CN107937525B - NRAS mutation detection kit and extension primer based on liquid chip method - Google Patents
NRAS mutation detection kit and extension primer based on liquid chip method Download PDFInfo
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Abstract
The invention discloses a NRAS gene detection specific primer and a liquid chip, wherein the liquid chip mainly comprises: respectively designing ASPE extension primers aiming at 18 mutant gene types and wild gene types in codons 12 and 13 on exon2, codon 61 on exon3, codon 146 on exon4 of the NRAS gene, and magnetic beads which are coated by anti-tag sequences and have different color codes; and (3) a primer for amplifying a target sequence which needs to be detected and has a corresponding mutation site. The NRAS gene mutation detection liquid chip can be used for detecting 18 mutation types of NRAS genes, has more comprehensive detection sites, provides more reliable results, and provides more accurate diagnosis basis for doctors.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to an NRAS gene mutation detection kit and an extension primer, which can detect NRAS gene mutation related to cancer by using a liquid chip technology.
Background
Ras gene is a protooncogene, mainly activates kinases controlling gene transcription, regulates cell proliferation and differentiation, and is an important downstream regulatory gene in an EGFR signal transduction pathway in a cell signal transmission path. In normal cells, RAS exists in an inactive state (GDP-bound), and when stimulated by an exogenous signal, RAS is phosphorylated, conformationally altered to become activated GTP-bound, and activated GTP-RAS activates downstream RAF signaling proteins until degraded. The oncogenic point mutation of the RAS gene can reduce the capability of hydrolyzing GTP into GDP of the RAS protein, so that the RAS protein and the GTP are continuously combined, a downstream pathway is continuously activated, cells are subjected to malignant proliferation independent of growth factors, and cancer is caused.
Neuroblastoma murine sarcoma oncogene (NRAS) is one of the Ras gene family members. Mutations of the NRAS gene are mainly located in exons 2, 3 and 4, resulting in various malignant tumors, with a mutation rate of 1-6% in colorectal cancer (CRC) patients and a mutation probability of 20% in malignant melanoma (malignantmelanoma), which are common oncogenes in human tumors. Clinical studies find that NRAS-mutated colorectal cancer patients have significantly reduced sensitivity to EGFR monoclonal antibody drugs mainly comprising cetuximab, so NRAS gene mutation is an independent prediction factor of the treatment effect of the anti-epidermal growth factor receptor monoclonal antibody. NRAS gene detection is listed as a routine examination which must be done before the internal medicine treatment of colorectal cancer patients in European and American areas, and NCCN colon cancer clinical practice guideline and NCCN rectal cancer clinical practice guideline of the United states cancer comprehensive network (NCCN) are listed as necessary examination items of clinical medicines. In addition, there are related studies that indicate that NRAS mutations are associated with TKI resistance in the treatment of lung cancer. There are also clinical data indicating that inhibitors targeting Cdk4 in combination with MEK inhibitors can treat melanoma patients with NRAS mutations. In conclusion, there is increasing evidence that NRAS mutations are of great significance in the development of many tumors, such as human melanoma, colorectal cancer, and lung cancer. Therefore, in the process of treating related cancers, whether the patients have related functional mutation of the NRAS gene or not is detected, the effectiveness of molecular targeted drug treatment can be accurately predicted, accurate clinical medication is facilitated, the curative effect of the drug is obviously improved, the patients can benefit to the maximum extent, and the burden of medical expenses of the patients and the waste of social medical resources caused by unreasonable medication can be avoided.
At present, the kit aiming at the detection of human NRAS gene mutation is developed based on a fluorescent PCR technology platform, and the detection of most mutation types is only 9. Although both the fluorescent PCR and the liquid chip technology have the characteristics of high flux, high sensitivity and the like, the method has the advantages of shortening the detection period, reducing the cost, improving the efficiency and the like. Before PCR, the inventors developed specific primers and liquid phase chips (ZL2011102697755) for detecting NRAS gene mutation, and the detected sites of the primers comprise 13 gene mutation types on the No.2 exon, the No.3 exon and the No.4 exon of the NRAS gene. In practical clinical application, the detection site is found to be incapable of meeting the requirement of single-tube comprehensive detection and analysis.
