CN102994617B - HRAS gene mutation detection specificity primer and liquid chip thereof - Google Patents
HRAS gene mutation detection specificity primer and liquid chip thereof Download PDFInfo
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Abstract
The invention discloses an HRAS gene mutation detection specificity primer and a liquid chip thereof. The liquid chip mainly comprises: an ASPE primer composed of a 5'-terminal tag sequence and 3'-terminal specificity primer sequences focused on target gene mutation sites, wherein the specificity primer sequences comprise SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and/or SEQ ID NO.21 focused on a Codon12 site, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and/or SEQ ID NO.25 focused on a Codon13 site, and/or SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and/or SEQ ID NO.30 focused on a Codon61 site; a microsphere coated by an anti-tag sequence; and an amplimer. The consistency between the detection result of the detection liquid chip provided by the invention and the detection result of a sequencing method is high to 100%, and the wild-type and mutant parallel detection of a plurality of mutation sites is realized.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a HRAS gene mutation detection specific primer and a liquid chip.
Background
The ras gene is involved in regulation of cell growth and differentiation, and in the formation and development of various tumors. The ras gene family has three characteristic genes associated with human tumors, namely H-ras, K-ras and N-ras, which are located on chromosomes 11, 12 and 1, respectively. ras family oncogene activation is the molecular basis for the development of certain tumors, in which point mutations are an important mode of ras family gene activation, and point mutations at codons 12, 13 and 61 of the ras family gene confer the ability to transform cells. Therefore, the detection of the ras gene point mutation of the tumor cells has great significance for understanding the occurrence and development of tumors and monitoring the treatment effect of malignant tumors, and has important guiding significance for clinical work.
Wherein, the HRAS (v-Ha-ras Harvey rate viral oncogene homolog) gene is positioned on chromosome 11 and consists of 946 adenine, 2287 cytosine, 2113 guanine and 1107 thymine. The HRAS gene mutation and the mutation rate thereof are related to different geographic environmental conditions, ethnicities and carcinogens, and researches show that the HRAS point mutation is related to certain molecular biological behaviors of the gastric cancer and possibly becomes a useful index for judging the prognosis of patients with the gastric cancer. The mutation sites related to the HRAS gene mutation detection liquid phase chip developed by the invention are shown in the following table:
at present, few methods are used for detecting and analyzing the HRAS gene mutation, and a direct sequencing method and a PCR-RFLP analysis method are mainly used, wherein the PCR-RFLP analysis method is the most commonly used method. The PCR-RFLP method is based on the change of restriction enzyme recognition site caused by gene mutation, such as site loss or new site generation, a certain specific segment is amplified through PCR, the amplified product is cut by restriction enzyme, and the size of the segment is observed through electrophoresis. Thirdly, the methods have the limitation of detection flux, only one mutation type can be detected at a time, and the requirements of practical application cannot be met.
Disclosure of Invention
One of the purposes of the invention is to provide an HRAS gene mutation detection liquid chip which can be used for single or parallel detection of the normal genotype and five mutant types of the HRAS gene Codon 12: G12S, G12C, G12R, G12D, G12V, the normal genotype of Codon13 and three mutants: G13S, G13R, G13C, and the normal genotype and four mutants of Codon 61: Q61K, Q61R, Q61L, Q61H.
The technical scheme for realizing the purpose is as follows:
a liquid phase chip for detecting HRAS gene mutation comprises:
(A) wild type and mutant ASPE primers designed for different mutation sites of the HRAS gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequence is as follows: the gene sequence is shown in SEQ ID NO.16 and more than one of SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 aiming at the Codon12 site; more than one of SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25 for the Codon13 site; and/or SEQ ID NO.26 and one or more of SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30 for the Codon61 site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 15;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.31-SEQ ID NO.45, and the anti-tag sequence can be complementarily 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.
Preferably, the amplification primers are SEQ ID NO.46 and SEQ ID NO.47 aiming at the Codon12 and/or Codon13 site of the HRAS gene; and/or SEQ ID No.48 and SEQ ID No.49 for the Codon61 site.
