CN107354197B

CN107354197B - Kit for detecting human NRAS gene mutation

Info

Publication number: CN107354197B
Application number: CN201710304254.6A
Authority: CN
Inventors: 葛猛; 余倩; 王宏伟
Original assignee: Beijing FAH Biotech Co ltd
Current assignee: BEIJING GENOMEPRECISION TECHNOLOGY Co.,Ltd.
Priority date: 2015-11-25
Filing date: 2017-05-03
Publication date: 2020-10-27
Anticipated expiration: 2037-05-03
Also published as: CN107354196A; CN107354195A; CN107354197A; CN107338240B; WO2017088750A1; CN107354196B; CN107354195B; CN107338240A

Abstract

The invention discloses a kit for detecting human NRAS gene mutation. The kit comprises a component A and/or a component B; the component A comprises a closed sequence A and a primer pair A; the component B comprises a closed sequence B and a primer pair B; the closed sequence A and the closed sequence B are single-stranded DNA molecules, wherein a plurality of nucleotides are subjected to locked nucleic acid modification; the primer pair A consists of an upstream primer F1 and a downstream primer R1; the primer pair B consists of an upstream primer F2 and a downstream primer R2; the nucleotide sequences of the blocking sequence A, the upstream primer F1, the downstream primer R1, the blocking sequence B, the upstream primer F2 and the downstream primer R2 are sequentially shown as a sequence 2, a sequence 3, a sequence 4, a sequence 6, a sequence 7 and a sequence 8 in a sequence table. Experiments show that the kit provided by the invention has high accuracy and high sensitivity when used for detecting human NRAS gene mutation, thereby greatly reducing the false negative rate of detection and having important application value.

Description

Kit for detecting human NRAS gene mutation

Technical Field

The invention relates to the field of molecular biology, in particular to a kit for detecting human NRAS gene mutation.

Background

The RAS gene is a proto-oncogene, a key regulator in cell growth, proliferation and differentiation. The normal RAS protein localizes to the intracellular membrane, has high affinity for GTP and GDP, and has gtpase activity. RAS proteins function as molecular switches that, when expressed normally, regulate cell growth; when point mutations occur, abnormal conditions such as overexpression or gene translocation can lead to abnormal cell proliferation and ultimately to tumor formation. More than 30% of human tumors have mutations in the RAS gene. The RAS gene family includes KRAS, NRAS and HRAS genes. In recent years, abnormalities of RAS gene have been detected in bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, and hematopoietic tumors: KRAS gene mutations are commonly found in pancreatic, lung and colorectal cancer solid tumor cells, HRAS gene mutations are generally more common in bladder cancer tumors, while NRAS gene mutations are often found in hematologic, melanoma and thyroid cancer cells.

NRAS proteins encoded by the NRAS gene have up to 85% homology with other proteins of the RAS family, so functionally NRAS proteins also share many features common to RAS family proteins. The NRAS protein is positioned on the inner side of a cell membrane, belongs to a low molecular weight G protein, has strong affinity to guanylic acid and has GTPASe activity; (ii) an inactive state when the NRAS protein binds to GDP; when bound to GTP, it is in an active state, activating downstream signaling pathways and thus plays an extremely important switching role in signaling. When the NRAS protein is mutated, the mutation can cause abnormal activation of downstream RAF, MAPK and the like, thereby playing an important role in tumor malignant transformation. The NRAS protein mutation mainly occurs at codon 12 and codon 13 of exon 2, and at codon 61 of exon 3, wherein the mutation frequency is highest at codon 12 of exon 2 and codon 61 of exon 3.

Studies have shown that patients with metastatic colorectal cancer do not benefit from anti-EGFR targeting if mutations occur in exons 2 and 3 of the NRAS gene, and the 2017 version of the NCCN clinical practice guidelines for colorectal cancer states that only wild-type patients with NRAS genes are advised to receive treatment with EGFR inhibitors (e.g., cetuximab and panitumumab). Studies by DouillardJY (2013) et al show that other RAS mutations in patients receiving panitumumab in combination with FOLFOX4 can predict therapeutic efficacy, and treatment of metastatic colorectal cancer patients without RAS mutations with panitumumab in combination with FOLFOX4 can improve patient overall survival and progression-free survival.

