Primer probe combination and kit for detecting KRAS gene mutation and application of primer probe combination and kit

Abstract

The invention discloses a primer probe combination and a kit for detecting KRAS gene mutation and application of the primer probe combination and the kit, and belongs to the technical field of biology. By optimizing the sequence of the upstream primer (also called hairpin primer), the primer group capable of specifically closing wild type template amplification and highly selectively amplifying a KRAS gene mutation type template is obtained, the primer group has extremely high sensitivity when being used for detecting KRAS gene mutation, and the primer group does not need special modification of a basic group, so that the primer group can be applied to detection of KRAS gene mutation. The method has the advantages that the method is simple in operation and low in cost, rare mutation as low as one ten thousandth can be detected under qPCR conditions, the method is simple and convenient in operation process and easy to apply to conventional molecular detection laboratories, and the method is simple to operate and low in cost, and does not depend on expensive equipment; therefore, the method has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer-probe combination, kit, and application for detecting KRAS gene mutations. Background Technology

[0002] Colorectal cancer is one of the most common digestive tract tumors, and its incidence and mortality rates rank among the highest in my country. Cetuximab, as a targeted therapy, plays an important role in the treatment of colorectal cancer. Multiple clinical studies have shown that only patients with advanced colorectal cancer of the KRAS wild-type type can clearly benefit from cetuximab treatment, while there is no clear benefit for patients with KRAS gene mutations.

[0003] Therefore, multiple treatment guidelines and expert consensus both domestically and internationally clearly recommend that KRAS gene mutation testing must be performed on patients before using cetuximab. Currently, in clinical practice, the main sources of specimens for KRAS testing in colorectal cancer are tumor tissue and peripheral blood. Among these, tissue testing is considered the "gold standard" for genotyping, but in some patients, obtaining qualified tissue samples is extremely difficult due to the difficulty in puncturing the tumor at its location and insufficient sample volume. In such cases, circulating tumor DNA (ctDNA) testing based on peripheral blood shows important significance. However, ctDNA accounts for a very low percentage of total cell-free DNA in blood, placing extremely high demands on the sensitivity of the detection method. Currently, common mutation detection methods mainly include ARMS-PCR, digital PCR, and high-throughput detection technologies based on next-generation sequencing (NGS). ARMS technology is characterized by its simplicity, speed, low cost, and high technological availability, but its limited detection sensitivity restricts its application to some extent. Digital PCR is particularly suitable for detecting extremely low abundance mutations and does not require complex data interpretation; however, its instruments and dedicated consumables are expensive, limiting its clinical adoption. The advantage of NGS technology lies in its high throughput, but its operation process is complex, the detection cycle is long, and it requires high levels of personnel skills and bioinformatics analysis capabilities.

[0004] Therefore, there is an urgent need in this field to develop a new detection method that can ensure high sensitivity while also being easy to operate and low in cost, so as to better meet the needs of clinical testing. Summary of the Invention

[0005] The purpose of this invention is to provide a primer-probe combination, kit, and application for detecting KRAS gene mutations. This addresses the problems of low sensitivity, high cost, and complex operation in existing methods for detecting KRAS gene mutations.

[0006] In a first aspect, the present invention provides a primer-probe combination for detecting KRAS gene mutations. The primer-probe combination includes a primer set, which includes a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, and a seventh primer pair. The nucleotide sequences of the upstream primers of the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, and the seventh primer pair are shown in SEQ ID NO. 1-7, respectively, and the nucleotide sequence of the universal downstream primer is shown in SEQ ID NO. 8.

[0007] In this invention, the inventors discovered that by optimizing the sequence of the upstream primer (also known as a hairpin primer), a primer set was obtained that can specifically block the amplification of wild-type templates and highly selectively amplify KRAS gene mutant templates. Using this primer set for the detection of KRAS gene mutations has extremely high sensitivity. Furthermore, this primer set does not require special base modification, is low in cost, and can detect rare mutations as low as one in ten thousand under qPCR conditions. It has the advantages of simple operation, low cost, and does not rely on expensive equipment. The operation process is simple and easy to apply in routine molecular detection laboratories.

