A primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations and its application

Abstract

The present invention discloses a primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations and its application. The primer-probe combination includes primers and probes for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations. The present invention designs a primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, and uses digital PCR to qualitatively detect 14 hotspot mutations of KRAS, NRAS, BRAF and PIK3CA genes in tumors, with good specificity and sensitivity, simple operation and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations and an application thereof. Background Art

[0002] KRAS, NRAS, BRAF and PIK3CA genes play a role as molecular checkpoints in the downstream RAS-RAF-MEK-ERK pathway and PI3K-AKT-mTOR pathway of the EGFR signal transduction pathway, and are closely related to the occurrence and development of tumors. Mutations in KRAS, NRAS, BRAF and PIK3CA genes directly affect the efficacy of anti-tumor drugs targeting the EGFR gene. Detection and analysis of gene mutation status in cancer patients is of great significance for clinical medication and treatment guidance.

[0003] At present, there are two main methods for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations: fluorescent quantitative PCR and sequencing. Direct sequencing has the disadvantages of insufficient detection sensitivity, cumbersome operation, and inability to detect multiple mutations simultaneously. Most commercial kits rely on ARMS-qPCR technology, but can only perform qualitative analysis when detecting multiple mutations, and cannot distinguish mutation subtypes in a single tube system. For example, CN106636442A discloses a human tumor gene mutation combined detection kit, including the following parts: EGFR gene primer set, KRAS gene primer set, BRAF gene primer set, NRAS gene primer set, PIK3CA gene primer set, HiFi enzyme, PCR reaction solution, digestion enzyme, ligase, connection buffer, connection adapter, fluorescent probe, Taq enzyme for QPCR reaction, QPCR primer and QPCR reaction solution, which can simultaneously detect mutation sites of multiple driver genes in human tumors, including one or more gene mutation sites in EGFR, NRAS, KRAS, PIK3CA and BRAF. Commercial gene mutation detection kits based on digital PCR technology are also unable to distinguish mutation subtypes in multi-site mutation detection. Although detection schemes that only target two or three hotspot mutations can determine mutation subtypes, they cannot meet the actual clinical needs for multi-mutation detection.

[0004] In summary, providing a kit for detecting multiple mutation hotspots in KRAS, NRAS, BRAF and PIK3CA genes is of great significance in the field of tumor gene detection. Summary of the invention

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides a primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations and applications thereof. The primer-probe combination of a specific sequence is designed to cooperate with each other and can be based on digital PCR to simultaneously detect 14 hotspot mutations of KRAS, NRAS, BRAF and PIK3CA genes, with good specificity and sensitivity, and is easy to operate and low in cost.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, the primer-probe combination comprising primers and probes for detecting KRAS, NRAS, BRAF and PIK3CA genes; the primers for the KRAS gene comprise 3 pairs of primers, and the nucleic acid sequences comprise the sequences shown in SEQ ID NO.1 to SEQ ID NO.6; the nucleic acid sequences of the wild-type probes for the KRAS gene comprise the sequences shown in SEQ ID NO.7 to SEQ ID NO.9; the nucleic acid sequences of the reference probes for the KRAS gene comprise the sequences shown in SEQ ID NO.10 to SEQ ID NO.11; the primers for the NRAS gene comprise 2 pairs of primers, and the nucleic acid sequences comprise the sequences shown in SEQ ID NO.12 to SEQ ID NO.15; the nucleic acid sequences of the wild-type probes for the NRAS gene comprise the sequences shown in SEQ ID NO.16 and SEQ ID NO.17; the nucleic acid sequences of the reference probes for the NRAS gene comprise the sequences shown in SEQ ID NO.18 and SEQ ID NO.19; the primers for the BRAF gene comprise 1 pair of primers, and the nucleic acid sequences comprise SEQ ID NO. ID NO.20 and the sequence shown in SEQ ID NO.21; the nucleic acid sequence of the wild-type probe of the BRAF gene includes SEQ ID NO.22; the nucleic acid sequence of the reference probe of the BRAF gene includes SEQ ID NO.23; the primers of the PIK3CA gene include a pair of primers, and the nucleic acid sequence includes the sequences shown in SEQ ID NO.24 and SEQ ID NO.25; the nucleic acid sequence of the wild-type probe of the PIK3CA gene includes SEQ ID NO.26; the nucleic acid sequence of the reference probe of the PIK3CA gene includes SEQ ID NO.27.

