CN113005183A - KRAS gene mutation detection kit and application thereof - Google Patents
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Abstract
The invention discloses a KRAS gene mutation detection kit and application thereof, wherein the KRAS gene mutation detection kit comprises: specific upstream primer, downstream primer and double quenching probe of mutation site; wherein the specific upstream primer comprises the following components from 5 'end to 3' end in sequence: a capture zone: the length is 12-21bp, and the target fragment is completely matched; mismatch recognition region: the length is 3-6bp, and the target fragment is mismatched; mutation protection region: comprises a mutation recognition site, and a 3' end which is matched with the mutation recognition site to form a hairpin structure; the downstream primer is paired with the downstream of the KRAS gene mutation site; the double quenching probe is provided with two quenching groups. According to the invention, the upstream primer is specially designed and combined with the double-quenching probe, so that the detection sensitivity and specificity are obviously improved, two sample types of tissues and blood plasma can be compatible, the clinical requirements are met, the operation process is simple and rapid, and the detection result is simple and visual.
Description
Technical Field
The invention belongs to the technical field of gene mutation detection, and particularly relates to a KRAS gene mutation detection kit and application thereof.
Background
The RAS gene family has three genes closely related to human tumors, namely HRAS, KRAS and NRAS, and the proteins encoded by the three genes have about 90% amino acid sequence homology and are also called RASp21 proteins. The KRAS signal pathway is a downstream pathway of EGFR and other signal transduction, and when RAS gene is mutated, the ability of the encoded RAS proteolysis GTP-RAS is reduced, so that the signal transduction pathway is always in a continuous activation state, cells are stimulated to continuously proliferate and differentiate, and finally the cells are malignant. The KRAS mutant gene is a common oncogene and is mostly found in malignant tumors, about 30-40% of colorectal cancer patients have KRAS gene mutation, and about 15-30% of lung adenocarcinoma patients have KRAS mutation.
Research shows that KRAS gene mutation is related to primary drug resistance of NSCLC to gefitinib, erlotinib and other targeted therapeutic drugs. It has been found that mutation of the KRAS gene can cause colorectal cancer patients to be resistant to cetuximab treatment, and NCCN (2016 (non-small cell lung cancer) clinical practice guideline) indicates that KRAS gene mutation can cause lung cancer patients to be resistant to EGFR tyrosine kinase inhibitor (EGFT-TKI), and colorectal cancer patients to be resistant to anti-EGFR antibody drugs. Therefore, the risk prediction of KRAS gene mutation is an important prediction index for the generation of EGFR targeted therapy drug resistance.
The current clinical detection methods for gene mutation in DNA mainly comprise: 1) the direct sequencing method is a detection method which is most applied at present, mainly Sanger sequencing is adopted, the sequencing can be carried out only after the mutant genes are effectively enriched, and the detection is inaccurate under the condition of low content of the mutant genes, namely the sensitivity is low. 2) The high-throughput sequencing technology method has large difference between enrichment specificity and effectiveness on different experimental platforms, and has complicated library establishing process, long period and high cost. 3) Conventional ARMS-PCR: the sensitivity of the kit can only stably reach 1% mutation detection, but free DNA in a plasma sample is usually fragmented and has very small total amount, so that the blood sample required to be collected is overlarge, the harm to a patient is large, and the operation is complicated. At present, the collection of 5-10ml of whole blood of a patient is clinically recommended for subsequent detection, namely, the concentration of extracted free DNA is limited, so that a detection method and a detection kit which have higher sensitivity and can be compatible with two sample types of tissues and blood plasma are urgently needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a KRAS gene mutation detection kit and application thereof, wherein the identification efficiency and specificity are improved by specially designing a fluorescence PCR detection upstream primer, the primer is further combined with a double-quenching probe, the combination capability of the primer probe and a target fragment is obviously improved, the amplification efficiency and the signal value of the detection are improved, so that the requirements of high sensitivity and high specificity of blood detection are met, two sample types of tissues and blood plasma can be compatible, 7 types of KRAS gene mutation can be simultaneously detected, the operation process is simple and rapid, the cost is low, and the detection result is clear and easy to analyze.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a KRAS gene mutation detection kit, which comprises: specific upstream primer, downstream primer and double quenching probe of mutation site;
wherein the specific upstream primer structure sequentially comprises: a capture zone: the length is 12-21bp, and the target fragment is completely matched; mismatch recognition region: the length is 3-6bp, and the target fragment is mismatched; mutation protection region: comprises a mutation recognition site, and a 3' end which is matched with the mutation recognition site to form a hairpin structure;
the downstream primer is paired with the downstream of the KRAS gene mutation site;
the double quenching probe is provided with two quenching groups.
