 0.4%, 1.2%, or 4%); alternatively, the low copy number is 3000-5000 copies (e.g., 4000 copies), and the low variation frequency is 0.075-1.25% (e.g., 0.1%, 0.3%, or 1%); alternatively, the low copy number is 7000-9000 copy numbers (e.g., 8000 copy numbers), and the low variation frequency is 0.03% -1%, e.g., (0.05%, 0.15%, or 0.5%); alternatively, the low copy number is 15000-17000 copies (e.g., 6000 copies) 1, and the low variation frequency is 0.05% to 0.3% (e.g., 0.075% or 0.25%).

In some embodiments, the sample has a copy number of 1000-16000 wild-type gene and 12-40 mutant genes.

The invention also relates to: a method for amplifying a variant target gene fragment, the method comprising the steps of: (1) preparing the mixed reaction system; (2) performing a PCR reaction, wherein the PCR reaction comprises denaturation, annealing and extension; wherein, upon annealing, the non-quenched oligonucleotide probe binds to the wild-type target gene fragment and the variant target gene fragment; upon extension, the non-quenched oligonucleotide probe binds only to the wild-type target gene fragment.

In some specific embodiments, the method is used for amplifying EGFR-T790M variant gene fragments, and the non-quenched nucleotide probe is probe 1, probe 2 or probe 3; the annealing temperature of the probes 1-3 is preferably 56-60 ℃, more preferably 56.5-57.5 ℃, and most preferably 57 ℃; the extension temperature is preferably 66 ℃ to 69 ℃, more preferably 67.5 ℃ to 68.5 ℃, and most preferably 68 ℃.

In some embodiments, the method is used for amplifying a K-ras variant gene fragment, the non-quenched oligonucleotide probe is probe 4, the annealing temperature is preferably 61-63 ℃, more preferably 61.5-62.5 ℃, most preferably 62 ℃, the extension temperature is preferably 71-73 ℃, more preferably 71.5-72.5 ℃, and most preferably 72 ℃; alternatively, the non-quenched oligonucleotide probe is probe 5, the annealing temperature is preferably 65 ℃ to 67 ℃, more preferably 65.5 ℃ to 66.5 ℃, and most preferably 66 ℃, and the extension temperature is preferably 67 ℃ to 69 ℃, more preferably 67.5 ℃ to 68.5 ℃, and most preferably 68 ℃.

In some embodiments, the method is used to amplify EGFR-L858R variation, the non-quenched oligonucleotide probe is Probe 6 or Probe 7, the probes 6-7 are preferably annealed at a temperature of 58-61 deg.C, more preferably at a temperature of 59.5-60.5 deg.C, and most preferably at a temperature of 60 deg.C, and the extension temperature is preferably 67-70 deg.C, more preferably at a temperature of 68.5-69.5 deg.C, and most preferably at a temperature of 69 deg.C.

In some embodiments, the method is used for amplification of EGFR-19-Del, the non-quenched oligonucleotide probe is probe 8 or probe 9, the annealing temperature of the probes 8-9 is preferably 59-61 ℃, more preferably 59.5-60.5 ℃, most preferably 60 ℃, and the extension temperature is preferably 65-68 ℃, more preferably 65.5-66.5 ℃, most preferably 66 ℃.

In some embodiments, the method is used to amplify ESR1-D538G variation, the non-quenched oligonucleotide probe is probe 18, and the annealing temperature of probe 18 is preferably 56 ℃ to 60 ℃, more preferably 56.5 ℃ to 57.5 ℃, and most preferably 57 ℃; the extension temperature is preferably 66 ℃ to 70 ℃, more preferably 67 ℃ to 69 ℃, and most preferably 68 ℃.

In some embodiments, the method is used to amplify a PIK3CA-E542K variation and a PIK3CA-E545K variation, the unquenched oligonucleotide probe is probe 19, and the annealing temperature of the probe is preferably 56 ℃ to 60 ℃, more preferably 56.5 ℃ to 57.5 ℃, and most preferably 57 ℃; the elongation temperature is preferably 56 to 60 ℃, more preferably 56 to 58 ℃, and most preferably 56 ℃.

