CN112592966A - Kit for rapidly detecting EGFR gene drug resistance mutation - Google Patents
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Abstract
The invention relates to a kit for rapidly detecting EGFR gene drug-resistant mutation, which comprises a probe combined premix combined with the T790M locus of EGFR gene, an enzymatic reaction premix and a signal amplification premix; the probe binding premix comprises a modified circular locking nucleic acid probe primer, RecA protein, single-stranded binding protein and a buffer solution for promoting probe binding, wherein the specific recognition is used for detecting the T790M mutation site on the exon 20 of the human EGFR gene; the primer of the ring-locked nucleic acid probe comprises a part capable of being combined with EGFR gene and a non-specific combining part, and the sequence of the primer of the ring-locked nucleic acid probe is shown as SEQ ID NO. 1; the enzymatic reaction premix solution comprises high-fidelity high-temperature ligase, four kinds of random deoxyribonucleotide dNTPs and an enzymatic reaction buffer solution; the signal amplification premix comprises isothermal amplification enzyme with strand displacement activity, primer dNTP for strand displacement isothermal amplification, and double-stranded DNA binding solution
Fluorescent dye and buffer solution for promoting enzyme reaction.
Description
Technical Field
The invention relates to a kit for rapidly detecting EGFR gene drug-resistance mutation, which can rapidly detect EGFR mutation sites within 2 hours, and belongs to the technical fields of common gene drug-resistance site mutation detection, isothermal amplification and the like.
Background
Epidermal Growth Factor Receptor (EGFR), a multifunctional glycoprotein widely distributed on cell membranes of various tissues of the human body, is an avian erythroblastic leukemia virus (v-erb-b) oncogene homolog, and is one of four members of the HER/ErbB family. EGFR and its ligands are part of the cell signaling system, and EGFR gene copy number is increased or overexpressed, both to promote transformation of normal cells and metastasis of malignant tumors. When threonine at the 790 th site of the EGFR20 exon is replaced by methionine, the affinity of ATP and an EGFR-TK binding domain is enhanced, so that EGFR-TKI cannot effectively block a signal pathway to generate drug resistance, and the treatment response effect of drugs such as receptor tyrosine kinase inhibitor EGFR-TKI is greatly reduced.
Currently, the most common methods for detecting EGFR gene mutation are Polymerase Chain Reaction (PCR) and sequencing; although the precision of polymerase chain reaction such as digital microdroplet PCR is high, the instrument is too expensive, the sample processing flow is troublesome, and the reaction time is long, wherein, Real-time fluorescent Real-time PCR (qPCR) also has similar problems; the Amplification and repression Mutation System PCR (Amplification Mutation System PCR, ARMS PCR) has the limitation of precision besides long time; NGS (Next-Generation Sequencing) is a high-throughput deep Sequencing technology developed in recent years, can perform parallel Sequencing on millions to billions of DNA molecules at a time, but is insensitive to rare mutation and structural variation and expensive, has the advantages of high throughput, can detect mutation at multiple sites and modes simultaneously, but consumes too much time and instruments are expensive, and most of the measured data are useless.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a kit for rapidly detecting EGFR gene drug-resistant mutation, which utilizes a designed loop-locked primer and an isothermal amplification signal amplification mechanism to realize a rapid, accurate, low-cost and simple-operation detection method, reduces the diagnosis cost and shortens the diagnosis time.
The technical scheme of the invention is as follows:
a kit for rapidly detecting EGFR gene drug-resistant mutation comprises a probe binding premix combined with the T790M locus of an EGFR gene, an enzymatic reaction premix and a signal amplification premix;
the probe binding premix comprises a modified circular locking nucleic acid probe primer, RecA protein, single-stranded binding protein and a promoting probe binding buffer solution, wherein the modified circular locking nucleic acid probe primer is used for specifically identifying and detecting a T790M mutation site on a No. 20 exon of a human EGFR gene; the primer of the ring-locked nucleic acid probe comprises a part capable of being combined with EGFR gene and a non-specific combining part, and the sequence of the primer of the ring-locked nucleic acid probe is shown as SEQ ID NO. 1;
the enzymatic reaction premix solution comprises high-fidelity high-temperature ligase, four random deoxyribonucleotide dNTPs and an enzymatic reaction buffer solution;
the signal amplification premix comprises isothermal amplification enzyme with strand displacement activity, primer dNTP for strand displacement isothermal amplification and double-stranded DNA (deoxyribonucleic acid) combined
Green fluorescent dye and buffer solution for promoting enzyme reaction activity.