In recent years, the NRAS gene has been identified as a driver gene of lung cancer development, and NRAS gene mutation has been associated with TKI resistance in the treatment of lung cancer. As compared with Gefitinib-sensitive PC-9 cells (C-9/WT), EGFR-TKIs-resistant genes such as NRAS gene and HER2 gene were not detected in Gefitinib-resistant PC-9 cells, and a mutation at codon 61 of NRAS gene was found. In addition, when the gefitinib or the AZD6244/CI1040 can not promote the apoptosis when being used alone, the gefitinib and the AZD6244/CI1040 can effectively promote the apoptosis when being used together. Therefore, NRAS gene mutation may play an important role in TKI resistance in lung cancer treatment, and provides new basis and possibility for lung cancer detection and treatment.

Melanoma has become the most rapidly growing malignant tumor in recent years, with annual growth rates of about 3% to 5%. The frequency of NRAS gene mutations in melanoma is typically 20-30%. The 2012 results of the anderson cancer center study show that a new drug combination, namely an inhibitor using CDK4 as a drug target, can be used in combination with a MEK inhibitor to treat melanoma patients with NRAS gene mutation. The same year of tumor gene analysis from the PRIME study of ESMO showed: the NRAS gene, NRAS gene and BRAF gene mutation can be used as a biological prediction index of the metastatic colon cancer receiving panitumumab + FOLFOX first-line treatment. In addition, a study of a phase ii clinical trial by Lancet Oncology in 2013 shows that MEK162 is the first effective targeted therapeutic for melanoma patients with NRAS gene variation and may provide a new treatment option for cancer patients with few effective treatment methods. Therefore, detection of human NRAS gene mutation can guide doctors to treat and prognose cancer patients such as melanoma.

In conclusion, the NRAS gene mutation has important significance in the occurrence and development of a plurality of tumors such as human melanoma, colorectal cancer, lung cancer and the like. The detection of the mutation can accurately predict the effectiveness of the corresponding targeted drug treatment, facilitate the selection of clinical medication, obviously improve the treatment effect and enable patients to benefit to the maximum extent; meanwhile, the burden of medical expenses of patients and the waste of social medical resources caused by unreasonable medication can be avoided, and unnecessary aging loss and economic loss are reduced.

Currently, Sanger sequencing method, high-resolution dissolution curve detection method, high performance liquid chromatography, ARMS-PCR method and the like are used as methods for detecting gene mutation. At present, an ARMS-PCR method, namely an amplification mutation system (amplification mutation system ARMS), is commonly used and established in 1989, and the method has high sensitivity and good specificity, but can only be used for detecting known mutant genes. The Sanger sequencing method is a gold standard for mutation detection, and through continuous development and improvement for 30 years, DNA fragments with the length of 1000bp can be sequenced at present, and the reading accuracy of each base is as high as 99.999%. Because of high reading accuracy, the Sanger sequencing method becomes a gold standard for gene analysis such as gene mutation, single nucleotide polymorphism and the like, and can effectively detect unknown mutation sites. In tumor tissues, NRAS wild-type cells and mutant cells are mixed, and the sensitivity of the Sanger sequencing method is only 10-20%, namely the mutant cells can be detected when the mutant cells account for 10-20% of the whole detection cells, so that the application of the Sanger sequencing method is greatly limited. Therefore, it is necessary to establish a sensitive, rapid and effective method for detecting NRAS gene mutation, which can detect multiple mutation sites at one time.

Disclosure of Invention

The technical problem to be solved by the invention is how to detect whether the human NRAS gene is mutated.

In order to solve the technical problems, the invention firstly provides a kit for detecting human NRAS gene mutation.

The kit for detecting human NRAS gene mutation provided by the invention can comprise a component A and/or a component B.