In some specific embodiments, the method is used to amplify a PIK3CA-H1047R variation and a PIK3CA-H1047L variation, and the non-quenched oligonucleotide probe is probe 20; the annealing temperature of the probe is preferably 56-60 ℃, more preferably 56.5-57.5 ℃, and most preferably 57 ℃; the extension temperature is preferably 60 ℃ to 68 ℃, more preferably 60 ℃ to 65 ℃, and most preferably 62 ℃.

In some specific embodiments, the method is for amplifying a Braf-V600E variation, and the non-quenched oligonucleotide probe is probe 21; the annealing temperature of the probe is preferably 56-60 ℃, more preferably 56-58 ℃ and most preferably 56 ℃; the elongation temperature is preferably from 58 ℃ to 62 ℃, more preferably from 59 ℃ to 61 ℃, and most preferably 60 ℃.

In some specific embodiments, the test sample of the method is a blood, body fluid, tissue, circulating tumor cells, cfDNA, or fetal early test sample.

In some specific embodiments, the fetal early detection sample is selected from maternal blood, a villus puncture sample, or an amniotic puncture sample.

In some specific embodiments, the method is for non-diagnostic purposes.

The invention also relates to: the oligonucleotide probe, the reagent and/or the kit, the mixed reaction system or the method can be used for detecting or enriching the target gene variation, and preferably, the enrichment is a database before sequencing.

Definition of terms

The term "match" as used herein means that the nucleotide sequences of the two are identical or satisfy a reverse complementary pair.

The non-quenched oligonucleotide probe refers to the probe which is not marked with a fluorescent group and a quenching group.

Variations described herein include, but are not limited to, point mutations, deletion mutations, frameshift mutations, and insertion mutations.

The probe 1, the Ins and the BL27 are the same probe; probe 2 and BL26 are the same probe; probe 3 and BL28 are the same probe; the probe 4 and the K-ras-bock-36 are the same probe; the probe 6 is the same as the L8-BL 3; the probe 7 is the same as the probe L8-BL 4; the probe 8 and the probe 19D-BL1 are the same; the probe 9 and the probe 19D-BL2 are the same; probe 16 is the same probe as Blocker 1; probe 17 is the same probe as Blocker 2; probe 18 and BL1-538 are the same probe; probe 19 and BL1-542 are the same probe; the probe 20 and BL1-1047 are the same probe; probe 21 and BL1-600 are the same probe.

Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a non-quenching oligonucleotide probe, a reagent and/or a kit, a mixed reaction system, an amplification method and application for targeted amplification of a target gene fragment containing single-base variation or multi-base variation, wherein the oligonucleotide probe balances the inhibition of wild-type gene amplification and the inhibition of mutant gene amplification, inhibits the wild-type gene amplification to the maximum limit, avoids influencing the mutant gene amplification, plays a role in high mutant gene enrichment efficiency and good detection sensitivity, and is particularly suitable for samples with low abundance, such as CTC cells, or samples with low variation rate (such as samples with variation rate of 0.05-0.5%).

(2) The probe is a non-quenching oligonucleotide probe, does not mark a fluorescent group and/or a quenching group, is modified by dideoxy, amino or phosphorylation at the 3' end, and the like, and has the advantages of low cost, high flexibility of an enriched detection mode and stable effect.