[0008] In some embodiments, the primer-probe combination also includes a universal probe, the nucleotide sequence of which is shown in SEQ ID NO.9; and the universal probe is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.

[0009] In some embodiments, the fluorescent group includes at least one of FAM, HEX, VIC, ROX, and CY5; and / or, the quenching group includes at least one of BHQ1, BHQ2, and MGB.

[0010] In a second aspect, the present invention provides the use of any of the primer-probe combinations described above in the preparation of products for detecting KRAS gene mutations.

[0011] In a third aspect, the present invention provides a kit for detecting KRAS gene mutations, the kit comprising any of the primer-probe combinations described above.

[0012] In some implementations, the kit also includes at least one of the following: reagents required for PCR amplification, a positive control, and a negative control.

[0013] In some implementations, the concentrations of the upstream primer, universal downstream primer, and universal probe in the primer-probe combination are all 0.2–0.4 μM.

[0014] In a fourth aspect, the present invention provides a method for detecting KRAS gene mutations, comprising the following steps: using DNA in the sample to be tested as a template, performing real-time PCR using any of the above-mentioned primer-probe combinations or any of the above-mentioned kits, and determining the results based on the real-time PCR results.

[0015] In some implementations, the reaction procedure for quantitative real-time PCR is as follows: 95°C for 5 min; 95°C for 15 s, 60°C for 60 s, 50 cycles.

[0016] In some implementation schemes, the result determination based on the quantitative real-time PCR results specifically includes: determining the result based on the amplification curve and Ct value; wherein, when there is a clear S curve and the Ct value is <39.0, it can be determined as KRAS mutation positive; when there is no clear S curve or the Ct value is ≥39.0, it can be determined as KRAS mutation negative.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention optimizes the sequence of the upstream primers (also known as hairpin primers) to obtain a primer set that can specifically block wild-type template amplification and highly selectively amplify KRAS gene mutant templates. Using this primer set for KRAS gene mutation detection has extremely high sensitivity. Furthermore, this primer set does not require special base modification, is low in cost, and can detect rare mutations as low as one in ten thousand under qPCR conditions. It has the advantages of simple operation, low cost, and does not rely on expensive equipment. The operation process is simple and easy to implement in routine molecular detection laboratories; therefore, it has good application prospects. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the detection principle of the hairpin primers designed for KRAS gene mutations in Example 1 of the present invention. Figure 2 The amplification curve of KRAS G12D detected by allele-specific PCR in Example 3 of this invention; Figure 3 The amplification curve of KRAS G12D detected using the kit of the present invention in Example 3 of the present invention; Figure 4 The amplification curve of KRAS G12C detected by allele-specific PCR in Example 3 of this invention; Figure 5 The amplification curve of KRAS G12C detected using the kit of the present invention in Example 3 of the present invention; Figure 6 The amplification curve of KRAS G12S detected by allele-specific PCR in Example 3 of this invention; Figure 7The amplification curve of KRAS G12S detected using the kit of the present invention in Example 3 of the present invention; Figure 8 The amplification curve of KRAS G12R detected by allele-specific PCR in Example 3 of this invention; Figure 9 The amplification curve of KRAS G12R detected using the kit of the present invention in Example 3 of the present invention; Figure 10 The amplification curve of KRAS G12V detected by allele-specific PCR in Example 3 of this invention; Figure 11 The amplification curve of KRAS G12V detected using the kit of the present invention in Example 3 of the present invention; Figure 12 The amplification curve of KRAS G12A detected by allele-specific PCR in Example 3 of this invention; Figure 13 The amplification curve of KRAS G12A detected using the kit of the present invention in Example 3 of the present invention; Figure 14 The amplification curve of KRAS G13D detected by allele-specific PCR in Example 3 of this invention; Figure 15 The amplification curve of KRAS G13D detected using the kit of the present invention in Example 3 of the present invention; Figure 16 This is the amplification curve of the KRAS G12D mutant template with different mutation ratios detected in Example 4 of the present invention; Figure 17 This is the amplification curve of the KRAS G12C mutant template with different mutation ratios detected in Example 4 of the present invention; Figure 18 This is the amplification curve of the KRAS G12S mutant template with different mutation ratios detected in Example 4 of the present invention; Figure 19 This is the amplification curve of the KRAS G12R mutant template with different mutation ratios detected in Example 4 of the present invention; Figure 20 This is the amplification curve of the KRAS G12V mutant template with different mutation ratios detected in Example 4 of the present invention; Figure 21 This is the amplification curve of the KRAS G12A mutant template with different mutation ratios detected in Example 4 of the present invention; Figure 22 This is the amplification curve of the KRAS G13D mutant template with different mutation ratios detected in Example 4 of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0021] Currently, existing methods for detecting KRAS gene mutations suffer from problems such as low sensitivity, high cost, and complex operation.