Disclosure of Invention
One of the purposes of the present invention is to provide a liquid chip for detecting NRAS gene mutation, which can be used to detect 18 types of gene mutations, i.e., G34A, G34T, G34C, G35A, G35C, G35T, G37C, G37T, G38A, G38T, G38C, C181A, a182C, a182T, a182G, a183T, G436A, and G437T, on exon2, exon3, and exon4 of NRAS gene, so as to provide a more comprehensive, accurate, and convenient detection method, overcome the disadvantages of single reaction, single detection site, low sensitivity evidence, and high detection cost of the current single tube detection, and provide a powerful clinical treatment.
The technical scheme for achieving the purpose is as follows.
A liquid chip kit for NRAS gene mutation detection comprises:
(A) designing ASPE extension primers aiming at 18 mutant gene types and wild gene types in No.12 codon, No.13 codon, No.3 codon, No. 146 codon on No.4 exon on the No.2 exon of the NRAS gene respectively, wherein each ASPE extension primer consists of a tag sequence at the 5 'end and a specific primer sequence aiming at a mutant site at the 3' end, and the specific primer sequence of the mutant gene type is selected from at least one of the following sequences: SEQ ID NO.22 for position G34A, SEQ ID NO.23 for position G34T, SEQ ID NO.24 for position G34C, SEQ ID NO.25 for position G35A, SEQ ID NO.26 for position G35C, SEQ ID NO.27 for position G35T, SEQ ID NO.28 for position G37C, SEQ ID NO.29 for position G37T, SEQ ID NO.30 for position G38A, SEQ ID NO.31 for position G38T, SEQ ID NO.32 for position G38C, SEQ ID NO.33 for position C181A, SEQ ID NO.34 for position A182C, SEQ ID NO.35 for position A182T, SEQ ID NO.36 for position A182 48, SEQ ID NO.37 for position A T, SEQ ID NO.37 for position G A, SEQ ID NO. 437, SEQ ID NO.2 for position G35 59638, SEQ ID NO. 59639;
the ASPE extension primer of the wild genotype is selected from the corresponding at least one of: SEQ ID NO.40 for exon2, SEQ ID NO.41 for exon3, and SEQ ID NO.73 for exon 4;
(B) magnetic beads coated with anti-tag sequences and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequences and the magnetic beads; the anti-tag sequence is selected from SEQ ID NO. 42-SEQ ID NO.62, and the anti-tag sequence can be completely complementary and paired with the tag sequence selected in the step (A) correspondingly;
(C) primers for amplifying a target sequence to be detected having a corresponding mutation site.
In one embodiment, the amplification primers are at least one pair selected from: SEQ ID NO.63 and SEQ ID NO.64 for exon2, SEQ ID NO.65 and SEQ ID NO.66 for exon3, and SEQ ID NO.67 and SEQ ID NO.68 for exon 4.
In one embodiment, the corresponding ASPE extension primers, the magnetic beads and the amplification primers with the 18 mutation sites as described in claim 1 are included.
In one embodiment, the spacer arm is 5-10T.
Another objective of the invention is to provide an ASPE extension primer for NRAS gene mutation detection.
The technical scheme for achieving the purpose is as follows.
ASPE extension primers for an NRAS gene mutation detection solution are designed aiming at 18 mutant gene types and wild gene types in No.12 codons and No.13 codons on No.2 exons, No. 61 codons on No.3 exons and No. 146 codons on No.4 exons of NRAS genes respectively, each ASPE extension primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a mutant site, and the specific primer sequence of the mutant gene type is selected from at least one of the following sequences: SEQ ID NO.22 for position G34A, SEQ ID NO.23 for position G34T, SEQ ID NO.24 for position G34C, SEQ ID NO.25 for position G35A, SEQ ID NO.26 for position G35C, SEQ ID NO.27 for position G35T, SEQ ID NO.28 for position G37C, SEQ ID NO.29 for position G37T, SEQ ID NO.30 for position G38A, SEQ ID NO.31 for position G38T, SEQ ID NO.32 for position G38C, SEQ ID NO.33 for position C181A, SEQ ID NO.34 for position A182C, SEQ ID NO.35 for position A182T, SEQ ID NO.36 for position A182 48, SEQ ID NO.37 for position A T, SEQ ID NO.37 for position G A, SEQ ID NO. 437, SEQ ID NO.2 for position G35 59638;
the ASPE extension primer of the wild genotype is selected from the corresponding at least one of: SEQ ID NO.40 for exon2, SEQ ID NO.41 for exon3, and SEQ ID NO.73 for exon 4;
the tag sequence is selected from: SEQ ID NO.1 to SEQ ID NO. 21.