(3) Aiming at EGFR-T790M, K-ras, EGFR-L858R, EGFR-19Del, ESR1-D538G, PIK3CA-E542K, PIK3CA-E545K, PIK3CA-H1047R, PIK3CA-H1047L and Braf-V600E variant genes, the invention designs and optimizes a probe sequence, a primer pair, extension and annealing temperature, realizes the enrichment and detection of the variant genes in a low-abundance sample or a sample with low variant rate (such as 0.1 percent of variant rate, even 0.05 percent of variant rate), and can be used for the construction of a library before sequencing and the improvement of the detection limit of a low-sensitivity PCR kit. By using the reagent and/or kit provided by the invention for enrichment, amplification of a wild type template can be blocked, so that a large amount of templates in a sample containing a small amount of mutant templates can be enriched for subsequent Sanger sequencing analysis, and a sample with 0.1% of mutations can be detected by Sanger sequencing after library construction.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of the principle of the oligonucleotide probe of the present invention for inhibiting the PCR amplification of wild-type gene (example EGFR-T790M), wherein the inhibitor is the oligonucleotide probe of the present invention;

FIG. 2 PCR amplification results for different cases (wild type sample or variant sample, with or without probe 1, 64 ℃/68 ℃/72 ℃/76 ℃) with the inhibitor, probe 1 described in example 1;

FIG. 3 shows the effect of probes 1-3 (i.e., BL26/27/28) described in example 1 on amplification of EGFR-T790M wild-type gene;

FIG. 4 shows the effect of probes 1-3 (i.e., BL26/27/28) described in example 1 on EGFR-T790M mutant gene amplification, where the curves represent, according to the Ct values from small to large, no probe control (i.e., no Block control), BL26 (i.e., probe 2), BL27 (i.e., probe 1), and BL28 (i.e., probe 3), respectively;

FIG. 5 shows the sequencing results of PCR products of EGFR-T790M template with different variation rates and different copy numbers (example 1);

FIG. 6 is a graph of the amplification effect of Probe 1 (i.e., BL27) on CTC recovered samples (example 1);

FIG. 7 is a graph showing the effect of probes 10-12 on the results of wild-type amplification of EGFR-T790M (example 1);

FIG. 8 is a graph showing the effect of probes 10-12 on the amplification results of EGFR-T790M variant (example 1);

FIG. 9 is a graph showing the effect of probe 4 (i.e., Kras-block-36) on the amplification result of a wild-type K-ras gene (example 2);

FIG. 10 is a graph showing the effect of probe 4 (i.e., Kras-block-36) on the amplification result of the mutant K-ras gene (G12V) (example 2);

FIG. 11 is a graph showing the effect of probe 4, Kras-block-36, on the amplification result of the mutant K-ras gene (G13D) (example 2);

FIG. 12 shows the amplification results of wild-type K-ras gene under the extension condition at 72 ℃ (example 2);

FIG. 13 shows the amplification results of wild-type K-ras gene under extension conditions at 73 ℃ (example 2);

FIG. 14 shows the amplification result of a mutant K-ras gene (G12V) under extension conditions at 73 ℃ (example 2);

FIG. 15 shows the amplification result of a mutant K-ras gene (G12V) under extension conditions at 73 ℃ (example 2);

FIG. 16 shows the amplification result of a mutant K-ras gene (G13D) under the extension condition at 72 ℃ (example 2);

FIG. 17 shows the amplification result of a mutant K-ras gene (G13D) under extension conditions at 73 ℃ (example 2);

FIG. 18 shows the results of fluorescent PCR amplification on the enriched K-ras gene variation sample (variation rate of 0.5%) (example 2);

FIG. 19 is the Ct value data of fluorescence PCR on the enriched K-ras gene variation sample (variation rate of 0.5%) (example 2);

FIG. 20 shows the results of fluorescent PCR amplification on the enriched K-ras gene variation sample (variation rate of 0.25%) (example 2);

FIG. 21 shows Ct value data of fluorescence PCR on enriched K-ras gene variation sample (variation rate of 0.25%) (example 2);

FIG. 22 shows the results of PCR amplification of plasmid S009 with different copy numbers (example 2);

FIG. 23 is a standard graph (example 2);

FIG. 24 shows the blocking effect of probes 13 to 15 on wild-type K-ras gene (example 2);

FIG. 25 shows the blocking effect of probe 5 on the wild-type K-ras gene (example 2);

FIG. 26 shows the blocking effect of probe 5 on variant G12V (example 2);

FIG. 27 shows the blocking effect of probe 5 on variant G13D (example 2);