[0022] To address the problems of low sensitivity, high cost, and complex operation in existing methods for detecting KRAS gene mutations, this invention provides a primer-probe combination, kit, and its application for detecting KRAS gene mutations.

[0023] In a first aspect, the present invention provides a primer-probe combination for detecting KRAS gene mutations. The primer-probe combination includes a primer set, which includes a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, and a seventh primer pair. The nucleotide sequences of the upstream primers of the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, and the seventh primer pair are shown in SEQ ID NO. 1-7, respectively, and the nucleotide sequence of the universal downstream primer is shown in SEQ ID NO. 8.

[0024] Specifically, in this invention, KRAS gene mutations include seven hotspot mutations in codons 12 and 13 of exon 2 of the KRAS gene. Preferably, the mutations specifically include G12D, G12C, G12S, G12R, G12V, G12A, and G13D. The first primer pair is used to detect the G12D mutation, the second primer pair is used to detect the G12C mutation, the third primer pair is used to detect the G12S mutation, the fourth primer pair is used to detect the G12R mutation, the fifth primer pair is used to detect the G12V mutation, the sixth primer pair is used to detect the G12A mutation, and the seventh primer pair is used to detect the G13D mutation.

[0025] In the primer-probe combination provided by this invention, the sequence of the upstream primer (also known as a hairpin primer) is optimized. Specifically, the hairpin primer consists of a hairpin sequence and an allele-specific primer sequence. The hairpin sequence further includes a blocking sequence and a stem sequence. The blocking sequence is designed to cover the KRAS gene mutation site and can pair complementaryly with the wild-type template, but does not bind to the mutant template. For the mutant template, since the blocking sequence does not pair with it, the allele-specific primer sequence can successfully complement and initiate the extension reaction. For the wild-type template, the blocking sequence preferentially binds to it, thereby spatially blocking the possibility of the allele-specific primer sequence pairing with the wild-type template and effectively inhibiting non-specific amplification. Therefore, the primer set designed as described above can both specifically block the amplification of wild-type templates and highly selectively amplify KRAS gene mutant templates. When using this primer set for KRAS gene mutation detection, it has extremely high sensitivity. Furthermore, this primer set does not require special base modification, is low in cost, and can detect rare mutations as low as one in ten thousand under qPCR conditions. It has the advantages of simple operation and low cost, does not rely on expensive equipment, has a simple operation procedure, and is easy to apply in routine molecular detection laboratories.

[0026] In some embodiments, the primer-probe combination also includes a universal probe, the nucleotide sequence of which is shown in SEQ ID NO.9; and the universal probe is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.

[0027] In this invention, a specific universal probe is designed and used in conjunction with a primer set to achieve efficient and rapid detection of KRAS gene mutations under qPCR detection conditions.

[0028] In some embodiments, the fluorescent group includes at least one of FAM, HEX, VIC, ROX, and CY5; and / or, the quenching group includes at least one of BHQ1, BHQ2, and MGB.

[0029] Understandably, the types of fluorescent groups and quencher groups can be conventionally selected according to actual usage needs, as long as they can efficiently detect KRAS gene mutations. For example, in this invention, the fluorescent group preferably includes at least one of FAM, HEX, VIC, ROX, and CY5; the quencher group preferably includes at least one of BHQ1, BHQ2, and MGB.

[0030] In a second aspect, the present invention provides the use of any of the primer-probe combinations described above in the preparation of products for detecting KRAS gene mutations.

[0031] In a third aspect, the present invention provides a kit for detecting KRAS gene mutations, the kit comprising any of the primer-probe combinations described above.