The component A can comprise a closed sequence A and a primer pair A;

the closed sequence A can be a single-stranded DNA molecule, wherein a plurality of nucleotides are subjected to locked nucleic acid modification;

the primer pair A can consist of two primers for amplifying a specific DNA fragment A; the specific DNA fragment A has a target sequence A; the target sequence A can be a target sequence of a primer pair A consisting of an upstream primer F1 and a downstream primer R1 in a human genome;

the upstream primer F1 can be a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 3 of the sequence table;

a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;

the downstream primer R1 can be a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

a4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;

the blocking sequence A and the target sequence A can satisfy the following relations (c1) or (c2) or (c3) or (c4) or (c5) or (c 6):

(c1) more than 50 contiguous nucleotides in one strand of the blocking sequence A and the target sequence A are the same;

(c2) more than 50 consecutive nucleotides in the sense strand of the blocking sequence A and the target sequence A are the same;

(c3) more than 50 consecutive nucleotides in the antisense strand of the blocking sequence A and the target sequence A are the same;

(c4) one strand of the blocking sequence A and the target sequence A is identical;

(c5) the sense strand of the blocking sequence A and the sense strand of the target sequence A are identical;

(c6) the antisense strand of the blocking sequence A and the target sequence A are identical.

The component B can comprise a closed sequence B and a primer pair B;

the closed sequence B can be a single-stranded DNA molecule, wherein a plurality of nucleotides are subjected to locked nucleic acid modification;

the primer pair B can be composed of two primers for amplifying a specific DNA fragment B; the specific DNA fragment B has a target sequence B; the target sequence B can be a target sequence of a primer pair B consisting of an upstream primer F2 and a downstream primer R2 in a human genome;

the upstream primer F2 can be a5) or a6) as follows:

a5) a single-stranded DNA molecule shown in sequence 7 of the sequence table;

a6) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and having the same functions as the sequence 7;

the downstream primer R2 can be a7) or a8) as follows:

a7) a single-stranded DNA molecule shown in sequence 8 of the sequence table;

a8) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 8 and have the same functions as the sequence 8;

the blocking sequence B and the target sequence B can satisfy the following relations (d1) or (d2) or (d3) or (d4) or (d5) or (d 6):

(d1) more than 50 consecutive nucleotides in one strand of the blocking sequence B and the target sequence B are the same;

(d2) more than 50 continuous nucleotides in the sense strand of the blocking sequence B and the target sequence B are the same;

(d3) more than 50 continuous nucleotides in the antisense strand of the blocking sequence B and the target sequence B are the same;

(d4) one strand of the blocking sequence B is identical to that of the target sequence B;

(d5) the sense strand of the blocking sequence B is identical to the sense strand of the target sequence B;

(d6) the antisense strand of the blocking sequence B and the target sequence B are identical.

In the above kit, the phrase "a plurality of nucleotides are modified by locked nucleic acid" can mean that there is one modified locked nucleic acid every 2 to 5 bases (i.e., the modified locked nucleic acid is substantially uniformly distributed), for example, the distribution of the modified locked nucleic acid is such that there is one modified base every 2, 3, 4 or 5 unmodified bases.

In the kit, the length of the blocking sequence A is about 20 to 40 bases shorter than that of the target sequence A, and both ends of the blocking sequence A are respectively overlapped with the upstream primer F1 and the downstream primer R1 by about 3 to 5 bases. The length of the blocking sequence B is about 20-40 bases shorter than that of the target sequence B, and both ends of the blocking sequence B are respectively overlapped with the upstream primer F2 and the downstream primer R2 by about 3-5 bases.

In the kit, the blocking sequence A or the blocking sequence B can be chemically modified. The blocking sequence A comprises at the 3' end chemical modifications that prevent the blocking sequence A from undergoing extension in a PCR reaction, such as phosphorylation modifications, C3-spacer modifications, C6-spacer modifications. The blocking sequence B comprises chemical modifications at the 3' end which prevent the blocking sequence B from extending in the PCR reaction, such as phosphorylation modification, C3-spacer modification, C6-spacer modification.

In the above kit, the blocking sequence a and the target sequence a may specifically satisfy the following relationship: the 69 contiguous nucleotides in one strand of the blocking sequence A and the target sequence A are the same. The blocking sequence B and the target sequence B can specifically satisfy the following relationship: the blocking sequence B and the target sequence B have the same 69 consecutive nucleotides in one strand.

In the kit, the nucleotide sequence of the blocking sequence A can be shown as a sequence 2 in a sequence table.

In the kit, the primer pair A can be composed of the upstream primer F1 and the downstream primer R1.