The specific upstream primer comprises a mutation protection region of a hairpin structure, and when the temperature is lower than the melting temperature, the hairpin structure of the primer is closed, so that mutation identification is protected from being combined with other sequence mismatching; when the denaturation temperature is reached, the hairpin structure opens, and as the primer capture region binds to the target fragment, the double recognition of the formation of the target mutation site by the mismatch recognition region and the open hairpin structure begins to play a role: during detection, if the segment to be detected is wild, the mismatch region, the mutation site and the 3' end region of the hairpin structure cannot be combined with the target segment to form a super-strong mismatch, so that amplification cannot proceed; when the target fragment is the mutant type, the capture region and the mutant site region on the primer can be combined with the target, the amplification reaction can be smoothly carried out, and further, the double-fluorescence quenching probe is combined, so that the mutant type can be successfully detected.
Further, the upstream primer specific to the mutation site comprises:
specific upstream primer of Gly12Cys mutation site: the sequence is shown as SEQ ID NO. 1;
specific upstream primer of Gly12Ser mutation site: the sequence is shown in SEQ ID NO. 2;
③ Gly12Arg mutation site specific upstream primer: the sequence is shown in SEQ ID NO. 3;
specific upstream primer of Gly12Val mutation site: the sequence is shown in SEQ ID NO. 4;
fifth, specific upstream primer of Gly12Asp mutation site: the sequence is shown as SEQ ID NO. 5;
sixthly, specific upstream primer of Gly12Ala mutation site: the sequence is shown in SEQ ID NO. 6;
seventhly 13Asp mutation site specific upstream primer: the sequence is shown in SEQ ID NO. 7.
Further, the downstream primer is a universal downstream primer, and the sequence of the downstream primer is shown in SEQ ID NO. 8.
Furthermore, the 5' end of the double-quenching probe sequence is marked with a fluorescence reporter group, and the 3 end is marked with a fluorescence quenching group connected with an MGB modifying group. Wherein, the MGB modified group does not generate fluorescence, thereby greatly reducing the intensity of background signals and further improving the detection specificity.
Further, the fluorescent reporter group is selected from any one of FAM, VIC, ROX, Cy3, Cy5, HEX and JOE;
the fluorescence quenching group is selected from any one of TAMRA, BHQ1, BHQ2, NFQ and LFN.
Further, the sequence of the double-quenching probe is as follows:
5’FAM-CAAGAGTGC-LFN-CTTGACGATA-MGB 3’。
further, the detection kit further comprises: DNA polymerase, UNG enzyme, dNTPs and Mg2+。
Further, the PCR reaction system in the detection kit contains every 25 mu L: 0.5U-1.0U of DNA polymerase, 0.1U-0.5U of UNG enzyme, MgCl21-5mM, 0.4-0.4mM dNTPs, 0.1-0.5 μ M upstream primer, 0.1-0.5 μ M downstream primer, and 0.1-0.2 μ M double-quenching probe.
Further, in the PCR reaction system, the concentration ratio of the upstream primer to the downstream primer to the probe is 0.2. mu.M: 0.2. mu.M: 0.1. mu.M.
Further, in the PCR reaction system, MgCl2Was 3 mM.
Further, in the PCR reaction system, the DNA polymerase is Taq enzyme, and the total amount ratio of the Taq enzyme to the UNG enzyme is 0.5U: 0.1U, 0.5U: 0.5U, 1.0U: 0.1U or 1.0U: 0.5U.
