Kit for detecting KRAS and NRAS gene mutation and application

Abstract

The kit for detecting KRAS and NRAS gene mutation is characterized by comprising a KRAS detection reaction solution, an NRAS detection reaction solution, a KRAS positive control, an NRAS positive control, a negative control and a blank control, according to the present invention, based on the high multiplex PCR (Polymerase Chain Reaction) and the high precision capillary electrophoresis method, the kit for detecting the KRAS and NRAS gene mutation is developed, wherein the kit has characteristics of high sensitivity and high detection flux. The kit provided by the invention takes FFPE sample human genome DNA as a detection sample, and can be applied to preparation of a detection reagent by detecting KRAS and NRAS gene mutation. The method is high in sensitivity (can reach 0.5%), detection can be completed only in 150 min, and meanwhile the method has the advantages of being good in specificity, high in accuracy, low in price and easy to operate.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a kit for detecting KRAS and NRAS gene mutations and its application. Background Art

[0002] The RAS gene family is one of the common oncogenes in human tumors, including KRAS, NRAS, and HRAS. Mutations in the RAS gene family are observed in approximately 20% of tumors. Among them, the mutation frequency of KRAS in all tumors is 15.95%, the mutation frequency of NRAS in all tumors is 3.03%, and the mutation frequency of HRAS in all tumors is 0.94%. Clinical studies have shown that the RAS gene mutation status is related to the primary resistance of non-small cell lung cancer to targeted therapeutic drugs such as gefitinib and erlotinib, and the mutation of RAS in rectal cancer patients is related to the drug resistance to drugs such as cetuximab. At present, the detection of RAS gene (KRAS, NRAS) mutations has been listed as a mandatory item for clinical drug use in the "NCCN Clinical Practice Guidelines for Colon Cancer" and the "NCCN Clinical Practice Guidelines for Rectal Cancer".

[0003] Currently, the commonly used gene mutation detection methods mainly include sequencing method and fluorescence quantitative PCR method. These methods all have certain defects. The sequencing method has low sensitivity (only 20%), resulting in a high false negative rate, and has more steps and complex operations. The fluorescence quantitative PCR method is the current mainstream detection method, with high sensitivity and simple operation, but it also has the deficiencies of large demand for detection samples, low detection throughput, and low precision. Therefore, it is crucial to find a gene detection method with low demand for detection samples, high detection throughput, high sensitivity, and high precision. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a kit for detecting KRAS and NRAS gene mutations.

[0005] The second purpose of the present invention is to provide the application of the above kit for detecting KRAS and NRAS gene mutations in the preparation of detection reagents.

[0006] The technical solution of the present invention is outlined as follows:

[0007] The kit for detecting KRAS and NRAS gene mutations includes a KRAS detection reaction solution, an NRAS detection reaction solution, a KRAS positive control, an NRAS positive control, a negative control, and a blank control;

[0008] The KRAS detection reaction solution is composed of KRAS primers and a PCR buffer;

[0009] The KRAS primers are composed of PK-1213-M-F1, PK-12-M1-R1, PK-12-M2-R1, PK-12-M3-R1, PK-12-M4-R1, PK-12-M5-R1, PK-12-M6-R1, PK-13-M1-R1, PK-13-M2-R1, PK-61-M1-F1, PK-61-M2-F1, PK-61-M3-F1, PK-61-M-R1, PK-146-M-F1, PK-146-M-R1, PK-1-F and PK-1-R; or composed of PK-1213-M-F2, PK-12-M1-R2, PK-12-M2-R2, PK-12-M3-R2, PK-12-M4-R2, PK-12-M5-R2, PK-12-M6-R2, PK-13-M1-R2, PK-13-M2-R2, PK-61-M1-F2, PK-61-M2-F2, PK-61-M3-F2, PK-61-M-R2, PK-146-M-F2, PK-146-M-R2, PK-1-F and PK-1-R;