In one embodiment, the ASPE extension primer of a mutant gene type is selected from at least one of: the sequence consisting of SEQ ID NO.1 and SEQ ID NO.22 for position G34A, the sequence consisting of SEQ ID NO.2 and SEQ ID NO.23 for position G34T, the sequence consisting of SEQ ID NO.3 and SEQ ID NO.24 for position G34C, the sequence consisting of SEQ ID NO.4 and SEQ ID NO.25 for position G35A, the sequence consisting of SEQ ID NO.5 and SEQ ID NO.26 for position G35C, the sequence consisting of SEQ ID NO.6 and SEQ ID NO.27 for position G35T, the sequence consisting of SEQ ID NO.7 and SEQ ID NO.28 for position G37C, the sequence consisting of SEQ ID NO.8 and SEQ ID NO.29 for position G37T, the sequence consisting of SEQ ID NO.9 and SEQ ID NO.30 for position G38A, the sequence consisting of SEQ ID NO.10 and SEQ ID NO.31 for position SEQ ID NO.38, the sequence consisting of SEQ ID NO.31 and SEQ ID NO.31 for position G34, the sequence consisting of SEQ ID NO.12 and SEQ ID NO.33 for the C181A site, the sequence consisting of SEQ ID NO.13 and SEQ ID NO.34 for the A182C site, the sequence consisting of SEQ ID NO.14 and SEQ ID NO.35 for the A182T site, the sequence consisting of SEQ ID NO.15 and SEQ ID NO.36 for the A182G site, the sequence consisting of SEQ ID NO.16 and SEQ ID NO.37 for the A183T site, the sequence consisting of SEQ ID NO.17 and SEQ ID NO.38 for the G436A site, the sequence consisting of SEQ ID NO.18 and SEQ ID NO.39 for the G437T site;
wild type the ASPE extension primer is selected from the corresponding at least one of: the sequence consisting of SEQ ID NO.19 and SEQ ID NO.40 for exon2, the sequence consisting of SEQ ID NO.20 and SEQ ID NO.41 for exon3, and the sequence consisting of SEQ ID NO.21 and SEQ ID NO.73 for exon 4.
The NRAS gene mutation detection liquid chip mainly comprises the following advantages:
1 to meet the needs of clinical testing, new site tests were developed. Based on the accumulated experience, the inventor designs a proper ASPE extension primer for 18 sites of concurrent mutation detection, so that the specificity and the extension efficiency of primer extension are improved, and non-specific extension is reduced. Meanwhile, a proper tag sequence is selected, so that the cross reaction between the probe primer and the anti-tag sequence is reduced and avoided as much as possible; and the PCR amplification primer with reasonable design can efficiently amplify the segment to be detected, avoid non-specific amplification and the stability of a detection system.
2. In the liquid phase chip technology, magnetic beads which can be suitable for MAGPIX and luminex instruments are selected to replace microspheres which can only be detected in the luminex instrument before, and corresponding technical consideration is carried out when primers and probes are designed for the microspheres.
3. The NRAS gene mutation detection liquid chip can be used for detecting 18 mutation types of NRAS genes, has more comprehensive detection sites, provides more reliable results, and provides more accurate diagnosis basis for doctors.
Detailed Description
Example 1
An NRAS liquid chip mainly comprises:
1ASPE primer
The ASPE specific extension primers are designed aiming at the mutation gene types and the wild gene types in the No.12 codon and the No.13 codon on the No.2 exon, the No. 61 codon on the No.3 exon and the No. 146 codon on the No.4 exon of the NRAS gene respectively. The ASPE primer sequence comprises two parts including a sequence TAG TAG sequence and a specific extension primer. The primer sequence table is as follows:
table one: NRAS gene mutation ASPE primer sequence
Tag sequence marked by epidiasPE primer and anti-tag sequence information coated by magnetic beads
2: anti-tag coated magnetic beads
According to tag sequences selected by the designed ASPE specific primers, anti-tag sequences complementary to the tag sequences are selected to be coated on the surfaces of the magnetic beads, and the labels of the selected magnetic beads, the corresponding anti-tag sequences on the magnetic beads and the numbers of the anti-tag sequences are shown in Table 2.