In the kit, the nucleotide sequence of the blocking sequence B can be shown as a sequence 6 in a sequence table.

In the above kit, the primer pair B may consist of the upstream primer F2 and the downstream primer R2.

The preparation method of any one of the above kits also belongs to the protection scope of the invention. The method for preparing any one of the above kits may comprise the step of separately packaging the blocking sequence A, the forward primer F1, the reverse primer R1, the blocking sequence B, the forward primer F2 and the reverse primer R2.

The application of any one of the above kits in preparing products also belongs to the protection scope of the invention; the function of the product may be b1) or b2) as follows:

b1) identifying or assisting in identifying whether the tissue to be detected is a tumor tissue;

b2) identifying or assisting in identifying whether the test patient is a cancer patient.

In order to solve the technical problems, the invention also provides a method for detecting human NRAS gene mutation.

The method for detecting human NRAS gene mutation provided by the invention can comprise the following steps: carrying out biased amplification on the human NRAS gene by adopting any one of the kits to obtain an amplification product; identifying a mutation in the amplification product.

In the above method, the amplification bias reaction system (hereinafter referred to as amplification bias system) may include the forward primer F1, the reverse primer R1, and the blocking sequence a. The reaction system biased towards amplification can comprise the upstream primer F2, the downstream primer R2 and the blocking sequence B. The reaction system of the biased amplification can be 20 mu L, and consists of 10 mu L of LPremix Ex Taq (2X), 0.5 mu L of the upstream primer F1 with the concentration of 10 mu mol/L, 0.5 mu L of the downstream primer R1 with the concentration of 10 mu mol/L, 0.5 mu L of the blocking sequence A with the concentration of 2.5nM, 1 mu L of human genome DNA to be detected (about 50ng) and 7.5 mu L of nuclease-free water. The reaction system for biased amplification can be 20 μ L, and comprises 10 μ L of Premix Ex Taq (2 ×), 0.5 μ L of the upstream primer F2 with a concentration of 10 μmol/L, 0.5 μ L of the downstream primer R2 with a concentration of 10 μmol/L, 0.5 μ L of the blocking sequence B with a concentration of 2.5nM, 1 μ L of human genome DNA to be detected (about 50ng), and 7.5 μ L of nuclease-free water. The Premix Ex Taq (2X) may be a product of TAKARA, Cat No. RR 390A.

In the above method, the reaction conditions of the biased amplification system may specifically be: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 15s, annealing at 70.5 ℃ for 90s, denaturation at 84 ℃ for 20s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 3 min.

The invention also provides a method for identifying or assisting in identifying whether a tissue to be detected is a tumor tissue or is a candidate for the tumor tissue, which comprises the following steps: the kit is adopted to carry out biased amplification on the tissues to be detected and normal tissues respectively, and then the following judgment is carried out:

if the nucleotide sequence of the amplification product of the tissue to be detected is consistent with that of the amplification product of the normal tissue, the tissue to be detected is not or is not a candidate tumor tissue;

and if the nucleotide sequence of the amplification product of the tissue to be detected is not consistent with that of the amplification product of the normal tissue, determining that the tissue to be detected is or is selected as tumor tissue.

The invention also provides a method for identifying or assisting in identifying whether a patient to be tested is a cancer patient, which comprises the following steps: the kit is adopted to carry out biased amplification on the tissues of a patient to be detected and the tissues of a normal person respectively, and then the following judgment is carried out:

if the amplification product of the tissue of the test patient is identical with the nucleotide sequence of the amplification product of the tissue of the normal person, the test patient is not or is not a candidate for a cancer patient;

and if the nucleotide sequence of the amplification product of the tissue of the patient to be tested is not consistent with the nucleotide sequence of the amplification product of the tissue of the normal person, determining that the patient to be tested is or is selected as a cancer patient.

Any of the above mentioned tumor tissues may specifically be human thyroid cancer tissue.

Any of the above cancers may specifically be human thyroid cancer.

Experiments show that the kit provided by the invention has high accuracy and high sensitivity (the sensitivity can be as low as 0.5% -1%, and the sensitivity of the conventional Sanger sequencing method is only 10% -20%) when being used for detecting human NRAS gene mutation, so that the false negative rate of detection is greatly reduced. Therefore, the kit provided by the invention has important application value.