[0010] PK-1213-M-F1, PK-1213-M-F2, PK-12-M1-R1, PK-12-M1-R2, PK-12-M2-R1, PK-12-M2-R2, PK-12-M3-R1, PK-12-M3-R2, PK-12-M4-R1, PK-12-M4-R2, PK-12-M5-R1, PK-12-M5-R2, PK-12-M6-R1, PK-12-M6-R2, PK-13-M1-R1, PK-13-M1-R2, PK-13-M2-R1, PK-13-M2-R2, PK-61-M1-F1, PK-61-M1-F2, PK-61-M2-F1, PK-61-M2-F2, PK-61-M3-F1, PK-61-M3-F2, PK-61-M-R1, PK-61-M-R2, PK-146-M-F1, PK-146-M-F2, PK-146-M-R1, PK-146-M-R2, PK-1-F and PK-1-R are the sequences shown in SEQ ID NO.01 to 32 in sequence;

[0011] The NRAS detection reaction solution is composed of NRAS primers and PCR buffer;

[0012] The NRAS primers consist of PN-1213-M-F1, PN-12-M1-R1, PN-12-M2-R1, PN-12-M3-R1, PN-12-M4-R1, PN-12-M5-R1, PN-12-M6-R1, PN-13-M1-R1, PN-13-M2-R1, PN-61-M1-F1, PN-61-M2-F1, PN-61-M3-F1, PN-61-M4-F1, PN-61-M-R1, PN-1-F and PN-1-R; or consist of PN-1213-M-F2, PN-12-M1-R2, PN-12-M2-R2, PN-12-M3-R2, PN-12-M4-R2, PN-12-M5-R2, PN-12-M6-R2, PN-13-M1-R2, PN-13-M2-R2, PN-61-M1-F2, PN-61-M2-F2, PN-61-M3-F2, PN-61-M4-F2, PN-61-M-R2, PN-1-F and PN-1-R;

[0013] PN-1213-M-F1, PN-1213-M-F2, PN-12-M1-R1, PN-12-M1-R2, PN-12-M2-R1, PN-12-M2-R2, PN-12-M3-R1, PN-12-M3-R2, PN-12-M4-R1, PN-12-M4-R2, PN-12-M5-R1, PN-12-M5-R2, PN-12-M6-R1, PN-12-M6-R2, PN-13-M1-R1, PN-13-M1-R2, PN-13-M2-R1, PN-13-M2-R2, PN-61-M1-F1, PN-61-M1-F2, PN-61-M2-F1, PN-61-M2-F2, PN-61-M3-F1, PN-61-M3-F2, PN-61-M4-F1, PN-61-M4-F2, PN-61-M-R1, PN-61-M-R2, PN-1-F and PN-1-R are the sequences shown in SEQ ID NO.33 - 62 in sequence.

[0014] Preferably, the KRAS positive control is KRAS plasmid DNA; the NRAS positive control is NRAS plasmid DNA; the negative control is wild-type human genomic DNA; the blank control is ddH2O.

[0015] The application of the above kit in preparing a detection reagent includes the following steps:

[0016] (1) Extract human genomic DNA from the FFPE sample;

[0017] (2)Prepare the KRAS reaction system and the NRAS reaction system separately;

[0018] The KRAS reaction system is to add the human genomic DNA extracted from the FFPE sample in step (1), the KRAS positive control, the negative control, and the blank control as templates into the KRAS detection reaction solution respectively;

[0019] The NRAS reaction system is to add the human genomic DNA extracted from the FFPE sample in step (1), the NRAS positive control, the negative control, and the blank control as templates into the NRAS detection reaction solution respectively;

[0020] Perform PCR amplification on the KRAS reaction system and the NRAS reaction system respectively;

[0021] (3)Perform capillary electrophoresis detection on the PCR amplification products respectively;

[0022] (4)Judge the detection results according to the fragment size, fluorescence labeling color, and peak height of the amplification products shown in the capillary electrophoresis peak map;

[0023] The composition of the KRAS detection reaction solution is:

[0024]

[0025] The composition of the NRAS detection reaction solution is:

[0026]

[0027] The conditions for PCR amplification in step (2) are: pre-denaturation at 95°C for 3 minutes, 1 cycle; denaturation at 94°C for 5 seconds, annealing at 51°C for 10 seconds, extension at 68°C for 20 seconds, 2 cycles; denaturation at 94°C for 5 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 20 seconds, 40 cycles; extension at 72°C for 10 minutes, 1 cycle.