21 magnetic beads selected from the above table, the anti-tag sequence was coated on the surface, and 5-10T spacer sequences were attached between them. The spacer arm is used for spacing the anti-tag sequence from the surface of the magnetic bead and also ensuring that the anti-tag sequence is placed in a hydrophilic environment. In addition, the spacer arm sequence can reduce steric hindrance and improve the efficiency and specificity of hybridization reaction.
3: primer for amplifying to-be-detected site
The No.12 codon and the No.13 codon of the NRAS gene are both positioned on the No.2 EXON of the NRAS gene, so that primers can be designed at two ends aiming at the No.12 codon, the No.13 codon and a wild genotype, three genotypes of the No.2 EXON can be amplified, and the primer is marked as EXON2(NRAS-P1-F, NRAS-P1-R). Codon 61 of the NRAS gene is located on EXON3, and the PCR amplification primers designed for the wild type and mutant type of EXON3 are labeled EXON3(NRAS-P2-F, NRAS-P3-R). The PCR amplification primers designed for the wild type and mutant type of the NRAS gene at EXON4 of EXON 146 were labeled EXON4(NRAS-P3-F, NRAS-P4-R). The sequence information of the primers is as follows:
TABLE III NARS gene amplification primer sequence table
All primers were synthesized by Shanghai Biotechnology engineering services, Inc. Each primer after synthesis was prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer.
Example 2
The NRAS liquid phase chip is used for detecting samples
Firstly, DNA extraction of a sample:
the DNA to be detected is obtained by referring to the related method of DNA extraction in molecular cloning.
Second, PCR amplification of the sample to be tested
Three pairs of primer sequences are designed aiming at the amplification of the NRAS gene type, the sizes of amplification products are 198bp, 169bp and 161bp respectively aiming at the No.2 exon, the No.3 exon and the No.4 exon of the NRAS gene, and specific primer sequence information is shown in the table III. Firstly, preparing NRAS amplification primer mixed liquor: respectively taking 100ul of 3 pairs of primer stock solutions, adding the primer stock solutions into a 1.5ml centrifuge tube, and uniformly mixing to obtain the NRAS amplification primer mixed solution. The multiplex PCR reaction system is as follows:
PCR reaction system | Per reaction (ul) |
NF water | 20.6 |
5×Colorless GoTaq Flexi Buffer | 10 |
25mM MgCl2 | 7 |
NRAS amplification primer mixture | 6 |
2.5mM dNTP | 4 |
GoTaq Hot Start polymerase(5U/μl) | 0.4 |
Sample (I) | 2 |
Total volume | 50 |
The PCR reaction conditions were as follows: 3min at 95 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 40 s; 10min at 72 ℃; storing at 4 deg.C for use.
Thirdly, enzyme digestion treatment of PCR product
1. Taking 7.5ul of the product after PCR reaction, adding 1ul of 10 XSAP buffer solution, 1ul of SAP enzyme and 0.5ul of Exo-I enzyme;
incubate at 2.37 ℃ for 15min, incubate at 80 ℃ for 15min, inactivate excess enzyme. The product after enzyme digestion is directly used for the subsequent ASPE primer extension reaction.
Site-specific primer extension reaction (ASPE)
The primer extension reaction is carried out by using the ASPE primer designed above, and the biotin-labeled dCTP is incorporated during the reaction, so that the product after the reaction is labeled with a plurality of biotin.