Drawings

FIG. 1 shows the results of a biased expansion experiment of cell line 1.

FIG. 2 shows the results of a biased expansion experiment on cell line 2.

FIG. 3 shows the results of a biased expansion experiment of cell line 3.

FIG. 4 shows the results of a biased expansion experiment of cell line 4.

FIG. 5 shows the results of experiments on biased expansion of cell line 5.

FIG. 6 shows the results of experiments on biased expansion of cell line 6.

FIG. 7 shows the results of experiments on biased expansion of cell line 7.

FIG. 8 shows the results of experiments on biased expansion of cell line 8.

FIG. 9 shows the results of experiments on biased expansion of cell line 9.

FIG. 10 shows the results of part of the experiment in example 4.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The Light Cycler 480 fluorescent quantitative PCR instrument is a product of Roche. SYBR Premix Ex Taq (2X) and Premix Ex Taq (2X) are both products of TAKARA, having the product catalog numbers RR820A and RR390A in that order.

Example 1 design and Synthesis of blocking sequences and primers

First, design and synthesis of blocking sequence and primer of No. 2 exon of NRAS gene

The No. 2 exon and its peripheral sequence of normal human NRAS gene are shown in sequence 1 in the sequence table.

5’-TGATTATAGAAAGCTTTAAAGTACTGTAGATGTGGCTCGCCAATTAACCCTGATTACTGGTTTCCAACAGGTTCTTGCTGGTGTGAAATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGTGGTGTTGGGAAAAGCGCA CTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGTGAGGCCCAGTGGTAGCCCGCTGACCTGATCCTGTCTCTCACTTGTCGGATCATCTTTACCC-3' (SEQ ID NO: 1 in the sequence Listing)

Based on the mutation positions (codon 12 and codon 13 located in exon 2), a blocking sequence (single-stranded DNA molecule) of exon 2 of the NRAS gene was designed and synthesized. The closed sequence of the 2 nd exon of the NRAS gene is shown as a sequence 2 in a sequence table (the base with an asterisk in italics represents that the base is modified by locked nucleic acid).

5’-AAT^*GACT^*GAG^*TACAAAC^*TG^*GTGGT^*GGTT^*GGAGC^*AGGTGG^*TGT^*TGGG^*AAAAGC^*GCACT^*GACAA^*TCC^*AGCT-3' (SEQ ID NO: 2 in sequence table)

Based on the mutation positions (codon 12 and codon 13 located in exon 2), primer pair A for detecting exon 2 of NRAS gene was designed and synthesized. The primer pair A consists of an upstream primer F1 and a downstream primer R1.

The upstream primer F1: 5'-TTCTTGCTGGTGTGAAATGA-3' (SEQ ID NO: 3 in the sequence Listing).

The downstream primer R1: 5'-CAAAGTGGTTCTGGATTAGCT-3' (SEQ ID NO: 4 in the sequence Listing).

The target sequence of the primer pair A is a DNA molecule (namely, underlined part) (101bp) shown in 72 th to 172 th positions from the 5' tail end of a sequence 1 in a sequence table.

Design and synthesis of blocking sequence and primer of No. 3 exon of NRAS gene

The 3 rd exon and the peripheral sequence of the normal human NRAS gene are shown as a sequence 5 in a sequence table.

5’-GGTTTTTAATAAAAATTGAACTTCCCTCCCTCCCTGCCCCCTTACCCTCCACACCCCCAGGATTCTTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACAAGAAGAGTAC AGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATAGCAAGTCATTTGCGGATATTAACCTCTACAGGTACTAGGAGCA-3' (SEQ ID NO: 5 in the sequence listing).

Based on the mutation position (codon 61 located in exon 3), a blocking sequence (single-stranded DNA molecule) of exon 3 of the NRAS gene was designed and synthesized. The closed sequence of the 3 rd exon of the NRAS gene is shown as a sequence 6 in a sequence table (the base with an asterisk in italics represents that the base is modified by locked nucleic acid).