Further, the kit also contains a homozygote positive control substance, a homozygote negative control substance and a standard substance;
the homozygote positive control is a synthetic DNA double-stranded fragment with the concentration of 15 ng/mu L, wherein the synthetic DNA double-stranded fragment is combined with the circular locking nucleic acid probe in the probe-combined premix liquid but does not promote the circular locking probe to form a ring;
the negative control substance is a synthetic DNA double-stranded fragment which is at the concentration of 15 ng/mu L, is combined with the circular locking nucleic acid probe in the probe-combined premix solution and promotes the circular locking probe to form a ring;
the standard substance is a double-stranded DNA fragment for fluorometric detection and consists of two tube components of low-concentration DNA and high-concentration DNA.
Further, the pH of the probe binding promotion buffer in the probe binding premix is 7.5, and the probe binding promotion buffer includes, but is not limited to, Tris-HCl and MgCl2DTT, ATP, EDTA or glycerol.
Further, the pH of the enzymatic reaction buffer in the enzymatic reaction premix is 7.6, and the enzymatic reaction buffer includes but is not limited to one of Tris-HCl, potassium acetate, magnesium acetate, NAD I type coenzyme, DTT, Triton X-100 or glycerol.
Further, the pH of the enzyme reaction promoting buffer solution in the signal amplification premix is 7.5, and the enzyme reaction promoting buffer solution includes, but is not limited to, Tris-HCl and MgCl2、(NH4)2SO4DTT or glycerol.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a kit for rapidly detecting EGFR gene drug-resistant mutation, which utilizes a designed detection method which can specifically identify and detect T790M mutation site on exon 20 of human EGFR gene and is provided with a modified ring-locked probe primer and an isothermal amplification signal amplification mechanism to realize rapidness, accuracy, low cost and simple operation, wherein the ring-locked probe primer comprises a bindable part and a non-specific binding part on EGFR gene, the non-specific binding part is not complementary with any fragment of human genome, and the ring-locked probe primer can be combined with a specific region of EGFR gene of the genome to form a hybrid chain under the belt of RecA (recombination A) protein; in a wild type sample, the ring-locked probe primer cannot be completely complementary with a template, so that ring formation cannot be performed, and when a T790M mutant genotype exists, the ring-locked probe primer is complementary with the template, or is complementary with the template after an enzymatic reaction is completed, and can be further subjected to ending and connecting to form a ring, so that the wild type and the mutant type (T790M) at the 790 th site of the EGFR gene can be distinguished, and the rapid and accurate detection of the drug-resistant mutation of the EGFR gene can be realized.
2. The kit for rapidly detecting the drug-resistant mutation of the EGFR gene provided by the invention can react the whole detection process of the drug-resistant mutation of the EGFR gene at room temperature, and the whole operation time is finished within 2 hours.
Drawings
FIG. 1 is a schematic diagram of a primer sequence and structure of a ring-locked probe in an embodiment of the present invention;
Detailed Description
The invention is further described with reference to the drawings and the preferred embodiments.