[0028] The present invention develops a kit for detecting KRAS and NRAS gene mutations with high sensitivity and high detection throughput based on highly multiplexed PCR and high-precision capillary electrophoresis methods. The kit of the present invention uses the human genomic DNA of FFPE samples as the detection sample. By detecting KRAS and NRAS gene mutations, it can be applied in the preparation of detection reagents. This method has high sensitivity (can reach 0.5%), and the detection can be completed in only 150 minutes. At the same time, it also has the characteristics of good specificity, high accuracy, low price, and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the capillary electrophoresis diagram of the KRAS positive control result in Example 1.

[0030] Figure 2 It is the capillary electrophoresis diagram of the KRAS negative control result in Example 1.

[0031] Figure 3 It is the capillary electrophoresis diagram of the KRAS blank control result in Example 1.

[0032] Figure 4 It is the capillary electrophoresis diagram of the positive result of KRAS clinical sample G12D in Example 1.

[0033] Figure 5 It is the capillary electrophoresis diagram of the positive result of KRAS clinical sample G12R in Example 1.

[0034] Figure 6 It is the capillary electrophoresis diagram of the positive result of KRAS clinical sample G12V in Example 1.

[0035] Figure 7 It is the capillary electrophoresis diagram of the positive result of KRAS clinical sample G12C in Example 1.

[0036] Figure 8 It is the capillary electrophoresis diagram of the positive result of KRAS clinical sample G13D in Example 1.

[0037] Figure 9 It is the capillary electrophoresis diagram of the positive result of KRAS clinical sample Q61H in Example 1.

[0038] Figure 10 It is the capillary electrophoresis diagram of the experimental result of the sensitivity analysis of KRAS clinical samples G12D and G12V in Example 1.

[0039] Figure 11 It is the capillary electrophoresis diagram of the NRAS positive control result in Example 1.

[0040] Figure 12 It is the capillary electrophoresis diagram of the NRAS negative control result in Example 1.

[0041] Figure 13 It is the capillary electrophoresis diagram of the NRAS blank control result in Example 1.

[0042] Figure 14 It is the capillary electrophoresis diagram of the positive result of NRAS clinical sample Q61K in Example 1.

[0043] Figure 15 It is the capillary electrophoresis diagram of the positive result of NRAS clinical sample Q61R in Example 1.

[0044] Figure 16 It is the capillary electrophoresis diagram of the positive result of NRAS clinical sample G12D in Example 1.

[0045] Figure 17 It is the capillary electrophoresis diagram of the sensitivity analysis experiment results of G12D and Q61K in the NRAS clinical sample of Example 1.

[0046] Figure 18 It is the capillary electrophoresis diagram of the KRAS positive control result of Example 2.

[0047] Figure 19 It is the capillary electrophoresis diagram of the KRAS negative control result of Example 2.

[0048] Figure 20 It is the capillary electrophoresis diagram of the KRAS blank control result of Example 2.

[0049] Figure 21 It is the capillary electrophoresis diagram of the positive result of G12D in the KRAS clinical sample of Example 2.

[0050] Figure 22 It is the capillary electrophoresis diagram of the positive result of G12R in the KRAS clinical sample of Example 2.

[0051] Figure 23 It is the capillary electrophoresis diagram of the positive result of G12V in the KRAS clinical sample of Example 2.

[0052] Figure 24 It is the capillary electrophoresis diagram of the positive result of G12C in the KRAS clinical sample of Example 2.

[0053] Figure 25 It is the capillary electrophoresis diagram of the positive result of G13D in the KRAS clinical sample of Example 2.

[0054] Figure 26 It is the capillary electrophoresis diagram of the positive result of Q61H in the KRAS clinical sample of Example 2.

[0055] Figure 27 It is the capillary electrophoresis diagram of the sensitivity analysis experiment results of G12D and G12V in the KRAS clinical sample of Example 2.

[0056] Figure 28 It is the capillary electrophoresis diagram of the NRAS positive control result of Example 2.

[0057] Figure 29 It is the capillary electrophoresis diagram of the NRAS negative control result of Example 2.