FIG. 28 shows the blocking effect of probe 6 (i.e., L8-BL3) and probe 13(L8-BL4) on wild-type EGFR-L858R gene (example 3);

FIG. 29 shows the blocking effect of probes 6 (i.e., L8-BL3) and 13(L8-BL4) on the mutant EGFR-L858R gene (example 3);

FIG. 30 shows the blocking effect of probe 7(L8-BL4) on the mutant EGFR-L858R gene after optimization of reaction conditions (example 3);

FIG. 31 shows the sequencing results of PCR products of EGFR-L858R wild-type template (example 3);

FIG. 32 shows the sequencing results of PCR products of EGFR-L858R variant template with a variation rate of 0.1% (example 3);

FIG. 33 shows the sequencing results of PCR products of EGFR-L858R variant template with a variation rate of 0.2% (example 3);

FIG. 34 shows the sequencing results of PCR products of EGFR-L858R variant template with a variation rate of 0.5% (example 3);

FIG. 35 shows the sequencing results of PCR products of EGFR-L858R variant template with a variation rate of 1% (example 3);

FIG. 36 shows the effect of probes 16-17 (i.e., Blaker 1 and Blocker2) on amplification of EGFR-L858R wild-type template (example 3);

FIG. 37 shows the effect of probes 16-17 (i.e., Blaker 1 and Blocker2) on amplification of EGFR-L858R variant templates (example 3);

FIG. 38 shows the effect of probes 8-9 (i.e., 19D-BL1, 19D-BL2) on amplification of EGFR-19Del wild-type template (example 4);

FIG. 39 shows the effect of probes 8-9 (i.e., 19D-BL1, 19D-BL2) on amplification of EGFR-19Del variant template (example 4);

FIG. 40 is the sequencing result of PCR product of EGFR-19Del wild-type template (example 4);

FIG. 41 shows the sequencing results of PCR products of EGFR-19Del variant template with a variation rate of 0.1% (example 4);

FIG. 42 shows the sequencing results of PCR products of EGFR-19Del variant template with a variation rate of 0.2% (example 4);

FIG. 43 shows the sequencing results of PCR products of EGFR-19Del variant template with a variation rate of 0.5% (example 4);

FIG. 44 shows the sequencing results of PCR products of EGFR-19Del variant template with a variation rate of 1% (example 4);

FIG. 45 shows the blocking effect of probe 18 (i.e., BL1-538) on the wild-type ESR1-D538G gene (example 5);

FIG. 46 shows the effect of probe 18 (i.e., BL1-538) on the blocking of the variant ESR1-D538G gene (example 5);

FIG. 47 shows the sequencing results of PCR products of ESR1-D538G wild-type template (example 5);

FIG. 48 shows the sequencing results of the PCR products of the ESR1-D538G variant template with a variation rate of 0.5% (example 4);

FIG. 49 shows the sequencing results of PCR products from ESR1-D538G variant templates with a variation rate of 0.2% (example 4);

FIG. 50 shows the sequencing results of PCR products from ESR1-D538G variant templates with a variation rate of 0.1% (example 4);

FIG. 51 shows the effect of probe 19 (i.e., BL1-542) on the blockade of the wild-type PIK3CA gene (example 6);

FIG. 52 shows the blocking effect of probe 19 (i.e., BL1-542) on the variant PIK3CA-E545K gene (example 6);

FIG. 53 shows the blocking effect of probe 19 (i.e., BL1-542) on the variant PIK3CA-E542K gene (example 6);

FIG. 54 is the sequencing result of the PCR product of the wild-type template of PIK3CA-E545K (example 6);

FIG. 55 shows the sequencing results of the PCR products of the PIK3CA-E545K variant template with a variation rate of 0.5% (example 6);

FIG. 56 shows the sequencing results of the PCR products of the PIK3CA-E545K variant template with a variation rate of 0.2% (example 6);

FIG. 57 shows the sequencing results of the PCR products of the PIK3CA-E545K variant template with a variation rate of 0.1% (example 6);