Further, concentrations of dATP, dUTP, dGTP and dCTP in dNTP in the PCR reaction system were 0.15mM, 0.15mM and 0.3mM, respectively.
Further, the detection kit further comprises: positive control solution and/or blank control solution.
Further, the detection kit also comprises an internal standard primer which can be used for monitoring sample application, and the sequence of an upstream primer is as follows: GGGCCACTAGGCGCTCA, the sequence of the downstream primer is: AGCCACCCGCGAACTCA, the sequence of the fluorescent probe is: 5 'VIC-CTCTCCCTCCGCGCAGCCG-3' MGB
Further, in some embodiments of the present invention, the primer set further comprises a quality control primer for monitoring the quality of the sample, wherein the quality control primer has a base sequence, and the upstream primer sequence of the quality control primer is: GACTGAATATAAACTTGTGGTAGTTGGA, the sequence of the downstream primer is: CATATTCGTCCACAAAATGATTCTG, the sequence of the fluorescent probe is: 5 'FAM-CTGTATCGTCAAGGCACT-3' MGB
The invention also provides the application of the detection kit in the non-disease diagnosis and treatment purposes of detecting KRAS gene mutation.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a detection kit for detecting KRAS gene mutation and application, the invention designs a special upstream primer based on 7 mutation sites of KRAS gene, namely a mismatch identification region and a mutation protection region are added in the primer, thereby improving the identification efficiency and the identification specificity of the primer on a target fragment, further, the invention discovers that the primer is combined with a double-quenching probe, has a synergistic interaction effect, can obviously improve the combination capability of the primer probe and a target fragment, and improves the amplification efficiency and the signal value of detection, namely, the detection sensitivity and the specificity are obviously improved, the accurate detection of 1copy mutation (1/12000) in 40ng DNA sample can be realized, the high-sensitivity and high-specificity requirements of blood detection are met, and 2-5ml of plasma extracted ctDNA stock solution can be directly used for detection; 50ng of genomic DNA background interference can be tolerated for tissue samples. The detection kit can simultaneously detect 7 types of KRAS gene mutation, realize auxiliary diagnosis and treatment of EGFR targeted treatment drug resistance, remarkably improve sensitivity and specificity, and be compatible with two sample types of tissues and blood plasma so as to meet clinical requirements; meanwhile, the cost is low, the operation process is simple and quick, and the detection can be finished within 100 min; and the detection result can be directly judged according to the detection Ct value of the reaction solution, the quality control Ct value is not required to be additionally used for calculating the related delta Ct value, and the interpretation of the detection result is simple and visual.
Drawings
FIG. 1 is the structure of a specific forward primer in example 1 of the present invention;
FIG. 2 is the operation principle of the specific forward primer in example 1 of the present invention;
FIG. 3 is a graph showing amplification curves in the sensitivity tests of example 1 and comparative example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example provides a KRAS gene mutation detection kit, which includes a specific upstream primer, a universal downstream primer, a dual-quenching probe, Taq enzyme, UNG enzyme, dNTPs and Mg for 7 mutation sites of KRAS gene2+。
The 7 mutant sites and sequences of KRAS gene are shown in Table 1, wherein the bold bases are the mutant sites.
TABLE 1 7 mutation sites and sequences of KRAS Gene
According to the 7 mutation sites described in table 1, specific upstream primers for fluorescent quantitative PCR detection are respectively designed, and the structures thereof are sequentially from 5 'end to 3' end: a capture zone: the length is 12-21bp, and the target fragment is completely matched; mismatch recognition region: the length is 3-6bp, and the target fragment is mismatched; mutation protection region: comprises a mutation recognition site, and is paired with the mutation recognition site to form the 3' end of the hairpin structure (shown in figure 1). The working principle of the primer is as follows: (1) when the temperature is lower than the melting temperature, the hairpin structure of the primer is closed, and mutation identification is protected from being combined with other sequence mismatching; when the denaturation temperature is reached, the hairpin structure is opened, and the mismatch recognition region and the opened hairpin structure form double recognition on the target mutation site along with the combination of the primer capture region and the target fragment. (2) During detection, if the segment to be detected is wild, the mismatch region, the mutation site and the hairpin structure 3' end region cannot be combined with the target segment to form an ultra-strong mismatch, and amplification cannot be performed; when the target fragment is a mutant type, the mutation site on the primer can bind thereto and amplify the mutant gene. The 7 specific upstream primers and sequences based on this design are shown in Table 2, wherein the underlined is the mismatch sequence and the bolded base is the mutation recognition site.