5’-TGGT^*GAAA^*CCTGT^*TTGT^*TGGA^*CATA^*CTG^*GATAC^*AGCTG^*GACAA^*GAAGA^*GTACA^*GTGCC^*ATGA^*GAG^*ACCA-3' (SEQ ID NO: 6 in sequence Listing)

Based on the mutation position (codon 61 located at exon 3), primer pair B for detecting exon 3 of NRAS gene was designed and synthesized. Primer pair B consists of an upstream primer F2 and a downstream primer R2.

The upstream primer F2: 5'-AACAAGTGGTTATAGATGGTG-3' (SEQ ID NO: 7 in the sequence Listing).

The downstream primer R2: 5'-GCCTTCGCCTGTCCTCATGTA-3' (SEQ ID NO: 8 in the sequence Listing).

The target sequence of the primer pair B is a DNA molecule (namely, underlined part) shown in the 74 th to 180 th positions from the 5' tail end of the sequence 5 in the sequence table (107 bp).

Example 2 method for detecting mutation of human NRAS Gene

The locked nucleic acid has a stronger affinity for its reverse complementary DNA sequence than the native oligonucleotide sequence. Through repeated verification, the number and the positions of locked nucleic acids in the blocking sequence of the No. 2 exon of the NRAS gene and the blocking sequence of the No. 3 exon of the NRAS gene are enabled to be slightly higher than the Tm value of a PCR amplification product due to the fact that the key denaturation temperature Tc (lower than the Tm value between the blocking sequence and the target DNA).

1. Determination of the melting temperature (Tm) of the product

The genome DNA of normal human cells is taken as a template, a primer pair A and a primer pair B are respectively used for amplification on a LightCycler 480 fluorescent quantitative PCR instrument, the reaction system is shown in Table 1, and the reaction conditions are as follows: 2min at 95 ℃; 15s at 95 ℃, 30s at 56 ℃ and 40 cycles; 95 ℃ for 15s, 60 ℃ for 1min, 95 ℃ for 15s, 1 cycle. The final step of PCR is product melting curve analysis: 95 ℃ for 1min, 40 ℃ for 1min, 65-85 ℃.

TABLE 1

Components	Volume of
		SYBR Premix Ex Taq(2×)	10μL
Upstream primer (concentration 10. mu. mol/L)	0.5μL
		Downstream primer (concentration 10. mu. mol/L)	0.5μL
Form panel	1μL(50ng)
		Nuclease-free water	8μL

The result shows that the melting temperature (Tm) of the wild type product of the No. 2 exon of the NRAS gene is 82.5 ℃; the melting temperature (Tm) of the wild type product of exon 3 of the NRAS gene was 82 ℃.

2. Determination of the Critical denaturation temperature (Tc value)

Taking a closed sequence of the No. 2 exon of the NRAS gene as a template, and carrying out amplification on a LightCycler 480 fluorescent quantitative PCR instrument by using an upstream primer F1 and a downstream primer R1, wherein the reaction system is shown in Table 1, and the reaction conditions are as follows: 5min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 70.5 ℃ for 90s, Tx ℃ for 20s, 56 ℃ for 15s, 72 ℃ for 30s, 35 cycles; extension at 72 ℃ for 3 min. Where Tx (the temperature to be varied and fumered), generally measured from high to low, is determined by varying the Tx value and observing the experimental results to determine the key denaturation temperature (Tc value) in the biased amplification system: the initial Tx value is preferably 1-2 ℃ higher than the Tm value, if PCR amplification products can be amplified, the Tx value is further reduced by 0.5-l ℃ each time until the Tx value is reduced to the point that a fluorescent signal cannot be detected (namely, PCR amplification products cannot be amplified), and 1 temperature gradient on the Tx value is biased to the Tc value of an amplification system or the PCR amplification products.

The Tx value of this experiment started at 86 ℃. When the Tx value is set to 86 ℃, PCR amplification products can be amplified; further reducing Tx value to 85.5 ℃, 85 ℃, 84.5 ℃ and 84 ℃, and still amplifying PCR amplification products; however, when the Tx value is reduced to 83.5 ℃, PCR amplification products cannot be amplified. Thus, the critical denaturation temperature for the biased amplification system of exon 2 of the NRAS gene is 84 ℃.