FIG. 58 shows the sequencing results of the PCR products of the wild-type template of PIK3CA-E542K (example 6);

FIG. 59 shows the sequencing results of the PCR products of the PIK3CA-E542K variant template with a variation rate of 0.5% (example 6);

FIG. 60 shows the sequencing results of the PCR products of the PIK3CA-E542K variant template with a variation rate of 0.2% (example 6);

FIG. 61 shows the sequencing results of the PCR products of the PIK3CA-E542K variant template with a variation rate of 0.1% (example 6);

FIG. 62 shows the blocking effect of probe 20 (i.e., BL1-1047) on wild-type PIK3CA gene (example 7);

FIG. 63 shows the blocking effect of probe 20 (i.e., BL1-1047) on the variant PIK3CA-H1047R gene (example 7);

FIG. 64 shows the blocking effect of probe 20 (i.e., BL1-1047) on the variant PIK3CA-H1047L gene (example 7);

FIG. 65 shows the sequencing results of the PCR products of the wild-type template of PIK3CA-H1047R/L (example 7);

FIG. 66 shows the results of sequencing the PCR products of the PIK3CA-H1047R variant template with a variation rate of 0.5% (example 7);

FIG. 67 shows the sequencing results of the PCR products of the PIK3CA-H1047R variant template with a variation rate of 0.2% (example 7);

FIG. 68 shows the sequencing results of the PCR products of the PIK3CA-H1047R variant template with a variation rate of 0.1% (example 7);

FIG. 69 shows the results of sequencing the PCR products of the PIK3CA-H1047L variant template with a variation rate of 0.5% (example 7);

FIG. 70 shows the results of sequencing the PCR products of the PIK3CA-H1047L variant template with a variation rate of 0.2% (example 7);

FIG. 71 shows the results of sequencing the PCR products of the PIK3CA-H1047L variant template with a variation rate of 0.1% (example 7);

FIG. 72 is a graph showing the blocking effect of probe 21 (i.e., BL1-600) on the wild-type Braf-V600E gene (example 8);

FIG. 73 shows the blocking effect of probe 21 (i.e., BL1-600) on the variant Braf-V600E gene (example 8);

FIG. 74 is the sequencing result of the PCR product of the wild-type template of Braf-V600E (example 8);

FIG. 75 shows the sequencing results of the PCR products of the Braf-V600E variant template with a variation rate of 0.5% (example 8);

FIG. 76 shows the sequencing results of the PCR products of the Braf-V600E variant template with a variation rate of 0.2% (example 8);

FIG. 77 shows the sequencing results of the PCR products of the Braf-V600E variant template with a variation rate of 0.1% (example 8);

FIG. 78 shows the blocking effect of BL2-538 on the wild-type ESR1-D538G gene (comparative example);

FIG. 79 shows the blocking effect of BL2-538 on the variant ESR1-D538G gene (comparative example);

FIG. 80 shows the sequencing results of PCR products from ESR1-D538G variant templates with a variation rate of 0.5% (comparative example);

FIG. 81 shows the blocking effect of BL2-542 on the wild-type PIK3CA gene (comparative example);

FIG. 82 shows the blocking effect of BL2-542 on the variant PIK3CA-E542K gene (comparative example);

FIG. 83 is a graph showing the blocking effect of BL2-542 on the variant PIK3CA-E545K gene (comparative example);

FIG. 84 is a graph showing the effect of BL2-1047 in blocking the wild-type PIK3CA-H1047R/L gene (comparative example);

FIG. 85 shows the blocking effect of BL2-1047 on the variant PIK3CA-H1047R gene (comparative example);

FIG. 86 is a graph showing the blocking effect of BL2-1047 on the mutant PIK3CA-H1047L gene (comparative example);

FIG. 87 shows the blocking effect of the probe Blocker BL2-600 on the wild-type Braf-V600E gene (comparative example);

FIG. 88 shows the blocking effect of the probes Block BL2-600 on the variant Braf-V600E gene (comparative example).

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.