[0032] The kit provided by this invention has the advantages of high sensitivity, simple operation and low cost, and therefore has good application prospects in the detection of KRAS gene mutations.

[0033] In some implementations, the kit also includes at least one of the following: reagents required for PCR amplification, a positive control, and a negative control.

[0034] In some implementations, the reagents required for PCR amplification include at least one of PCR buffer, hot-start Taq DNA polymerase, and sterile water; preferably, the PCR buffer includes 2×PCR buffer (containing MgCl2), and the hot-start Taq DNA polymerase includes 0.02-0.04 U / μL hot-start Taq DNA polymerase.

[0035] Understandably, the components in the kit can be routinely adjusted according to actual usage needs, as long as they can efficiently detect KRAS gene mutations.

[0036] In some implementations, the concentrations of the upstream primer, universal downstream primer, and universal probe in the primer-probe combination are all 0.2-0.4 μM, for example, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, or other values ​​within this range.

[0037] In this invention, by controlling the concentrations of the upstream primer, universal downstream primer, and universal probe in the primer-probe combination within a specific range, highly sensitive detection of KRAS gene mutations can be achieved.

[0038] In a fourth aspect, the present invention provides a method for detecting KRAS gene mutations, comprising the following steps: using DNA in the sample to be tested as a template, performing real-time PCR using any of the above-mentioned primer-probe combinations or any of the above-mentioned kits, and determining the results based on the real-time PCR results.

[0039] The method provided by this invention is simple, low-cost, and the detection results have good reliability.

[0040] In some implementations, the reaction procedure for quantitative real-time PCR is as follows: 95°C for 5 min; 95°C for 15 s, 60°C for 60 s, 50 cycles.

[0041] Understandably, the reaction procedure parameters for quantitative real-time PCR can be routinely adjusted according to actual needs, as long as efficient amplification can be achieved.

[0042] In some implementations, the sample to be tested includes at least one of paraffin-embedded tissue specimens, freshly frozen tissue specimens, and plasma specimens.

[0043] In some implementation schemes, the result determination based on the quantitative real-time PCR results specifically includes: determining the result based on the amplification curve and Ct value; wherein, when there is a clear S curve and the Ct value is <39.0, it can be determined as KRAS mutation positive; when there is no clear S curve or the Ct value is ≥39.0, it can be determined as KRAS mutation negative.

[0044] The result determination method provided by this invention is simple and can efficiently and quickly detect KRAS gene mutations in the test sample.

[0045] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0046] Example 1: Design of Primer-Probe Combinations In this embodiment, corresponding detection primer-probe combinations were designed for seven hotspot mutations (specifically G12D, G12C, G12S, G12R, G12V, G12A, and G13D) in codons 12 and 13 of exon 2 of the KRAS gene.

[0047] Specifically, the primer-probe combination consists of an upstream primer (hairpin primer) for detecting the above mutations, a universal downstream primer, and a universal probe.

[0048] Among them, hairpin primers (detection principle as follows) Figure 1 (As shown) It consists of a hairpin sequence and an allele-specific primer sequence, with the hairpin sequence composed of a blocking sequence and a stem sequence. The blocking sequence covers the KRAS gene mutation site, is complementary to the wild-type template, and is 18-35 bases in length, with a Tm value 5-10°C higher than that of the allele-specific primer sequence. The stem sequence is located at both ends of the blocking sequence and can pair with each other to form a double-stranded stem structure, with a length of 5-7 bases. The allele-specific primer sequence can have an additional mismatched base added at the 2nd-3rd position from the end of the 3' end of the primer to increase specificity, with a Tm value of 57-63°C. The universal downstream primer is located downstream of the hairpin primer, with a Tm value of 57-63°C. The universal fluorescent probe has a Tm value of 65-70°C, with fluorescent and quenching groups labeled at the 5' and 3' ends, respectively.