TABLE 2 specific upstream primers
Further, according to the sequence described in table 1, a universal downstream primer KRAS-R is designed, and the sequence is: TTCGTCCACAAAATGATTCTGAAT (shown in SEQ ID NO. 8);
furthermore, the double-quenching probe adopted in this embodiment is KRAS-P1, and its sequence is specifically: 5 'FAM-CAAGAGTGC-LFN-CTTGACGATA-MGB 3'.
Comparative example 1
This comparative example differs from example 1 in that: the probe adopted in the comparative example is a conventionally designed probe, only has one quenching group, and has the specific sequence as follows:
KRAS-P2:5’FAM-CAAGAGTGCCTTGACGATA-MGB 3’
comparative example 2
This comparative example differs from example 1 in that: the upstream primers used in the comparative example are conventional ARMS primers, and the sequence table 3 of each upstream primer is shown.
TABLE 3 conventional ARMs primers
Comparative example 3
This comparative example differs from example 1 in that: the upstream primer used in this comparative example was a conventional ARMS primer whose sequence is shown in Table 3 in comparative example 2; meanwhile, the probe adopted in the comparative example is a conventionally designed probe, only has one quenching group, and the sequence of the quenching group is the same as that of the probe in the comparative example 1, namely: KRAS-P2 ' 5 ' FAM-CAAGAGTGCCTTGACGATA-MGB 3 '
Evaluation method
To verify the detection effect of each primer set and probe in example 1 and comparative examples 1-3, a reference system was first established, i.e., DNA fragments of 7 mutant genes shown in Table 1 were cloned into pEASY-T1 plasmid vectors and transformed into DH 5. alpha. strain, respectively, by a conventional method, and plasmids A of the plasmids were extracted from the cultured strain260/A280The value is between 1.6 and 2.0, and the plasmid with the sequence confirmed to be error-free is used as a reference.
And further extracting genomic DNA of the HT-29 cell line, quantifying and diluting the mutant plasmids obtained by the method in a gradient manner, and mixing the mutant plasmids with the genomic DNA to form mixed samples containing mutant plasmids with different proportions.
(1) Evaluation of specificity
Genomic DNA was used as test samples (without mutant plasmid) at concentrations of 10 ng/. mu.L and 20 ng/. mu.L, respectively. The detection systems of example 1 and comparative examples 1-3 were respectively subjected to fluorescent quantitative PCR detection to verify the detection specificity of the different detection systems. The PCR assay system is shown in Table 4.
TABLE 4 fluorescent quantitative PCR detection system
The PCR amplification conditions were: 37 ℃ for 10 min; 95 deg.C for 5 min; 95 ℃, 15s, 60 ℃, 60s, 45 cycles. The real-time fluorescent quantitative PCR instrument was used to read the cycle numbers, i.e., CT values, of samples of different concentrations and different detection systems, and the results are shown in Table 5.
TABLE 5 evaluation of specificity test results
According to the specificity detection results in table 5, the comparative examples 2 and 3 can still read the CT value under the condition that the sample to be detected does not contain the mutant plasmid, which indicates that the specificity is poor and the detection result is inaccurate, while the comparative examples 1 and 1 can not read the corresponding CT value, i.e. the amplification is not performed under the condition that the sample to be detected does not contain the mutation, which indicates that the detection specificity can be enhanced by using the specially designed upstream primer of the present invention, the interference of the background DNA up to 20 ng/. mu.L can be tolerated, and the false positive result caused by the non-specific amplification can be avoided.