Firstly, preparing mixed ASPE primer working solution: respectively taking the corresponding wild type and mutant ASPE of the gene to be detected
Adding 10ul of primer stock solution into a 1.5ml microcentrifuge tube, adding 10mmol/L Tris Buffer to supplement to 200ul, and uniformly mixing to obtain the ASPE mixed primer working solution. The system for the ASPE reaction is as follows:
ASPE reaction system | Per reaction (ul) |
NF water | 1.55 |
NRAS extension primer mixture | 5 |
10 XPCR Buffer (Mg-free)2+) | 2 |
25mM MgCl2 | 1 |
Biotin-dCTP(400uM) | 0.25 |
TspDNA polymerase | 0.2 |
EXO/SAP product | 10 |
Total volume | 20 |
The reaction procedure is as follows: 2min at 96 ℃; 30 cycles of 94 ℃ for 30s, 54 ℃ for 1min, 72 ℃ for 2 min; storing at 4 deg.C for use.
Fifthly, hybridization reaction
1. According to the designed ASPE primers, each group selects 21 kinds of corresponding coated magnetic beads (as described in example 1), each magnetic bead concentration is 500 per reaction, and a magnetic bead mixed solution is prepared
2. The magnetic bead mixed liquid is swirled for 30s and mixed evenly;
3. taking 45ul of the above magnetic bead mixed solution, placing in the corresponding hole of 96-hole filter plate
4. 5ul of ASPE reaction solution is put into the corresponding hole and evenly mixed by a pipette
5, wrapping a 96-well plate by using tin foil paper to avoid light, and incubating and hybridizing at 95 ℃ for 60s and 37 ℃ for 15 min;
6 the hybridized 96-well plate is placed on a magnetic plate and stands for 1min, the liquid in the reaction hole is poured out in a turning way, and the 96-well plate is tightly attached to the magnetic plate in the process
7 resuspend the beads in 75ul of 1 XTM hybridization buffer;
placing the 8.96-well plate on a magnetic plate, standing for 2min, turning over to pour out liquid in the reaction well, and allowing the 96-well plate to cling to the magnetic plate in the process
9. Taking the 96-well plate off the magnetic plate, adding 75ul of 1 XTm hybridization buffer solution into the reaction hole to resuspend the magnetic beads, and adding 15ul of streptavidin-phycoerythrin (SA-PE) with the concentration of 10 ug/ml;
incubate at 12.37 ℃ for 15min, and detect on Luminex/MAGPIX instrument.
Sixthly, result detection and data analysis
And detecting the product after reaction by a Luminex series analytical instrument. And taking the mutant fluorescence value (MFI) of more than 100 as a cut-off value, judging that the sample has the mutant type when the MFI value detected by the mutant is more than 100, and otherwise, judging that the sample is the corresponding wild type.
The invention detects the cross reaction rate between genes of the ASPE primer for NRAS gene mutation detection, and the experimental result shows (see table four and table five); the ASPE primers aiming at the specific extended NRAS gene types (wild type and various mutation types) can specifically amplify the corresponding detection types, and the amplification efficiency is high. And for other mutation types, non-specific extension can not be carried out, and even if partial non-specific amplification exists, the cross amplification reaction rate among genes is lower than 3%, so that the ASPE primers in the research have high specificity and the detection result is more reliable.
This example was analyzed for 18 samples and the results showed (see tables six and seven): the coincidence rate of the NRAS genotype detection result and the sequencing result of 18 detected samples reaches 100%. Therefore, the NRAS gene mutation detection liquid chip provided by the invention can accurately detect the mutation type of the NRAS gene, and the result is stable and reliable.
TABLE four ASPE primer Cross-reaction MFI values
TABLE V ASPE primer Cross-reactivity
Table six NRAS test sample results
TABLE seven samples NRAS genotype results
Sample number | Liquid phase chip detection result | Sequencing results |
1 | A182G | A182G |
2 | Wild type | Wild type |
3 | G34C | G34C |
4 | Wild type | Wild type |
5 | Wild type | Wild type |
6 | G34A | G34A |
7 | Wild type | Wild type |
8 | Wild type | Wild type |
9 | Wild type | Wild type |
10 | Wild type | Wild type |
11 | Wild type | Wild type |
12 | Wild type | Wild type |
13 | Wild type | Wild type |
14 | Wild type | Wild type |
15 | Wild type | Wild type |
16 | Wild type | Wild type |
17 | Wild type | Wild type |
18 | Wild type | Wild type |
EXAMPLE 3 selection of primer and Probe sequences specific for detection of polymorphisms in the NRAS Gene
Firstly, the method comprises the following steps: preparation of liquid chip (ASPE extension primer screening and tag sequence screening)
Taking the common gene mutation site G34A of codon 12 on exon2 of NRAS gene mutation as an example, designing specific primer sequences respectively, and designing specific probe sequences aiming at wild types and mutant types of the sites respectively by taking a forward complementary sequence or a reverse complementary sequence of a target sequence where the mutation site is located as a template, wherein the specific probe sequences comprise the preferred specific primer and probe sequences in the embodiment 1 of the invention and 2 (or more than 2) alternative specific primer and probe sequences, and the sequences are shown in tables 8 and 9.