Example 1
A kit for rapidly detecting EGFR gene drug-resistant mutation comprises a probe binding premix combined with the T790M locus of an EGFR gene, an enzymatic reaction premix and a signal amplification premix;
the probe binding premix comprises a modified circular locking nucleic acid probe primer, RecA protein, single-stranded binding protein and a promoting probe binding buffer solution, wherein the modified circular locking nucleic acid probe primer is used for specifically identifying and detecting a T790M mutation site on a No. 20 exon of a human EGFR gene; the primer of the ring-locked nucleic acid probe comprises a part capable of being combined with EGFR gene and a non-specific combining part, and the sequence of the primer of the ring-locked nucleic acid probe is shown as SEQ ID NO. 1; GAATGAATGGGTGAAAGAAGATAAGCCTCATGCTTCTTCGGTGCCCATTACGAGCGCGCAGACACGATAGTCTGACTAATACTGCTGAATGAATGAATGAA, respectively;
wherein, a modification group exists between the first position G and the second position A, a modification group exists between the fifth position G and the sixth position A, a modification group exists between the thirteenth position G and the fourteenth position A, a modification group exists between the eighty-fifth position G and the eighty-sixth position T, a modification group exists between the ninety-fifth position G and the ninety-sixth position A, a modification group exists between the ninety-ninth position G and the hundred-first position A, a modification group exists between the hundred-first position A and the hundred-zero position A, and the modification group is not limited to chemical structure information such as phosphate group, incomplete phosphate group, hydroxyl group, incomplete hydroxyl group, mutant base, damaged base or chemical group, abnormal DNA double-strand structure and the like; the unshaded area on the probe represents a part which can be combined with the human gene, and the shaded area represents a part which can not be combined with the human gene;
the pH of the probe binding promoting buffer is 7.5, and the buffer comprises but is not limited to Tris-HCl and MgCl2One of DTT, ATP, EDTA or glycerol;
the enzymatic reaction premix solution comprises high-fidelity high-temperature ligase, four random deoxyribonucleotide dNTPs and an enzymatic reaction buffer solution; the enzymatic reaction buffer has a pH of 7.6 and includes, but is not limited to, one of Tris-HCl, potassium acetate, magnesium acetate, NAD I type coenzyme, DTT, Triton X-100, or glycerol;
the signal amplification premix comprises isothermal amplification enzyme with strand displacement activity, primer dNTP for strand displacement isothermal amplification and double-stranded DNA (deoxyribonucleic acid) combined
Green fluorescent dye and buffer solution for promoting enzyme reaction activity; the pH of the buffer for promoting the enzyme reaction activity is 7.5, and the buffer comprises but is not limited to Tris-HCl and MgCl2、(NH4)2SO4DTT or glycerol.
Further, the kit also contains a homozygote positive control substance, a homozygote negative control substance and a standard substance;
the homozygote positive control is a synthetic DNA double-stranded fragment which has a specific concentration of 15 ng/mu L (can be detected within 100 percent of the detection range), is combined with a ring-locked nucleic acid probe in a probe-combined premix solution, and does not promote the ring formation of the ring-locked probe;
the negative control substance is a synthetic DNA double-stranded fragment which has a specific concentration of 15 ng/mu L (can be detected within 100 percent of the detection range), is combined with the circular locking nucleic acid probe in the probe-combined premix solution and promotes the circular locking probe to form a ring;
the standard substance is a double-stranded DNA fragment for fluorometric detection and consists of two tube components of low-concentration DNA and high-concentration DNA.
The invention also discloses an operation method of the kit for rapidly detecting the drug resistance mutation of the EGFR gene, which comprises the following steps:
(1) and (3) probe binding reaction: adding 30-100ng of purified human genome DNA or control group sample into a clean nuclease-free reaction tube by a pipette, combining with a probe required for one-time reaction respectively to obtain a premixed solution (2 x), reacting at 37 deg.C (metal bath available) for 15min, wherein the human genome DNA is sampled from somatic cells, such as genomic DNA extracted from whole blood leukocytes, oral epithelial cell DNA (collected by oral swab), etc.;
(2) enzymatic reaction: adding enzymatic reaction premixed liquid (2 x) required by one reaction into each reaction tube by using a pipettor in equal volume, uniformly blowing and stirring by using the pipettor for at least 10 times, and reacting for 25min at the temperature of 37 ℃;
(3) signal amplification: diluting the corresponding system according to the dilution times (1:10, 1:100, 1:1000, 1:10000, 1:100000), adding a signal amplification premixed solution (5 x) required by one-time reaction into each reaction tube by using a pipette, and reacting for 2 hours at 37 ℃;
(4) signal detection: life corporation under the Saimer Fei flag
3.0 fluorometer operating instructions, preparing working solution, measuring the fluorescence value of the standard substance, determining the standard curve of the two-point method, reading the fluorescence value of each sample (including a control sample), and judging whether the detection result has T790M mutation on the EGFR gene according to the fluorescence value.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Shanghai Share Biotech Co., Ltd
<120> kit for rapidly detecting EGFR gene drug resistance mutation
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Claims (5)
1. A kit for rapidly detecting EGFR gene drug-resistant mutation is characterized in that: the kit contains a probe binding premix, an enzymatic reaction premix and a signal amplification premix which are bound with the T790M site of the EGFR gene;
the probe binding premix comprises a modified circular locking nucleic acid probe primer, RecA protein, single-stranded binding protein and a promoting probe binding buffer solution, wherein the modified circular locking nucleic acid probe primer is used for specifically identifying and detecting a T790M mutation site on a No. 20 exon of a human EGFR gene; the primer of the ring-locked nucleic acid probe comprises a part capable of being combined with EGFR gene and a non-specific combining part, and the sequence of the primer of the ring-locked nucleic acid probe is shown as SEQ ID NO. 1;
the enzymatic reaction premix solution comprises high-fidelity high-temperature ligase, four random deoxyribonucleotide dNTPs and an enzymatic reaction buffer solution;
the signal amplification premix comprises isothermal amplification enzyme with strand displacement activity, primer dNTP for strand displacement isothermal amplification and double-stranded DNA (deoxyribonucleic acid) combined
Green fluorescent dye and buffer solution for promoting enzyme reaction activity.