[0058] Figure 30 It is the capillary electrophoresis diagram of the NRAS blank control result of Example 2.

[0059] Figure 31It is the capillary electrophoresis diagram of the positive result of Q61K in the NRAS clinical sample of Example 2.

[0060] Figure 32 It is the capillary electrophoresis diagram of the positive result of Q61R in the NRAS clinical sample of Example 2.

[0061] Figure 33 It is the capillary electrophoresis diagram of the positive result of G12D in the NRAS clinical sample of Example 2.

[0062] Figure 34 It is the capillary electrophoresis diagram of the experimental result of the sensitivity analysis of G12D and Q61K in the NRAS clinical sample of Example 2. Detailed implementation manners

[0063] The present invention will be further described below through specific examples.

[0064] The invention adopts specific fluorescent primers, and can realize the rapid detection of KRAS and NRAS gene mutations in FFPE samples.

[0065] Components of 2×PCR buffer

[0066] Table 1 Composition of PCR buffer

[0067]

[0068] The PCR buffers of Example 1 and Example 2 take the second PCR buffer as an example, and the use effects of the first PCR buffer and the third PCR buffer are similar to those of the second.

[0069] SEQ ID numbers of each primer of KRAS:

[0070] PK-1213-M-F1, PK-1213-M-F2, PK-12-M1-R1, PK-12-M1-R2, PK-12-M2-R1, PK-12-M2-R2, PK-12-M3-R1, PK-12-M3-R2, PK-12-M4-R1, PK-12-M4-R2, PK-12-M5-R1, PK-12-M5-R2, PK-12-M6-R1, PK-12-M6-R2, PK-13-M1-R1, PK-13-M1-R2, PK-13-M2-R1, PK-13-M2-R2, PK-61-M1-F1, PK-61-M1-F2, PK-61-M2-F1, PK-61-M2-F2, PK-61-M3-F1, PK-61-M3-F2, PK-61-M-R1, PK-61-M-R2, PK-146-M-F1, PK-146-M-F2, PK-146-M-R1, PK-146-M-R2, PK-1-F and PK-1-R are the sequences shown in SEQ ID NO.01 to 32 in turn;

[0071] NRAS primer sequence numbers:

[0072] PN-1213-M-F1, PN-1213-M-F2, PN-12-M1-R1, PN-12-M1-R2, PN-12-M2-R1, PN-12-M2-R2, PN-12-M3-R1, PN-12-M3-R2, PN-12-M4-R1, PN-12-M4-R2, PN-12-M5-R1, PN-12-M5-R2, PN-12-M6-R1, PN-12-M6-R2, PN-13-M1-R1, PN-13-M1-R2, PN-13-M2-R1, PN-13-M2-R2, PN-61-M1-F1, PN-61-M1-F2, PN-61-M2-F1, PN-61-M2-F2, PN-61-M3-F1, PN-61-M3-F2, PN-61-M4-F1, PN-61-M4-F2, PN-61-M-R1, PN-61-M-R2, PN-1-F and PN-1-R are the sequences shown in SEQ ID NO.33 to 62 in turn.

[0073] The KRAS positive control, NRAS positive control, negative control and blank control in Example 1 and Example 2 are the same, as follows

[0074] The KRAS positive control is KRAS plasmid DNA;

[0075] The KRAS plasmid DNA consists of KRAS G12D, G12A, G12V, G12S, G12R, G12C, G13C, G13D, Q61H, Q61R, Q61L, A146T positive plasmid DNA;

[0076] The NRAS positive control is NRAS plasmid DNA;

[0077] The NRAS plasmid DNA consists of NRAS G12A, G12V, G12D, G12S, G12R, G12C, G13D, G13R, Q61R, Q61H, Q61L, Q61K positive plasmid DNA;

[0078] The negative control is wild-type human genomic DNA;

[0079] The blank control is ddH2O;

[0080] Example 1

[0081] A kit for detecting KRAS and NRAS gene mutations, comprising:

[0082] The first KRAS detection reaction solution, the first NRAS detection reaction solution, the KRAS positive control, the NRAS positive control, the negative control and the blank control;