According to the method, the blocking sequence of the 2 nd exon of the NRAS gene is replaced by the blocking sequence of the 3 rd exon of the NRAS gene, the upstream primer F1 is replaced by the upstream primer F2, the downstream primer R1 is replaced by the upstream primer R2, and other steps are not changed, so that the key denaturation temperature of the biased amplification system of the 3 rd exon of the NRAS gene is 84 ℃.

In summary, the specific steps for detecting human NRAS gene mutation are as follows: taking the genome DNA of a patient to be detected as a template, and performing biased amplification by adopting a primer pair A or a primer pair B (the PCR reaction system of the primer pair A is shown in table 2, and the PCR reaction system of the primer pair B is shown in table 3) to obtain a PCR amplification product; and sequencing the PCR amplification product so as to judge whether the NRAS gene of the patient to be detected is mutated.

Reaction conditions biased toward amplification system: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 15s, annealing at 70.5 ℃ for 90s, denaturation at 84 ℃ for 20s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 3 min.

TABLE 2

TABLE 3

Example 3 sensitivity test

The NRAS gene of human thyroid cancer WRO cell line was subjected to point mutation to obtain 9 cell lines containing different constitutive mutations of exon 2 and exon 3 of the NRAS gene. Details of the 9 cell lines are shown in Table 4.

TABLE 4

The sensitivity experiment is carried out by adopting the 9 cell lines, and the specific steps are as follows:

1. the culture method of the 9 cell lines was established, and the whole genomic DNA of the 8 cell lines was extracted.

2. The mixed DNA is obtained by mixing the whole genome DNA of 8 cell lines with the whole genome DNA of normal human cells according to a certain ratio (1: 5, 1:10, 1:20, 1:100 or 1:200) by a gradient dilution method.

3. Performing biased amplification by using a primer pair A or a primer pair B by using the mixed DNA as a template (a PCR reaction system with a mutation region of a No. 2 exon is shown in Table 5, and a PCR reaction system with a mutation region of a No. 3 exon is shown in Table 6) to obtain a PCR amplification product; then Sanger sequencing was performed on the PCR amplification products by Beijing catalpi-xi Bio Inc., and the sensitivity was calculated.

TABLE 5

TABLE 6

The experimental results are shown in fig. 1 to 9. The result shows that the ratio of the lowest mutation DNA of the cell line 1, the cell line 2, the cell line 3, the cell line 4, the cell line 6 and the cell line 7 detected by the method provided by the invention is 1:200 (0.5%), which is 20 times higher than that of the traditional sanger sequencing method; the method provided by the invention detects that the lowest mutation DNA proportion of the cell line 5, the cell line 8 and the cell line 9 is 1:100 (1%), which is 10 times higher than that of the traditional sanger sequencing method.

Example 4 validation of clinical samples

1. Template preparation

Paraffin-embedded tissue section samples of 112 thyroid cancer patients (all patients with thyroid cancer informed consent) were collected, and genomic DNA was extracted (concentration and purity of genomic DNA were determined, requiring concentration of more than 10 ng/. mu.L; OD_260nm/OD_280nmBetween 1.8-2.0).

2. Respectively taking the genomic DNA extracted in the step 1 as a template, and performing biased amplification by using a primer pair A or a primer pair B (the PCR reaction system by using the primer pair A is shown in table 2, and the PCR reaction system by using the primer pair B is shown in table 3) to obtain a PCR amplification product; and sequencing the PCR amplification product so as to judge whether the NRAS gene of the patient to be detected is mutated.

The results of part of the experiment are shown in FIG. 10(A is the result of detection of the mutation type Q61K, B is the result of detection of the mutation type Q61R, C is the result of detection of the mutation type G12D, and D is the result of detection of the mutation type G13D) and Table 7. The results showed that, among 112 thyroid cancer patients, 3 patients had mutations in exon 2 of the NRAS gene, wherein the mutation region was 2 of codon 12 and 1 of codon 13, and 6 patients had mutations in exon 3 of the NRAS gene, with a total detection rate of 8%.

TABLE 7

NRAS genes of 112 thyroid cancer patients were detected by ARMS-PCR. The test result of the ARMS-PCR method is completely consistent with the test result of the method provided by the invention.

Therefore, the method provided by the invention is used for detecting whether the NRAS gene of the patient to be detected is mutated or not, has high accuracy and has important application value.