[0008] In the present invention, primers, wild-type probes and reference probes are designed for different exons of KRAS gene (Exon2, Exon3, Exon4), two exons of NRAS gene (Exon2, Exon3), BRAF gene and PIK3CA gene, which can cooperate and simultaneously detect 14 hotspot mutations of KRAS, NRAS, BRAF and PIK3CA genes based on digital PCR, including KRAS mutant gene p.G12S, p.G12D, p.G12 C, p.G12V, p.G12A, p.G13D, p.Q61H, p.K117N (19940), p.K117N (28519); NRAS mutant gene p.G12D, p.Q61R, p.Q61K; BRAF mutant gene p.V600E; PIK3CA mutant gene p.H1047R, the targets of different exons are respectively recorded as KRAS-E2, KRAS-E3, KRAS-E4, NRAS-E2 and NRAS-E3.

[0009] KRAS-E2 forward primer (SEQ ID NO. 1): 5′-AAGGCCTGCTGAAAATGACT-3′.

[0010] KRAS-E2 reverse primer (SEQ ID NO. 2): 5'-GTCCTGCACCAGTAATATGC-3'.

[0011] KRAS-E3 forward primer (SEQ ID NO. 3): 5′-TCTCCCTTCTCAGGATTCCT-3′.

[0012] KRAS-E3 reverse primer (SEQ ID NO. 4): 5'-TACACAAAGAAAGCCCTCCC-3'.

[0013] KRAS-E4 forward primer (SEQ ID NO.5):

[0014] 5'-GGACTCTGAAGATGTACCTATGG-3'.

[0015] KRAS-E4 reverse primer (SEQ ID NO.6):

[0016] 5'-TCAGTGTTACTTACCTGTCTTGT-3'.

[0017] NRAS-E2 forward primer (SEQ ID NO. 12): 5'-GGTTTCCAACAGGTTCTTGC-3'.

[0018] NRAS-E2 reverse primer (SEQ ID NO. 13): 5'-ACTGGGCCTCACCTCTATG-3'.

[0019] NRAS-E3 forward primer (SEQ ID NO. 14): 5'-CACACCCCCAGGATTCTTAC-3'.

[0020] NRAS-E3 reverse primer (SEQ ID NO. 15): 5'-ATTGGTCTCTCATGGCACTG-3'.

[0021] BRAF forward primer (SEQ ID NO.20):

[0022] 5'-TCTTCATGAAGACCTCACAGTAAA-3'.

[0023] BRAF reverse primer (SEQ ID NO. 21): 5'-AGCCTCAATTCTTACCATCCA-3'.

[0024] PIK3CA forward primer (SEQ ID NO.24):

[0025] 5'-AGAGGCTTTGGAGTATTTCATGA-3'.

[0026] PIK3CA reverse primer (SEQ ID NO. 25): 5'-GTGGAATCCAGAGTGAGCTT-3'.

[0027] KRAS-E2 wild-type probe (SEQ ID NO.7):

[0028] 5′-TGGAGCTGGTGGCGT-3′.

[0029] KRAS-E3 wild-type probe (SEQ ID NO.8):

[0030] 5′-CACAGCAGGTCAAGAGGA-3′.

[0031] KRAS-E3 reference probe (SEQ ID NO.10):

[0032] 5'-CCTCATGTACTGGTCCCTCATTGCACTG-3'.

[0033] KRAS-E4 wild-type probe (SEQ ID NO.9):

[0034] 5'-AGGAAATAAATGTGATTTGCCTTC-3'.

[0035] KRAS-E4 reference probe (SEQ ID NO.11):

[0036] 5'-TGCTAAGTCCTGAGCCTGTTTTGTGTCT-3'.

[0037] NRAS-E2 wild-type probe (SEQ ID NO.16):

[0038] 5′-TGGTTGGAGCAGGTGG-3′.

[0039] NRAS-E2 reference probe (SEQ ID NO.18):

[0040] 5'-CTGGATTAGCTGGATTGTCAGTGCGCT-3'.