TABLE 8 screening List of primer sequences at position G12S
TABLE 9 screening of site-specific probes in G12S sequence listing
II, secondly: sample testing
The liquid phase chip prepared above was used to test sample nos. 19-40 according to the test procedures and methods described in example 2, with the following test results:
table 10: analysis of screening results of G12S site primer sequence
According to the experimental result, the elongation efficiency of the mutant ASPE extension primer-SEQ ID NO.22 at the G12S site and the elongation efficiency of the wild ASPE extension primer-SEQ ID NO.40 are the highest.
Table 11: analysis of screening results of G12S site Probe
According to the experimental result, the TAG label sequences SEQ ID NO.1 and SEQ ID NO.2 have the best effect when being respectively used as TAG sequence labels of G12S site mutant ASPE extension primer-SEQ ID NO.22 and wild type ASPE extension primer SEQ ID NO. 40.
In conclusion, SEQ ID NO.22 and SEQ ID NO.40 were selected as the G12S site mutant ASPE extension primers, and SEQ ID NO.1 and SEQ ID NO.2 were selected as the TAG TAG sequences, respectively. The same method is adopted to select primers and tag sequences of other sites, and specific experimental data is omitted.
Example 4 selection of magnetic beads or microspheres for detection of polymorphisms of the NRAS Gene
The liquid phase chip technology utilizes fluorescent coded microspheres or magnetic beads to covalently cross-link anti-tag sequences, and the sequences can be specifically complementary with tag sequences at the 5' ends of allele specific extension primers. After the ASPE primer is combined with the determined target molecule, the target gene is specifically extended to form an extension product containing a tag sequence, then the tag sequence on the ASPE primer is specifically combined with an anti-tag sequence on a magnetic bead or a microsphere through hybridization reaction, finally, the fluorescence value of each sample is analyzed by using an instrument luminex (both the magnetic bead and the microsphere can be read) or MAGPX (only the signal of the magnetic bead can be read), and the mutation condition of the target gene in the sample is judged according to the reading height of the fluorescence value. The detection of NRAS gene mutation by the liquid phase chip technology is carried out by microspheres, and data can only be read by a luminex instrument. According to the research, the magnetic beads are used for replacing microspheres, the magnetism of the magnetic beads is utilized to increase the plate washing process after hybridization reaction, and the fluorescence signal value is greatly improved, so that the detection sensitivity is further improved, the signal-to-noise ratio is enhanced, and the detection result is more accurate and reliable.
Taking the common gene mutation site G34A of codon 12 on exon2 of NRAS gene mutation as an example, anti-tag sequences coated with magnetic beads and microspheres are respectively used for detection of the NRAS gene G34A site extension primer, as shown in Table 12.
TABLE 12 screening List of the G12S locus detection method
II, secondly: sample testing
The liquid phase chip prepared above was used to test sample nos. 41-60 according to the test procedures and methods described in example 2, with the following test results:
according to the experimental results, the fluorescent signal value of the magnetic beads coated with the anti-tag sequence is greatly improved compared with that of microspheres coated with the anti-tag sequence, so that the detection sensitivity is further improved, the signal-to-noise ratio is enhanced, and the detection result is more accurate and reliable. The primer and the probe designed by the invention are more suitable for using the magnetic beads as the coating carrier of the anti-tag sequence, and the effect is better. The same method is adopted to detect other mutation types of the NRAS gene, specific data are omitted, and the result also shows that the detection effect of the magnetic bead as an anti-tag coated carrier is better.