Preferably, the ASPE primers are: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.16 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.17, a sequence consisting of SEQ ID NO.3 and SEQ ID NO.18, a sequence consisting of SEQ ID NO.4 and SEQ ID NO.19, a sequence consisting of SEQ ID NO.5 and SEQ ID NO.20, and/or a sequence consisting of SEQ ID NO.6 and SEQ ID NO.21 for the Codon12 site; a sequence consisting of SEQ ID No.7 and SEQ ID No.22 and a sequence consisting of SEQ ID No.8 and SEQ ID No.23, a sequence consisting of SEQ ID No.9 and SEQ ID No.24, and/or a sequence consisting of SEQ ID No.10 and SEQ ID No.25 for the Codon13 site; and/or the sequence consisting of SEQ ID NO.11 and SEQ ID NO.26 and the sequence consisting of SEQ ID NO.12 and SEQ ID NO.27, the sequence consisting of SEQ ID NO.13 and SEQ ID NO.28, the sequence consisting of SEQ ID NO.14 and SEQ ID NO.29, and/or the sequence consisting of SEQ ID NO.15 and SEQ ID NO.30 for the Codon61 site.
Another objective of the invention is to provide specific primers for HRAS gene mutation detection.
The technical scheme for realizing the purpose is as follows:
specific primers for detecting HRAS gene mutation are as follows: the specific primer sequence is as follows: the sequence is shown in SEQ ID NO.16 and more than one of SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 aiming at the Codon12 site; more than one of SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25 for the Codon13 site; and/or SEQ ID NO.26 and one or more of SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30 directed to the Codon61 site.
The main advantages of the invention are:
1. the coincidence rate of the detection method provided by the invention and the sequencing method is up to 100%. And the time required by detection is far shorter than that of the common sequencing technology, and the method particularly meets the requirement of practical application. The prepared HRAS gene mutation detection liquid phase chip has very good signal-to-noise ratio, no cross reaction exists between the designed probe and the anti-tag sequence basically, the tag sequence, the anti-tag sequence are selected, and the tag sequence is combined with a specific ASPE primer, so that the cross reaction can be avoided, and the parallel detection of a plurality of mutation sites can be realized.
2. The invention selects the optimal combination from a plurality of specific primers through the long-term accumulated design experience and a large amount of experimental operation of the inventor. The ASPE primer specific primer designed by the invention can sensitively and specifically identify the mutation site of target detection and accurately distinguish genotypes of various types; in the same reaction system, cross reaction basically does not exist among different specific primers and between the specific primers and a PCR amplification product for non-target detection, the detection specificity is good, and the cross reaction rate is lower than 3%; besides the mutation condition of a single site, the mutation conditions of a plurality of mutation sites can be simultaneously detected in parallel, and the detection effects are consistent.
3. The detection method has simple steps, can complete the amplification of 2 target sequences containing the mutation sites by one-step PCR for the detection of 12 mutation sites, and avoids a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like, thereby greatly improving the detection accuracy and embodying the accurate and qualitative and quantitative analysis characteristics.
4. The invention not only overcomes the defects of low sensitivity and poor repeatability of detection results of the traditional solid phase chip, but also improves the existing liquid phase chip technology, so that the prepared microspheres can be suitable for different detection items and have strong expansibility. The detected 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.
Detailed Description
Example 1
The liquid phase chip for detecting HRAS gene mutation described in this embodiment mainly includes:
first, ASPE primer
Normal genotype and five mutants for HRAS gene Codon 12: G12S, G12C, G12R, G12D, G12V, the normal genotype of Codon13 and three mutants: G13S, G13R, G13C, and the normal genotype and four mutants of Codon 61: Q61K, Q61R, Q61L and Q61H, respectively, and specific primer sequences were designed. The ASPE primer consists of a tag sequence and a specific primer sequence. The ASPE primer sequences are shown in the following table:
TABLE 1 ASPE primer sequences (tag sequence + specific primer sequence) of HRAS genes
Each ASPE primer comprises two parts, the 5 'end is a specific tag sequence aiming at an anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant type or wild type specific primer segment (as shown in the table 1). All ASPE 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.