2. The kit for rapidly detecting drug-resistant mutation of the EGFR gene according to claim 1, wherein: the kit also contains a homozygote positive reference substance, a homozygote negative reference substance and a standard substance;
the homozygote positive control is a synthetic DNA double-stranded fragment with the concentration of 15 ng/mu L, wherein the synthetic DNA double-stranded fragment is combined with the circular locking nucleic acid probe in the probe-combined premix liquid but does not promote the circular locking probe to form a ring;
the negative control substance is a synthetic DNA double-stranded fragment which is at the concentration of 15 ng/mu L, is combined with the circular locking nucleic acid probe in the probe-combined premix solution and promotes the circular locking probe to form a ring;
the standard substance is a double-stranded DNA fragment for fluorometric detection and consists of two tube components of low-concentration DNA and high-concentration DNA.
3. The kit for rapidly detecting drug-resistant mutation of the EGFR gene according to claim 1, wherein: the pH of the probe binding promoting buffer solution in the probe binding premix is 7.5, and the probe binding promoting buffer solution comprises but is not limited to Tris-HCl and MgCl2DTT, ATP, EDTA or glycerol.
4. The kit for rapidly detecting drug-resistant mutation of the EGFR gene according to claim 1, wherein: the pH of the enzymatic reaction buffer solution in the enzymatic reaction premix is 7.6, and the enzymatic reaction buffer solution comprises but is not limited to one of Tris-HCl, potassium acetate, magnesium acetate, NAD I type coenzyme, DTT, Triton X-100 or glycerol.
5. The kit for rapidly detecting drug-resistant mutation of the EGFR gene according to claim 1, wherein: the pH of the buffer solution for promoting the enzyme reaction activity in the signal amplification premix solution is 7.5, and the buffer solution for promoting the enzyme reaction activity comprises but is not limited to Tris-HCl and MgCl2、(NH4)2SO4DTT or glycerol.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728536A (en) * | 2018-05-25 | 2018-11-02 | 广州永诺生物科技有限公司 | Primer, probe, PCR reaction solution and the kit of Human epidermal growth factor receptor gene T790M site primers |
| CN111206100A (en) * | 2020-02-28 | 2020-05-29 | 宁波胤瑞生物医学仪器有限责任公司 | Kit and method for detecting mutation of T790M site of EGFR gene |
| CN111705120A (en) * | 2019-03-18 | 2020-09-25 | 上海市精神卫生中心（上海市心理咨询培训中心） | Kit and steps for detecting homozygote of human MIF gene CATT repetitive sequence |
| CN112029837A (en) * | 2020-10-13 | 2020-12-04 | 济南国益生物科技有限公司 | Kit for detecting SNP (Single nucleotide polymorphism) sites based on locked nucleic acid modified recombinase-mediated isothermal amplification method and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728536A (en) * | 2018-05-25 | 2018-11-02 | 广州永诺生物科技有限公司 | Primer, probe, PCR reaction solution and the kit of Human epidermal growth factor receptor gene T790M site primers |
| CN111705120A (en) * | 2019-03-18 | 2020-09-25 | 上海市精神卫生中心（上海市心理咨询培训中心） | Kit and steps for detecting homozygote of human MIF gene CATT repetitive sequence |
| CN111206100A (en) * | 2020-02-28 | 2020-05-29 | 宁波胤瑞生物医学仪器有限责任公司 | Kit and method for detecting mutation of T790M site of EGFR gene |
| CN112029837A (en) * | 2020-10-13 | 2020-12-04 | 济南国益生物科技有限公司 | Kit for detecting SNP (Single nucleotide polymorphism) sites based on locked nucleic acid modified recombinase-mediated isothermal amplification method and detection method thereof |
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