[0083] The first KRAS detection reaction solution consists of the first KRAS primer and the second PCR buffer;

[0084] The composition of the first KRAS detection reaction solution is:

[0085]

[0086] In the above sequences,

[0087] Experimental results show that the concentration of each primer can also be selected as 0.1 μM or 0.8 μM, and it can form a KRAS detection reaction solution with the second PCR buffer;

[0088] The first NRAS detection reaction solution consists of the first NRAS primer and the second PCR buffer;

[0089] The composition of the first NRAS detection reaction solution is:

[0090]

[0091] Experimental results show that the concentration of each primer can also be selected as 0.1 μM or 0.8 μM, and it can form an NRAS detection reaction solution with the second PCR buffer;

[0092] The application of the above kit in the preparation of detection reagents includes the following steps:

[0093] (1) DNA extraction;

[0094] 1) Plasmid treatment and extraction: The plasmids were extracted using a plasmid extraction kit. The specific extraction procedures were carried out according to the kit instructions. The extracted plasmids were dissolved in Tris-EDTA (10 mmol / L, pH 8.0). The extraction quality was detected by Nanodrop to determine the concentration, and then the plasmid concentration was adjusted to different copy numbers with Tris-EDTA (10 mmol / L, pH 8.0).

[0095] 2) Extraction of human genomic DNA from FFPE samples (samples to be tested): The paraffin tissue sections were processed using a microtome, with a section thickness of approximately 10 μm (it can also be 5 - 20 μm). The number of sections used for each sample was approximately 5. The processed paraffin sections were placed in centrifuge tubes, and then the DNA was extracted using an FFPE DNA extraction kit. The specific extraction procedures were carried out according to the kit instructions. The extracted DNA was dissolved in Tris-EDTA (10 mmol / L, pH 8.0). The extraction quality was detected by Nanodrop to determine the concentration, and then the DNA concentration was adjusted to different concentrations as the PCR template.

[0096] (2) Prepare the first KRAS reaction system and the first NRAS reaction system respectively;

[0097] The first KRAS reaction system was prepared by adding 25 ng of human genomic DNA extracted from FFPE samples in step (1), 5 μL of KRAS positive control, 25 ng of negative control, and 5 μL of blank control as templates into 20 μL of the first KRAS detection reaction solution respectively, and adding water to make up to 25 μL.

[0098] The first NRAS reaction system was prepared by adding 25 ng of human genomic DNA extracted from FFPE samples in step (1), 5 μL of NRAS positive control, 25 ng of negative control, and 5 μL of blank control as templates into 20 μL of the first NRAS detection reaction solution respectively, and adding water to make up to 25 μL.

[0099] PCR amplification was carried out on the first KRAS reaction system and the first NRAS reaction system respectively;

[0100] PCR amplification conditions: Pre-denaturation at 95 °C for 3 minutes, 1 cycle; Denaturation at 94 °C for 5 seconds, annealing at 51 °C for 10 seconds, extension at 68 °C for 20 seconds, 2 cycles; Denaturation at 94 °C for 5 seconds, annealing at 56 °C for 10 seconds, extension at 72 °C for 20 seconds, 40 cycles; Extension at 72 °C for 10 minutes, 1 cycle.

[0101] (3) Using ABI 3500, the PCR amplification products were respectively detected by capillary electrophoresis using fragment analysis; PCR products, denaturant (HI-DI), and fragment size markers (Rox350) were used for detection, and the detection system is shown in Table 2.

[0102] Table 2 Capillary Electrophoresis Detection System

[0103]

[0104] (4) According to the capillary electrophoresis peaks Figure 1 and Figure 11 The detection results were judged based on the fragment size, fluorescence label color, and peak height of the amplified products shown.

[0105] (5) Among all 104 clinical samples tested, 10 cases of KRAS G12D mutation, 7 cases of KRAS G12V mutation, 6 cases of KRAS G13D mutation, 2 cases of KRAS G12C mutation, 1 case of KRAS G12R mutation, 1 case of KRAS Q61H mutation, 1 case of NRAS G12D mutation, 1 case of NRAS Q61R mutation, 2 cases of NRAS Q61K mutation, and 73 negative samples were detected. A blank control was set for each group, and the results are shown in Figures 2 - 9 Figures 12 - 16.