Two, anti-tag sequence coated microsphere
Selecting tag sequences according to the designed ASPE specific primer fragments, and reducing secondary structures possibly formed between anti-tag sequences of the microspheres and tag and the ASPE specific primer fragments to the maximum extent, wherein the numbers of the selected 15 microspheres and the corresponding anti-tag sequences on the microspheres are shown in Table 2:
TABLE 2 numbering of microspheres and corresponding anti-tag sequences on microspheres
Selected 15 microspheres were purchased from Luminex, usa and were coated with the anti-tag sequence. 5-10T spacer arm sequences are connected between the anti-tag sequences and the microspheres, namely a 5-10T spacer arm sequence is added in front of each anti-tag sequence, and the anti-tag sequences are synthesized by Shanghai Bioengineering technology service GmbH. The synthetic anti-tag sequence was treated with sterile ddH2O is prepared into a 100nmol/ml stock solution. The spacer arm is a sequence for spacing the anti-tag from the surface of the microsphere or placing the anti-tag in a hydrophilic environment. By passingAnd a spacer arm sequence with proper length is arranged between the anti-tag sequence and the microsphere, so that the steric hindrance can be reduced, and the efficiency and specificity of the hybridization reaction can be improved. Common spacer sequences include poly-dT, i.e., poly (dT), oligo-tetrapolyethylene glycol, and (CH2) n spacers (n.gtoreq.3), such as (CH2)12, (CH2)18, and the like. In addition, if a poly (dA) interference is present, poly (TTG) may also be used as a spacer. The spacer arm of the invention is preferably 5-10T, and the process of coating the microspheres is as follows:
respectively taking 5 × 106Each of the above numbered carboxylated microspheres (from Luminex) was suspended in 50. mu.l of 0.1mol/L MES solution (pH4.5) and 10. mu.l of synthetic anti-tag molecule (100nmol/m1) was added. 10ng/ml of EDC (N- (3-methylenepropyl-N-ethylenecarboxyl) working solution (available from Pierce Chemical Co.) 2.5ul of EDC working solution was added to the microsphere suspension, incubated at constant temperature for 30 minutes, 2.5ul of EDC working solution was added, incubated at constant temperature for 30 minutes, washed once with 0.02% Tween-20 and then 0.1% SDS after the completion of the reaction, and the washed microspheres coated with the anti-tag sequence were resuspended in 100ul of Tris-EDTA solution [10mmol/L Tris (pH8.0) ]]In 1mmol/LEDTA, and storing at 2-8 deg.C in dark.
Thirdly, amplifying the primer of the target sequence containing the mutation site
Aiming at Codon12, Codon13 and Codon61 of HRAS gene, an amplification primer pair (see Table 3) is designed, and 2 target sequences containing 12 mutation sites are amplified, wherein Codon12 and Codon13 are located in the same amplification product.
TABLE 3 primers for amplifying target sequences with mutation sites
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 detection of samples Using the liquid phase chip for HRAS Gene mutation detection described in example 1
The formulations of the various solutions are as follows:
50mM MES buffer (pH5.0) formulation (250 ml):
2 XTM hybridization buffer
| Reagent | Origin of origin | Final concentration | The dosage of each 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5M NaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
After filtration, the mixture was stored at 4 ℃.
The ExoSAP-IT kit was purchased from U.S. USB.
Biotin-labeled dCTP was purchased from Shanghai Biotechnology engineering services, Inc.
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
2 pairs of primers are designed, and 2-item target sequences of 12 common genotypes containing HRAS genes Codon12, Codon13 and Codon61 are amplified in one step through multiplex PCR, wherein Codon12 and Codon13 are located in the same amplification product, the sizes of the products are 320bp and 282bp respectively, and the primer sequences (SEQ ID NO.46-49) are shown in the table 3.
Firstly, preparing a multiplex PCR primer working solution: 100ul of the primer stock solution of SEQ ID NO.46-49 is respectively taken and put in a 1.5ml microcentrifuge tube, and the multiple PCR primer working solution is obtained after uniform mixing. The multiplex PCR reaction system is as follows:
the PCR amplification procedure was: 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 10ul of wild type and mutant ASPE primer stock solution corresponding to the gene to be detected, 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:
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. Based on the designed ASPE primers, 15 corresponding coated microspheres (as described in example 1) were selected per set, each at a concentration of 2.5X 105Per ml;
2. 1ul of microspheres with each number are respectively taken and put in a 1.5ml microcentrifuge tube;
3. centrifuging the microspheres at a speed of more than or equal to 10000g for 1-2 min;
4. discarding the supernatant, resuspending the microspheres in 100ul of 2 XTM hybridization buffer, and mixing by vortex;
5. 25ul of the microsphere suspension was placed in the corresponding well of a 96-well filter plate, and 25ul of ddH was added to the control well2O；
6. Taking 5-25ul ASPE reaction solution into corresponding holes, and using ddH2O is complemented to 50 ul;
7. wrapping a 96-well plate with tin foil paper to avoid light, and incubating and hybridizing at 95 ℃ for 60s and 37 ℃ for 15 min;
8. centrifuging the hybridized microspheres for 2-5min at a speed of more than or equal to 3000 g;
9. removing supernatant, and suspending the microspheres in 75ul of 1 XTM hybridization buffer;
10. centrifuging the microspheres at a speed of more than or equal to 3000g for 2-5 min;
11. resuspend the microspheres in 75ul of 1 XTM hybridization buffer, add 15ul of streptavidin-phycoerythrin (SA-PE) at 10 ug/ml;
incubate at 12.37 ℃ for 15min and detect on Luminex instruments.