(2) Evaluation of sensitivity
The 7 kinds of mutant plasmids and the genome DNA are mixed according to certain concentration to form mixed samples containing mutant plasmids with different proportions. Each mutant plasmid contained 3 concentrations: 20 ng/. mu.L samples contained 1%, 0.1% or 0.01% copy number of the mutated plasmid (wherein the DNA copy number in 20 ng/. mu.L samples was 6000 copies/. mu.L). The detection systems of example 1 and comparative examples 1 to 3 were used for fluorescent quantitative PCR detection, respectively, to verify the detection sensitivity of the different detection systems. The PCR detection system and amplification conditions were the same as in the specific detection, and the CT values of different samples and different detection systems were read, respectively, and the results are shown in Table 6 and FIG. 3.
TABLE 6 evaluation of sensitivity test results
According to the test results of Table 6, in the case where 40ng of the sample contains 0.01% copy number (about 1copy) of the mutant plasmid, there are cases where CT values cannot be read partially or completely, i.e., the sensitivity is low, and high sensitivity test of plasma samples in clinical practice cannot be satisfied, in comparative examples 2 and 3. Namely, the detection sensitivity can be enhanced by adopting the specially designed upstream primer of the invention. Furthermore, according to table 6, the CT values of example 1 and comparative example 1 of the present invention are also different, and example 1 is advanced by about 1 CT value compared to comparative example 1, and the amplification efficiency is higher, and according to fig. 3, the Gly12Val site is taken as an example, and example 1 is higher in signal height and more obvious in curve linear plateau phase compared to comparative example 1, which indicates that the specially designed primer and the double-quenching probe of the present invention have a synergistic effect, can significantly increase the detection sensitivity, can realize accurate detection of 1copy mutation (1/12000) in 40ng of DNA sample, and meets the high sensitivity requirement of blood detection, and 2-5ml of ctDNA stock solution extracted from plasma can be directly used for detection.
(3) Evaluation of precision
The 7 kinds of mutant plasmids prepared above were mixed with genomic DNA to prepare mixed samples of mutant plasmids, each sample having a concentration of 5% of the mutant plasmids contained in 10 ng/. mu.L, and the samples were repeatedly tested 10 times using the test system of example 1, and the CT values were read to calculate the mean value, standard deviation, and coefficient of variation CV values, and the precision of the test system of example 1 was evaluated by the CV values. The results are shown in Table 7.
TABLE 7 evaluation of precision test results
| Repetition of | Gly12Cys | Gly12Ser | Gly12Arg | Gly12Val | Gly12Asp | Gly12Ala | Gly13Asp |
| 1 | 36.75 | 36.52 | 36.89 | 36.21 | 36.55 | 36.89 | 36.14 |
| 2 | 36.56 | 36.48 | 39.94 | 36.23 | 36.51 | 36.81 | 36.19 |
| 3 | 36.96 | 36.45 | 36.95 | 36.19 | 36.59 | 36.94 | 36.28 |
| 4 | 36.47 | 36.38 | 37.04 | 39.14 | 36.64 | 36.97 | 36.01 |
| 5 | 36.84 | 36.61 | 37.10 | 36.21 | 36.37 | 36.68 | 36.38 |
| 6 | 36.88 | 36.57 | 36.88 | 36.15 | 36.31 | 36.87 | 36.21 |
| 7 | 36.71 | 36.71 | 36.79 | 36.04 | 36.69 | 36.81 | 36.11 |
| 8 | 36.72 | 36.47 | 36.90 | 36.01 | 36.47 | 36.99 | 36.23 |
| 9 | 36.46 | 36.33 | 36.85 | 36.37 | 36.61 | 36.86 | 36.31 |
| 10 | 36.59 | 36.29 | 37.01 | 36.40 | 36.14 | 36.71 | 36.18 |
| Mean value of | 36.69 | 36.48 | 37.24 | 36.50 | 36.49 | 36.85 | 36.20 |
| Standard deviation of | 0.17 | 0.13 | 0.96 | 0.94 | 0.17 | 0.10 | 0.11 |
| CV value | 0.47％ | 0.35％ | 2.56％ | 2.57％ | 0.47％ | 0.28％ | 0.29％ |
According to the detection results in table 7, when the detection system of the embodiment 1 of the present invention is used for detecting mutant genes, the CV values are all lower than 3%, which shows that the detection system has good repeatability and high precision, and can meet the clinical use requirements.