[0049] Furthermore, the hairpin primer sequences for detecting the G12D mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcAACTTGTGGTAGTTGGAGCGGA-3' (SEQ ID NO. 1); The hairpin primer sequences for detecting the G12C mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcGAATATAAACTTGTGGTAGTTGGAGCAT-3' (SEQ ID NO. 2); The hairpin primer sequences for detecting the G12S mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcGACTGAATATAAACTTGTGGTAGTTGGAGATA-3' (SEQ ID NO. 3); The hairpin primer sequences for detecting the G12R mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcCTGAATATAAACTTGTGGTAGTTGGAGATC-3' (SEQ ID NO. 4); The hairpin primer sequences for detecting the G12V mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcGAATATAAACTTGTGGTAGTTGGAGCTCT-3' (SEQ ID NO. 5); The hairpin primer sequences for detecting the G12A mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcGAATATAAACTTGTGGTAGTTGGAGCTTC-3' (SEQ ID NO. 6); The hairpin primer sequences for detecting the G13D mutation are shown below: 5'-gcaccgagttggagctggtggcgtaggcaacggtgcGTGGTAGTTGGAGCTGGGGA-3' (SEQ IDNO.7); In the hairpin primer sequences mentioned above, lowercase base letters represent hairpin sequences, and uppercase base letters represent allele-specific primer sequences.

[0050] The universal downstream primer sequence is shown below: 5'-TGGATCATATTCGTCCACAAAATG-3' (SEQ ID NO. 8); The universal probe sequence is shown below: 5'-TGCCTTGACGATACAGC-3' (SEQ ID NO.9); The universal probe is labeled with a FAM fluorescent group at its 5' end and an MGB quencher group at its 3' end.

[0051] Example 2: Construction of the kit and method for detecting KRAS gene mutations using the kit. In this embodiment, a kit for detecting KRAS gene mutations was further constructed based on the primer-probe combination designed in Example 1.

[0052] Specifically, the kit includes the following components: 2×PCR Buffer (containing MgCl2), 0.2-0.4μM hairpin primer, 0.2-0.4μM universal downstream primer, 0.2-0.4μM universal fluorescent probe, 0.02-0.04U / μL hot-start Taq DNA polymerase, sterile water, positive control, and negative control.

[0053] Furthermore, a method for detecting KRAS gene mutations is provided, comprising the following steps: 1) Using DNA (extracted using conventional methods) from the sample to be tested (paraffin-embedded tissue specimen, fresh frozen tissue specimen, or plasma specimen) as a template, dispense the prepared PCR reaction mixture into PCR reaction tubes at a volume of 30 μL per well; add the DNA of the sample to be tested, the positive control, and the negative control to the PCR reaction tubes containing the PCR reaction mixture, at a volume of 10 μL / well, with the DNA concentration of the sample to be tested being 15 ng / μL; carefully cap the PCR tubes and centrifuge briefly for a few seconds.

[0054] 2) Place the above PCR reaction tubes in a real-time PCR instrument. The total reaction volume of qPCR is 40 μL. Set the amplification reaction program as follows: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 60 s (during which fluorescence signal is collected), for 50 cycles.

[0055] 3) After the reaction is complete, determine the appropriate fluorescence threshold based on the amplification curve and perform result analysis. When there is an obvious S curve and the Ct value is <39.0, it can be determined as KRAS mutation positive; when there is no obvious S curve or the Ct value is ≥39.0, it can be determined as KRAS mutation negative.

[0056] Example 3: Specificity verification of the kit To verify the specificity of the kit of the present invention, it was subjected to parallel experiments with the allele-specific PCR method.

[0057] Specifically, the negative reference was wild-type HT-29 cell genomic DNA at a concentration of 15 ng / μL, and the positive reference was a simulated sample containing 1% mutation ratio against a wild-type background at a concentration of 15 ng / μL. The simulated sample was a mixture of mutant plasmid DNA and wild-type HT-29 cell genomic DNA in a specific ratio. Detection was performed according to the method described in Example 2. After detection, the specificity of the kit was assessed by calculating the ΔCt value.

[0058] The formula for calculating the ΔCt value is: ΔCt = Ct value of negative reference sample - Ct value of positive reference sample. A higher ΔCt value indicates a stronger inhibitory effect of the kit on the amplification of non-target sequences, i.e., better specificity. Furthermore, if a sample is not detected, its Ct value is substituted with the maximum cycle number.