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 method is used for detecting HRAS gene mutation of a large number of samples, and the coincidence rate of the detection results of the method provided by the invention is calculated by comparing the detection result of a sequencing method with the result of a liquid chip. The HRAS genotype detection result of 20 samples detected by the method is matched with the sequencing result by 100%. Therefore, the HRAS gene mutation detection liquid-phase chip provided by the invention can accurately detect the mutation type of the HRAS gene, and the result is stable and reliable.
TABLE 4 one of the sample test results (MFI)
TABLE 5 two sample test results (MFI)
TABLE 6 results of analysis of HRAS Gene mutation types in samples
| Sample number | Liquid phase chip detection result | Sequencing results |
| 1 | Wild type | Wild type |
| 2 | Wild type | Wild type |
| 3 | Wild type | Wild type |
| 4 | Wild type | Wild type |
| 5 | Wild type | Wild type |
| 6 | G13R mutation | G13R mutation |
| 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 | G12C mutation | G12C mutation |
| 13 | Wild type | Wild type |
| 14 | Wild type | Wild type |
| 15 | G12D mutation | G12D mutation |
| 16 | Q61K mutation | Q61K mutation |
| 17 | Wild type | Wild type |
| 18 | Wild type | Wild type |
| 19 | Wild type | Wild type |
| 20 | Wild type | Wild type |
EXAMPLE 3 detection of HRAS Gene mutation site by liquid chip of different ASPE primers
Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
Taking HRAS gene G12S and G13S site mutation detection liquid phase chip as an example, specific primer sequences at the 3 'end of ASPE primers are designed aiming at the wild type and the mutant type of G12S and G13S respectively, the Tag sequence at the 5' end of the ASPE primers is selected from SEQ ID NO.1-SEQ ID NO.15, and correspondingly, the anti-Tag sequence coated on the microspheres and complementarily paired with the corresponding Tag sequence is selected from SEQ ID NO.31-SEQ ID NO. 45. The specific design is shown in the following table (table 7). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 7 design of liquid phase chip preparation
Second, sample detection
The liquid phase chip prepared by the design is adopted to detect the samples 21-40 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 8 sample test results and Gene mutation analysis
TABLE 9 sample test results and Gene mutation analysis
It can be seen from this example that when the tag sequence in example 1 is selected as the primer for ASPE to match with the specific primer sequence, the effect is better (the signal to noise ratio is better), see test group 1 and test group 4 in this example. Other different tag sequences are matched with the specific primer sequences, the results are the same as those of the example 2 and the example, and specific data are omitted.
In other liquid phase chips aiming at different mutation sites, different Tag sequences are applied to the ASPE primers, the result is still stable and reliable, and when the Tag sequences and the specific primer sequences in the embodiment 1 are selected as the ASPE primers to be matched, the effect is better (the signal-to-noise ratio is better), and specific data are omitted.
Example 4 selection of primer sequences specific for detection of mutation in HRAS Gene
Design of liquid phase chip preparation (selection of wild type and mutant type specific primer sequences)
Taking mutation site detection liquid phase chips of HRAS genes G12R and Q61K as examples, taking a forward or reverse complementary sequence of a target sequence where the mutation site is located as a template, designing specific primer sequences at the 3' end of an ASPE primer aiming at a wild type and a mutant type of G12R and Q61K respectively, wherein the specific primer sequences comprise a preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment 1 of the invention, and the sequences are shown in Table 10. Wherein,the internal base is a mutation site.
TABLE 10 specific primer sequences
Taking mutation site detection liquid phase chip of HRAS genes G12R and Q61K as an example, different specific primer sequences are selected for G12R and Q61K, and a Tag sequence at the 5' end of an ASPE primer is fixed as the best effect sequence in example 1, and an anti-Tag sequence corresponding to the best effect sequence is selected, and the specific design is shown in the following table (Table 11). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 11 design two for liquid phase chip preparation
Second, sample detection
The liquid phase chip prepared by the design is adopted to detect the samples 41-60 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 12 sample test results and Gene mutation analysis
TABLE 13 sample test results and Gene mutation analysis
In this example, the specific primer sequence in example 1 is selected as the ASPE primer to match with the tag sequence, which is better (better signal to noise ratio), see test group 7 and test group 10 in this example. Other different specific primer sequences derived from the forward or reverse complementary sequence of the target detection site are matched with the tag sequence, which is the same as the results of the embodiment 2 and the embodiment, i.e., the specific primer sequence described in the embodiment 1 is still better matched with different tag sequences, and specific data are omitted.