<110> Beijing FuanHua Biotech Co., Ltd

<120> a kit for detecting human NRAS gene mutation

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<223>

<400>3

ttcttgctgg tgtgaaatga 20

<210>4

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>4

caaagtggtt ctggattagc t 21

<210>5

<211>251

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

ggtttttaat aaaaattgaa cttccctccc tccctgcccc cttaccctcc acacccccag 60

gattcttaca gaaaacaagt ggttatagat ggtgaaacct gtttgttgga catactggat 120

acagctggac aagaagagta cagtgccatg agagaccaat acatgaggac aggcgaaggc 180

ttcctctgtg tatttgccat caataatagc aagtcatttg cggatattaa cctctacagg 240

tactaggagc a 251

<210>6

<211>69

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>6

tggtgaaacc tgtttgttgg acatactgga tacagctgga caagaagagt acagtgccat 60

gagagacca 69

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>7

aacaagtggt tatagatggt g 21

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>8

gccttcgcct gtcctcatgt a 21

Claims

1. A kit for detecting human NRAS gene mutation comprises a component A and/or a component B;

the component A comprises a closed sequence A and a primer pair A;

the closed sequence A is a single-stranded DNA molecule, wherein a plurality of nucleotides are subjected to locked nucleic acid modification;

the nucleotide sequence of the closed sequence A is shown as a sequence 2 in a sequence table;

the plurality of nucleotides are subjected to locked nucleic acid modification, namely, 4 th, 8 th, 11 th, 18 th, 20 th, 25 th, 29 th, 34 th, 40 th, 43 th, 47 th, 53 th, 58 th, 63 th and 66 th bases of the nucleotide sequence shown in the sequence 2 are subjected to locked nucleic acid modification from the 5' end;

the primer pair A consists of two primers for amplifying a specific DNA fragment A; the specific DNA fragment A has a target sequence A; the target sequence A is a target sequence of a primer pair A consisting of an upstream primer F1 and a downstream primer R1 in a human genome;

the upstream primer F1 is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

the downstream primer R1 is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

the component B comprises a closed sequence B and a primer pair B;

the closed sequence B is a single-stranded DNA molecule, wherein a plurality of nucleotides are subjected to locked nucleic acid modification;

the nucleotide sequence of the closed sequence B is shown as a sequence 6 in the sequence table; the plurality of nucleotides are subjected to locked nucleic acid modification, namely 5 th, 9 th, 14 th, 18 th, 22 th, 26 th, 29 th, 34 th, 39 th, 44 th, 49 th, 54 th, 59 th, 63 th and 66 th bases of the nucleotide sequence shown in the sequence 6 are subjected to locked nucleic acid modification from the 5' end;

the primer pair B consists of two primers for amplifying a specific DNA fragment B; the specific DNA fragment B has a target sequence B; the target sequence B is a target sequence of a primer pair B consisting of an upstream primer F2 and a downstream primer R2 in the human genome;

the upstream primer F2 is a single-stranded DNA molecule shown in a sequence 7 in a sequence table;

the downstream primer R2 is a single-stranded DNA molecule shown in a sequence 8 of a sequence table.

2. The kit of claim 1, wherein: the primer pair A consists of the upstream primer F1 and the downstream primer R1.

3. The kit of claim 1, wherein: the primer pair B consists of the upstream primer F2 and the downstream primer R2.

4. The method for preparing the kit according to any one of claims 1 to 3, comprising the step of packaging the blocking sequence A, the forward primer F1, the reverse primer R1, the blocking sequence B, the forward primer F2 and the reverse primer R2 in the kit according to claims 1 to 3 separately.

5. Use of a kit according to any one of claims 1 to 3 in the manufacture of a product; the function of the product is b1) or b2) as follows:

6. A method of identifying or aiding in identifying whether a test tissue is or is a candidate for a neoplastic tissue, comprising the steps of: the kit according to any one of claims 1 to 3 is used for performing biased amplification on a tissue to be detected and a normal tissue respectively, and then the following judgment is performed:

if the nucleotide sequence of the amplification product of the tissue to be detected is not consistent with that of the amplification product of the normal tissue, the tissue to be detected is or is selected as a tumor tissue;

the methods are non-disease diagnostic and therapeutic methods.