The technical features of the embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the description in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
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Claims (6)
1. A liquid chip kit for NRAS gene mutation detection is characterized by comprising:
(A) designing ASPE extension primers aiming at 18 mutant gene types and wild gene types in No.12 codon, No.13 codon, No.3 codon, No. 146 codon on No.4 exon on the No.2 exon of the NRAS gene respectively, wherein each ASPE extension primer consists of a tag sequence at the 5 'end and a specific primer sequence aiming at a mutant site at the 3' end, and the specific primer sequence of the mutant gene type comprises: SEQ ID NO.22 for position G34A, SEQ ID NO.23 for position G34T, SEQ ID NO.24 for position G34C, SEQ ID NO.25 for position G35A, SEQ ID NO.26 for position G35C, SEQ ID NO.27 for position G35T, SEQ ID NO.28 for position G37C, SEQ ID NO.29 for position G37T, SEQ ID NO.30 for position G38A, SEQ ID NO.31 for position G38T, SEQ ID NO.32 for position G38C, SEQ ID NO.33 for position C181A, SEQ ID NO.34 for position A182C, SEQ ID NO.35 for position A182T, SEQ ID NO.36 for position A182 48, SEQ ID NO.37 for position A T, SEQ ID NO.37 for position G A, SEQ ID NO. 437, SEQ ID NO.2 for position G5838, and SEQ ID NO. 59639;
the ASPE extension primer of the wild genotype comprises: SEQ ID NO.40 for exon2, SEQ ID NO.41 for exon3, and SEQ ID NO.73 for exon 4;
(B) magnetic beads coated with anti-tag sequences and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequences and the magnetic beads; the anti-tag sequence comprises SEQ ID NO. 42-SEQ ID NO.62, and the anti-tag sequence can be completely complementary and paired with the tag sequence selected in the step (A) correspondingly;
(C) an amplification primer for amplifying a target sequence to be detected having a corresponding mutation site.
2. The liquid phase chip kit for detecting NRAS gene mutation of claim 1, wherein said amplification primers are at least one pair selected from the group consisting of: SEQ ID NO.63 and SEQ ID NO.64 for exon2, SEQ ID NO.65 and SEQ ID NO.66 for exon3, and SEQ ID NO.67 and SEQ ID NO.68 for exon 4.
3. The NRAS gene mutation detection liquid chip kit of claim 1, wherein the ASPE extension primer of a mutant gene type comprises: the sequence consisting of SEQ ID NO.1 and SEQ ID NO.22 for position G34A, the sequence consisting of SEQ ID NO.2 and SEQ ID NO.23 for position G34T, the sequence consisting of SEQ ID NO.3 and SEQ ID NO.24 for position G34C, the sequence consisting of SEQ ID NO.4 and SEQ ID NO.25 for position G35A, the sequence consisting of SEQ ID NO.5 and SEQ ID NO.26 for position G35C, the sequence consisting of SEQ ID NO. 356 and SEQ ID NO.27 for position G35T, the sequence consisting of SEQ ID NO.7 and SEQ ID NO.28 for position G37C, the sequence consisting of SEQ ID NO.8 and SEQ ID NO.29 for position G37T, the sequence consisting of SEQ ID NO.9 and SEQ ID NO.30 for position G38A, the sequence consisting of SEQ ID NO.10 and SEQ ID NO. C for position G38, the sequence consisting of SEQ ID NO.31 and SEQ ID NO.31 for position SEQ ID NO.38, a sequence consisting of SEQ ID NO.12 and SEQ ID NO.33 for the C181A site, a sequence consisting of SEQ ID NO.13 and SEQ ID NO.34 for the A182C site, a sequence consisting of SEQ ID NO.14 and SEQ ID NO.35 for the A182T site, a sequence consisting of SEQ ID NO.15 and SEQ ID NO.36 for the A182G site, a sequence consisting of SEQ ID NO.16 and SEQ ID NO.37 for the A183T site, a sequence consisting of SEQ ID NO.17 and SEQ ID NO.38 for the G436A site, a sequence consisting of SEQ ID NO.18 and SEQ ID NO.39 for the G437T site;
the wild genotype ASPE extension primer comprises: the sequence consisting of SEQ ID NO.19 and SEQ ID NO.40 for exon2, the sequence consisting of SEQ ID NO.20 and SEQ ID NO.41 for exon3, and the sequence consisting of SEQ ID NO.21 and SEQ ID NO.73 for exon 4.