[0106] Sensitivity analysis: The plasmid DNA of KRAS G12D and G12V was diluted in different gradients, and 5 μL of template was added to each reaction for amplification. The results showed that the sensitivity of the detection system of the present invention for these two mutations of KRAS G12D and G12V could reach 0.5%, and the results are shown in Figure 10 Figure. Similarly, the plasmid DNA of NRAS G12D and Q61K was diluted in different gradients, and 5 μL of template was added to each reaction for amplification. The results showed that the sensitivity of the detection system of the present invention for these two mutations of NRAS G12D and Q61K could reach 0.5%, and the results are shown in Figure 17 Figure.

[0107] Example 2

[0108] A kit for detecting KRAS and NRAS gene mutations, comprising:

[0109] A second KRAS detection reaction solution, a second NRAS detection reaction solution, a KRAS positive control, an NRAS positive control, a negative control, and a blank control;

[0110] The second KRAS detection reaction solution is composed of a second KRAS primer and a second PCR buffer;

[0111] The composition of the second KRAS detection reaction solution is as follows:

[0112]

[0113] Experimental results show that the concentration of each primer can also be selected as 0.1 μM or 0.8 μM, and the second PCR buffer is used to form the KRAS detection reaction solution.

[0114] The second NRAS detection reaction solution is composed of the second NRAS primer and the second PCR buffer.

[0115] The composition of the second NRAS detection reaction solution is as follows:

[0116]

[0117] Experimental results show that the concentration of each primer can also be selected as 0.1 μM or 0.8 μM, and the second PCR buffer is used to form the NRAS detection reaction solution.

[0118] The application of the above kit in the preparation of detection reagents includes the following steps:

[0119] (1) Extract DNA;

[0120] 1) Plasmid treatment and extraction: The extraction of each plasmid is carried out using a plasmid extraction kit, and the specific extraction operation steps are carried out according to the kit instructions. The extracted plasmid is dissolved in Tris-EDTA (10 mmol / L, pH 8.0), and the extraction quality is detected by Nanodrop to determine its concentration, and then the plasmid concentration is adjusted to different copy numbers with Tris-EDTA (10 mmol / L, pH 8.0).

[0121] 2) Extract human genomic DNA from FFPE samples (samples to be tested): Use a microtome to process paraffin tissue sections, with a section thickness of about 10 μm (it can also be 5 - 20 μm), and about 5 sections are used for each sample. Place the processed paraffin sections in a centrifuge tube, and then the DNA extraction is carried out using an FFPE DNA extraction kit, and the specific extraction operation steps are carried out according to the kit instructions. The extracted DNA is dissolved in Tris-EDTA (10 mmol / L, pH 8.0), and the extraction quality is detected by Nanodrop to determine its concentration, and then the DNA concentration is adjusted to different concentrations as the PCR template.

[0122] (2) Prepare the second KRAS reaction system and the second NRAS reaction system respectively;

[0123] The second KRAS reaction system is prepared by respectively adding 25 ng of human genomic DNA extracted from the FFPE sample, 5 μL of KRAS positive control, 25 ng of negative control, and 5 μL of blank control as templates into 20 μL of the second KRAS detection reaction solution, and adding water to make up to 25 μL.

[0124] The second NRAS reaction system is prepared by respectively adding 25 ng of human genomic DNA extracted from the FFPE sample, 5 μL of NRAS positive control, 25 ng of negative control, and 5 μL of blank control as templates into 20 μL of the second NRAS detection reaction solution, and adding water to make up to 25 μL.

[0125] Perform PCR amplification on the second KRAS reaction system and the second NRAS reaction system respectively;

[0126] PCR amplification conditions: pre-denaturation at 95°C for 3 minutes, 1 cycle; denaturation at 94°C for 5 seconds, annealing at 51°C for 10 seconds, extension at 68°C for 20 seconds, 2 cycles; denaturation at 94°C for 5 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 20 seconds, 40 cycles; extension at 72°C for 10 minutes, 1 cycle.