Other specific primer sequences aiming at the same mutation site or different mutation sites are matched with the tag sequence, and the result is the same as that of the embodiment 2 and the embodiment, namely the specific primer selected in the embodiment 1 has better signal to noise ratio and better detection effect, and specific data are omitted.
The above is a detailed description of possible embodiments of the present invention, but the embodiments are not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the technical spirit of the present invention are intended to be included in the scope of the present invention.
Claims (4)
1. A liquid phase chip for HRAS gene mutation detection is characterized by comprising:
(A) wild type and mutant ASPE primers designed for different mutation sites of the HRAS gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequence is as follows: SEQ ID NO.16 and one or more of SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 for the Codon12 site; and at least one selected from the group consisting of specific primers for the Codon13 site and for the Codon61 site, said specific primers for the Codon13 site being one or more of SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO. 25; the specific primer aiming at the Codon61 site is more than one of SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO. 30; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 15;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.31-SEQ ID NO.45, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) a primer for amplifying a target sequence to be detected having a corresponding mutation site, wherein the amplification primer is: SEQ ID NO.46 and SEQ ID NO.47 for positions Codon12 and Codon 13; or the amplification primers are: SEQ ID NO.46 and SEQ ID NO.47 and SEQ ID NO.48 and SEQ ID NO.49 for the Codon61 site.
2. The liquid phase chip for detecting HRAS gene mutation as claimed in claim 1, wherein the ASPE primers are: the sequence consisting of SEQ ID No.1 and SEQ ID No.16 and the sequence consisting of SEQ ID No.2 and SEQ ID No.17, the sequence consisting of SEQ ID No.3 and SEQ ID No.18, the sequence consisting of SEQ ID No.4 and SEQ ID No.19, the sequence consisting of SEQ ID No.5 and SEQ ID No.20, and/or the sequence consisting of SEQ ID No.6 and SEQ ID No.21 for the Codon12 site; and a sequence selected from the group consisting of SEQ ID No.7 and SEQ ID No.22 and the sequence consisting of SEQ ID No.8 and SEQ ID No.23, the sequence consisting of SEQ ID No.9 and SEQ ID No.24, and/or the sequence consisting of SEQ ID No.10 and SEQ ID No.25 for the Codon13 site; and at least one of a sequence consisting of SEQ ID NO.11 and SEQ ID NO.26 and a sequence consisting of SEQ ID NO.12 and SEQ ID NO.27, a sequence consisting of SEQ ID NO.13 and SEQ ID NO.28, a sequence consisting of SEQ ID NO.14 and SEQ ID NO.29, and/or a sequence consisting of SEQ ID NO.15 and SEQ ID NO.30 for the Codon61 site.
3. The liquid phase chip for HRAS gene mutation detection according to claim 1,
(A) the ASPE primers are as follows: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.16 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.17, a sequence consisting of SEQ ID NO.3 and SEQ ID NO.18, a sequence consisting of SEQ ID NO.4 and SEQ ID NO.19, a sequence consisting of SEQ ID NO.5 and SEQ ID NO.20, and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.21 for the Codon12 site; a sequence consisting of SEQ ID NO.7 and SEQ ID NO.22 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.23, a sequence consisting of SEQ ID NO.9 and SEQ ID NO.24, and a sequence consisting of SEQ ID NO.10 and SEQ ID NO.25 for the Codon13 site; and a sequence consisting of SEQ ID NO.11 and SEQ ID NO.26 and a sequence consisting of SEQ ID NO.12 and SEQ ID NO.27, a sequence consisting of SEQ ID NO.13 and SEQ ID NO.28, a sequence consisting of SEQ ID NO.14 and SEQ ID NO.29, and a sequence consisting of SEQ ID NO.15 and SEQ ID NO.30 for the Codon61 site;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.31-SEQ ID NO.45, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) the amplification primers are as follows: SEQ ID NO.46 and SEQ ID NO.47 for Codon12, Codon13 site; and SEQ ID NO.48 and SEQ ID NO.49 for position Codon 61.
4. The liquid phase chip for detecting HRAS gene mutation according to any one of claims 1-3, wherein the spacer arm has 5-10T.
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| CN104774954A (en) * | 2015-04-23 | 2015-07-15 | 上海允英医疗科技有限公司 | Primers, probes and detection kit for HRAS mutation detection |
| CN108796052A (en) * | 2018-06-29 | 2018-11-13 | 上海赛安生物医药科技股份有限公司 | Detect the detection architecture and its kit of HRAS gene mutations |
| CN112725422A (en) * | 2021-02-26 | 2021-04-30 | 山东康华生物医疗科技股份有限公司 | Primer, probe and kit for HRAS G13R mutation detection |
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