By combining the detection results, the KRAS gene detection system and the detection kit provided by the invention can be used for simultaneously detecting 7 types of mutation of KRAS gene, and by combining the specially designed upstream primer with the double quenching probes, a synergistic effect is generated, so that the detection sensitivity and specificity are obviously improved, two sample types of tissues and blood plasma can be compatible, the clinical requirements are met, the auxiliary diagnosis and treatment of EGFR targeted treatment drug resistance are realized, the operation process is simple and quick, and the detection can be completed within 100 min; and the interpretation of the detection result is simple and intuitive.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
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Claims (10)
1. A KRAS gene mutation detection kit is characterized by comprising: specific upstream primer, downstream primer and double quenching probe of mutation site;
wherein the specific upstream primer comprises the following components from 5 'end to 3' end in sequence: a capture zone: the length is 12-21bp, and the target fragment is completely matched; mismatch recognition region: the length is 3-6bp, and the target fragment is mismatched; mutation protection region: comprises a mutation recognition site, and a 3' end which is matched with the mutation recognition site to form a hairpin structure;
the downstream primer is paired with the downstream of the KRAS gene mutation site;
the double quenching probe is provided with two quenching groups.
2. The detection kit according to claim 1, wherein the upstream primer specific to the mutation site comprises:
specific upstream primer of Gly12Cys mutation site: the sequence is shown as SEQ ID NO. 1;
specific upstream primer of Gly12Ser mutation site: the sequence is shown in SEQ ID NO. 2;
③ Gly12Arg mutation site specific upstream primer: the sequence is shown in SEQ ID NO. 3;
specific upstream primer of Gly12Val mutation site: the sequence is shown in SEQ ID NO. 4;
fifth, specific upstream primer of Gly12Asp mutation site: the sequence is shown as SEQ ID NO. 5;
sixthly, specific upstream primer of Gly12Ala mutation site: the sequence is shown in SEQ ID NO. 6;
seventhly 13Asp mutation site specific upstream primer: the sequence is shown in SEQ ID NO. 7.
3. The detection kit according to claim 1, wherein the downstream primer is a universal downstream primer, and the sequence of the universal downstream primer is shown as SEQ ID No. 8.
4. The detection kit of claim 1, wherein the double-quenching probe sequence is labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group linked with an MGB modifier group at the 3 end.
5. The detection kit according to claim 2, wherein the fluorescent reporter group is selected from any one of FAM, VIC, ROX, Cy3, Cy5, HEX, and JOE;
the fluorescence quenching group is selected from any one of TAMRA, BHQ1, BHQ2, NFQ and LFN.
6. The detection kit according to claim 3, wherein the sequence of the double-quenching probe is: 5 'FAM-CAAGAGTGC-LFN-CTTGACGATA-MGB 3'.
7. The detection kit according to claim 1, further comprising: DNA polymerase, UNG enzyme, dNTPs and Mg2+。
8. The detection kit according to claim 7, wherein the PCR reaction system in the detection kit comprises, per 25 μ L: 0.5U-1.0U of DNA polymerase, 0.1U-0.5U of UNG enzyme, MgCl21-5mM, 0.1-0.4mM dNTPs, 0.1-0.5. mu.M upstream primer, 0.1. mu.M-0.5. mu.M downstream primer, double quenching probe0.1μM-0.2μM。
9. The detection kit according to claim 1, further comprising: positive control solution and/or blank control solution.
10. Use of the test kit according to any one of claims 1 to 9 for non-disease diagnostic and therapeutic purposes for the detection of KRAS gene mutations.
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