4. The liquid phase chip kit for detecting NRAS gene mutation according to any one of claims 1 to 3, wherein said spacer arm has 5 to 10T.
5. ASPE extension primers for NRAS gene mutation detection liquid are characterized in that the ASPE extension primers are respectively designed aiming at 18 mutant gene types and wild gene types in No.12 codons and No.13 codons on No.2 exons, No. 61 codons on No.3 exons and No. 146 codons on No.4 exons of NRAS genes, each ASPE extension primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a mutant site, and the specific primer sequences of the mutant gene types comprise: SEQ ID NO.22 for position G34A, SEQ ID NO.23 for position G34T, SEQ ID NO.24 for position G34C, SEQ ID NO.25 for position G35A, SEQ ID NO.26 for position G35C, SEQ ID NO.27 for position G35T, SEQ ID NO.28 for position G37C, SEQ ID NO.29 for position G37T, SEQ ID NO.30 for position G38A, SEQ ID NO.31 for position G38T, SEQ ID NO.32 for position G38C, SEQ ID NO.33 for position C181A, SEQ ID NO.34 for position A182C, SEQ ID NO.35 for position A182T, SEQ ID NO.36 for position A182 48, SEQ ID NO.37 for position A T, SEQ ID NO.37 for position G A, SEQ ID NO. 437, SEQ ID NO.2 for position G35 59638;
the ASPE extension primer of the wild genotype comprises: SEQ ID NO.40 for exon2, SEQ ID NO.41 for exon3, and SEQ ID NO.73 for exon 4;
the tag sequence comprises: SEQ ID NO.1 to SEQ ID NO. 21.
6. The ASPE extension primer for NRAS gene mutation detection solution according to claim 5, wherein said ASPE extension primer of mutant gene type comprises: the sequence consisting of SEQ ID NO.1 and SEQ ID NO.22 for position G34A, the sequence consisting of SEQ ID NO.2 and SEQ ID NO.23 for position G34T, the sequence consisting of SEQ ID NO.3 and SEQ ID NO.24 for position G34C, the sequence consisting of SEQ ID NO.4 and SEQ ID NO.25 for position G35A, the sequence consisting of SEQ ID NO.5 and SEQ ID NO.26 for position G35C, the sequence consisting of SEQ ID NO.6 and SEQ ID NO.27 for position G35T, the sequence consisting of SEQ ID NO.7 and SEQ ID NO.28 for position G37C, the sequence consisting of SEQ ID NO.8 and SEQ ID NO.29 for position G37T, the sequence consisting of SEQ ID NO.9 and SEQ ID NO.30 for position G38A, the sequence consisting of SEQ ID NO.10 and SEQ ID NO.31 for position SEQ ID NO.38, the sequence consisting of SEQ ID NO.31 and SEQ ID NO.31 for position G38, the sequence consisting of SEQ ID NO.31 and SEQ ID NO.32 for position G34 82923, the sequence consisting of SEQ ID NO.12 and SEQ ID NO.33 for the C181A site, the sequence consisting of SEQ ID NO.13 and SEQ ID NO.34 for the A182C site, the sequence consisting of SEQ ID NO.14 and SEQ ID NO.35 for the A182T site, the sequence consisting of SEQ ID NO.15 and SEQ ID NO.36 for the A182G site, the sequence consisting of SEQ ID NO.16 and SEQ ID NO.37 for the A183T site, the sequence consisting of SEQ ID NO.17 and SEQ ID NO.38 for the G436A site, the sequence consisting of SEQ ID NO.18 and SEQ ID NO.39 for the G437T site;
the wild genotype ASPE extension primer comprises: the sequence consisting of SEQ ID NO.19 and SEQ ID NO.40 for exon2, the sequence consisting of SEQ ID NO.20 and SEQ ID NO.41 for exon3, and the sequence consisting of SEQ ID NO.21 and SEQ ID NO.73 for exon 4.
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