[0127] (3) Use ABI 3500 to perform capillary electrophoresis detection on the PCR amplification products respectively by the method of fragment analysis; detect using PCR products, denaturant (HI-DI), and fragment size marker (Rox350), and the detection system is shown in Table 2.

[0128] (4) Based on the capillary electrophoresis peaks Figure 18 and Figure 28 judge the detection results according to the fragment size, fluorescence label color, and peak height of the amplified products shown.

[0129] (5) Among all 104 clinical samples detected, 10 cases of KRAS G12D mutation, 7 cases of KRAS G12V mutation, 6 cases of KRAS G13D mutation, 2 cases of KRAS G12C mutation, 1 case of KRAS G12R mutation, 1 case of KRAS Q61H mutation, 1 case of NRAS G12D mutation, 1 case of NRAS Q61R mutation, 2 cases of NRAS Q61K mutation, and 73 negative samples were detected. A blank control was set for each group at the same time, and the results are shown in Figures 19 - 26 and 29 - 33.

[0130] Sensitivity analysis: Dilute plasmid DNA of KRAS G12D and G12V with different gradients, and add 5 μL of template for amplification reaction each time. The results show that the sensitivity of the detection system of the present invention for these two mutations of KRAS G12D and G12V can reach 0.5%, and the results are as shown in Figure 27Similarly, plasmid DNA of NRAS G12D and Q61K was diluted in different gradients, and 5 μL of the template was added to each reaction for amplification. The results showed that the sensitivity of the detection system of the present invention for these two mutations, NRAS G12D and Q61K, could reach 0.5%. The results are as shown in Figure 34 .

[0131] Information on all mutation types detected by the kit of the present invention is shown in Table 3. These clinical samples were detected by fluorescence quantitative PCR for comparison. The results of Examples 1 and 2 showed that the coincidence rate between the system of the present invention and the fluorescence quantitative PCR method reached 100%, as shown in Table 4, further demonstrating the accuracy, rapidity, and low cost of the detection by the system of the present invention. It can be seen that when using the present invention to detect KRAS and NRAS gene mutations in FFPE samples, compared with the fluorescence quantitative PCR method, it has the same accuracy, but is faster and has lower cost, and can meet the rapid detection of KRAS and NRAS gene mutations.

[0132] Table 3 Information on all mutation types detected by the kit

[0133]

[0134] Table 4 Comparison results between capillary electrophoresis method and fluorescence quantitative PCR method

[0135]

[0136] Table 5 Primer sequences of SEQ ID NO.01 - 32

[0137]

[0138] Table 6 Primer sequences of SEQ ID NO.33 - 62

[0139]