[0059] Test results show that, when testing the same negative reference sample, compared to allele-specific PCR methods (see...), Figure 2 , 4 (6, 8, 10, 12, 14), the kits in this invention may not exhibit obvious nonspecific amplification signals (see [reference]). Figure 3 , 5 , 7), or their non-specific amplification curves are significantly slower than those of allele-specific PCR methods (see Figure 9 , 11 (13, 15). The above results demonstrate that the kit of the present invention is significantly superior to the allele-specific PCR method in terms of detection specificity.

[0060] Furthermore, the statistical results of △Ct values ​​are shown in Table 1 below.

[0061] Table 1. Specificity test results of the kit

[0062] As can be seen from Table 1, when using the kit of the present invention for detection, the ΔCt value is significantly higher than that of the allele-specific PCR detection method, which further indicates that the kit of the present invention has good specificity.

[0063] Example 4: Sensitivity Verification of the Reagent Kit To verify the limit of detection of the kit of the present invention, mutant plasmid DNA and wild-type HT-29 cell genomic DNA were mixed in a certain proportion to prepare positive references at 15 ng / μL against a wild-type genomic background with mutation ratios of 10%, 1%, 0.1%, and 0.01%, respectively. Simultaneously, a mutant plasmid of 3333 copies / μL was used as a positive reference with a 100% mutation ratio, and detection was performed according to the method in Example 2. The results are as follows: Figure 16-22 As shown.

[0064] from Figure 16-22 As can be seen, the detection system of this kit can effectively detect G12D, G12C, G12S, G12R, G12V, G12A, and G13D mutations, with a sensitivity of 0.01% (one in ten thousand). The results indicate that the kit in this invention has high sensitivity.

[0065] In summary, this invention optimizes the sequence of the upstream primers (also known as hairpin primers) to obtain a primer set that can specifically block wild-type template amplification and highly selectively amplify KRAS gene mutant templates. Using this primer set for KRAS gene mutation detection has extremely high sensitivity. Furthermore, this primer set does not require special base modification, is low in cost, and can detect rare mutations as low as one in ten thousand under qPCR conditions. It has the advantages of simple operation and low cost.

[0066] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0067] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A primer probe combination for detecting a KRAS gene mutation, characterized by, The primer probe combination comprises a primer set comprising a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, and a seventh primer pair. The nucleotide sequences of the upstream primers of the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, and the seventh primer pair are shown in SEQ ID NO. 1-7, respectively, and the nucleotide sequence of the universal downstream primer is shown in SEQ ID NO.

8.

2. The primer probe combination according to claim 1, characterized in that, The primer probe combination further comprises a universal probe, and the nucleotide sequence of the universal probe is shown in SEQ ID NO. 9; and the 5' end of the universal probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

3. The primer probe combination of claim 2, wherein, The fluorescent group comprises at least one of FAM, HEX, VIC, ROX, and CY5; and / or the quenching group comprises at least one of BHQ1, BHQ2, and MGB.

4. Use of the primer probe combination according to any one of claims 1-3 in the preparation of a product for detecting KRAS gene mutation.

5. A kit for detecting a mutation in the KRAS gene, characterized in that, The kit comprises the primer probe combination according to any one of claims 1-3.

6. The kit of claim 5, wherein The kit further comprises at least one of reagents required for PCR amplification, a positive control, and a negative control.

7. The kit according to claim 5 or 6, characterized in that, The concentrations of the upstream primers, the universal downstream primer, and the universal probe in the primer probe combination are all 0.2-0.4 μM.

8. A method of detecting a mutation in the KRAS gene, characterized in that, The method comprises the following steps: DNA in the sample to be tested is used as a template, and the primer probe combination according to any one of claims 1-3 or the kit according to any one of claims 5-7 is used to perform fluorescent quantitative PCR, and the results are determined according to the results of the fluorescent quantitative PCR.

9. The method of claim 8, wherein, The reaction program of the fluorescent quantitative PCR is as follows: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 60 s, 50 cycles.

10. The method of claim 8, wherein, The determination of the results according to the results of the fluorescent quantitative PCR specifically comprises determining the results according to the amplification curve and the Ct value; When there is an obvious S curve and the Ct value is < 39.0, it is determined that the KRAS mutation is positive; when there is no obvious S curve or the Ct value is ≥ 39.0, it is determined that the KRAS mutation is negative.

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