Claims

1. A kit for detecting KRAS and NRAS gene mutations, characterized in that It includes a KRAS detection reaction solution, an NRAS detection reaction solution, a KRAS positive control, an NRAS positive control, a negative control, and a blank control; The KRAS detection reaction solution is composed of a KRAS primer and a PCR buffer; The KRAS primer is composed of PK-1213-M-F1, PK-12-M1-R1, PK-12-M2-R1, PK-12-M3-R1, PK-12-M4-R1, PK-12-M5-R1, PK-12-M6-R1, PK-13-M1-R1, PK-13-M2-R1, PK-61-M1-F1, PK-61-M2-F1, PK-61-M3-F1, PK-61-M-R1, PK-146-M-F1, PK-146-M-R1, PK-1-F, and PK-1-R; or it is composed of PK-1213-M-F2, PK-12-M1-R2, PK-12-M2-R2, PK-12-M3-R2, PK-12-M4-R2, PK-12-M5-R2, PK-12-M6-R2, PK-13-M1-R2, PK-13-M2-R2, PK-61-M1-F2, PK-61-M2-F2, PK-61-M3-F2, PK-61-M-R2, PK-146-M-F2, PK-146-M-R2, PK-1-F, and PK-1-R; PK-1213-M-F1, PK-1213-M-F2, PK-12-M1-R1, PK-12-M1-R2, PK-12-M2-R1, PK-12-M2-R2, PK-12-M3-R1, PK-12-M3-R2, PK-12-M4-R1, PK-12-M4-R2, PK-12-M5-R1, PK-12-M5-R2, PK-12-M6-R1, PK-12-M6-R2, PK-13-M1-R1, PK-13-M1-R2, PK-13-M2-R1, PK-13-M2-R2, PK-61-M1-F1, PK-61-M1-F2, PK-61-M2-F1, PK-61-M2-F2, PK-61-M3-F1, PK-61-M3-F2, PK-61-M-R1, PK-61-M-R2, PK-146-M-F1, PK-146-M-F2, PK-146-M-R1, PK-146-M-R2, PK-1-F, and PK-1-R are the sequences shown in SEQ ID NO.01 to 32 in sequence; The NRAS detection reaction solution is composed of an NRAS primer and a PCR buffer; The NRAS primers consist of PN-1213-M-F1, PN-12-M1-R1, PN-12-M2-R1, PN-12-M3-R1, PN-12-M4-R1, PN-12-M5-R1, PN-12-M6-R1, PN-13-M1-R1, PN-13-M2-R1, PN-61-M1-F1, PN-61-M2-F1, PN-61-M3-F1, PN-61-M4-F1, PN-61-M-R1, PN-1-F and PN-1-R; or consist of PN-1213-M-F2, PN-12-M1-R2, PN-12-M2-R2, PN-12-M3-R2, PN-12-M4-R2, PN-12-M5-R2, PN-12-M6-R2, PN-13-M1-R2, PN-13-M2-R2, PN-61-M1-F2, PN-61-M2-F2, PN-61-M3-F2, PN-61-M4-F2, PN-61-M-R2, PN-1-F and PN-1-R; PN-1213-M-F1, PN-1213-M-F2, PN-12-M1-R1, PN-12-M1-R2, PN-12-M2-R1, PN-12-M2-R2, PN-12-M3-R1, PN-12-M3-R2, PN-12-M4-R1, PN-12-M4-R2, PN-12-M5-R1, PN-12-M5-R2, PN-12-M6-R1, PN-12-M6-R2, PN-13-M1-R1, PN-13-M1-R2, PN-13-M2-R1, PN-13-M2-R2, PN-61-M1-F1, PN-61-M1-F2, PN-61-M2-F1, PN-61-M2-F2, PN-61-M3-F1, PN-61-M3-F2, PN-61-M4-F1, PN-61-M4-F2, PN-61-M-R1, PN-61-M-R2, PN-1-F and PN-1-R are the sequences shown in SEQ ID NO.33 - 62 in sequence.

2. The kit according to claim 1, characterized in that The KRAS positive control is KRAS plasmid DNA; the NRAS positive control is NRAS plasmid DNA; the negative control is wild-type human genomic DNA; the blank control is ddH2O.

3. Use of the kit of claim 1 or 2 in the preparation of a detection reagent, characterized in that It includes the following steps: (1) Extract human genomic DNA from FFPE samples; (2) Prepare KRAS reaction system and NRAS reaction system respectively; The KRAS reaction system is to add the human genomic DNA extracted from FFPE samples in step (1), KRAS positive control, negative control, and blank control as templates into the KRAS detection reaction solution respectively; The NRAS reaction system is to add the human genomic DNA extracted from the FFPE sample, the NRAS positive control, the negative control, and the blank control as templates into the NRAS detection reaction solution respectively; Perform PCR amplification on the KRAS reaction system and the NRAS reaction system respectively; (3) Perform capillary electrophoresis detection on the PCR amplification products respectively; (4) Judge the detection results according to the fragment size, fluorescence labeling color, and peak height of the amplification products shown in the capillary electrophoresis peak map; The composition of the KRAS detection reaction solution is: ; The composition of the NRAS detection reaction solution is:

4. The use according to claim 3, characterized in that The conditions for PCR amplification in step (2) are: pre-denaturation at 95°C for 3 minutes, 1 cycle; denaturation at 94°C for 5 seconds, annealing at 51°C for 10 seconds, extension at 68°C for 20 seconds, 2 cycles; denaturation at 94°C for 5 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 20 seconds, 40 cycles; extension at 72°C for 10 minutes, 1 cycle.

Images