[0041] NRAS-E3 wild-type probe (SEQ ID NO.17):

[0042] 5'-ATACAGCTGGACAAGAAGAGT-3'.

[0043] NRAS-E3 reference probe (SEQ ID NO.19):

[0044] 5'-CCAGTATGTCCAACAAACAGGTTTCACCA-3'.

[0045] BRAF wild-type probe (SEQ ID NO.22):

[0046] 5'-AGCTACAGTGAAATCTCGATGG-3'.

[0047] BRAF reference probe (SEQ ID NO.23):

[0048] 5'-ACTGTTCAAACTGATGGGACCCACTCCA-3'.

[0049] PIK3CA wild-type probe (SEQ ID NO.26):

[0050] 5′-TGCACATCATGGTGGCT-3′.

[0051] PIK3CA reference probe (SEQ ID NO.27):

[0052] 5'-TGCATGCTGTTTAATTGTGTGGAAGATCCA-3'.

[0053] Preferably, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group.

[0054] Preferably, the fluorescent group includes FAM, VIC, TET, JOE, HEX, TAMRA, ROX, CY5 or Texas Red.

[0055] Preferably, the quencher group comprises MGB, TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 or Eclipse.

[0056] In a second aspect, the present invention provides use of the primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations described in the first aspect in preparing products for detecting KRAS, NRAS, BRAF and PIK3CA genes.

[0057] In a third aspect, the present invention provides a kit for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, the kit comprising the primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations described in the first aspect.

[0058] Preferably, the kit further comprises a PCR reaction solution.

[0059] Preferably, the PCR reaction solution includes DNA polymerase, Mg 2+ , dNTPs and water.

[0060] In a fourth aspect, the present invention provides a method for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations for the purpose of non-disease diagnosis and / or treatment, the method comprising:

[0061] Using the genome in the sample to be tested as a template, digital PCR is performed using the primer probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations described in the first aspect, fluorescence signal detection is performed, and the results are interpreted based on the digital PCR results.

[0062] In the present invention, digital PCR (dPCR) is used to qualitatively detect 14 hot spot mutations of KRAS, NRAS, BRAF and PIK3CA genes in tumors, including KRAS mutant genes p.G12S, p.G12D, p.G12C, p.G12V, p.G12A, p.G13D, p.Q61H, p.K117N (19940), p.K117N (28519); NRAS mutant genes p.G12D, p.Q61R, p.Q61K; BRAF mutant gene p.V600E; PIK3CA mutant gene p.H1047R, which can be applied to the basic research on the functional mechanism of KRAS, NRAS, BRAF and PIK3CA genes.

[0063] Preferably, the reaction conditions of the digital PCR are:

[0064] Pre-denaturation at 93-98°C for 10 min;

[0065] 93-96°C, 25-35 seconds, 55-65°C, 25-35 seconds, for a total of 45-55 cycles.

[0066] Preferably, the digital PCR reaction system includes a KN multiplex digital PCR system and an NBP multiplex digital PCR system;

[0067] Preferably, the KN multiplex digital PCR reaction system comprises: PCR reaction solution, primers of KRAS and NRAS genes, KRAS-E2 wild-type probe, KRAS-E3 wild-type probe, KRAS-E3 reference probe, KRAS-E4 wild-type probe, KRAS-E4 reference probe, NRAS-E2 wild-type probe, NRAS-E2 reference probe and samples to be tested.

[0068] Preferably, the NBP multiplex digital PCR reaction system comprises: PCR reaction solution, primers of NRAS, BRAF and PIK3CA genes, NRAS-E3 wild-type probe, NRAS-E3 reference probe, BRAF wild-type probe, BRAF reference probe, PIK3CA wild-type probe, PIK3CA reference probe and samples to be tested.

[0069] Preferably, the PCR reaction solution is ddPCR Supermix for Probes (No dUTP) (Bio-Rad).

[0070] Preferably, the result interpretation includes classifying the droplets and judging whether a mutation occurs.

[0071] Preferably, the digital PCR may select multiplex droplet digital PCR (ddPCR).

[0072] In the present invention, according to the designed probe concentration, the wild-type target presents different wild-type droplet clusters in the digital PCR two-dimensional fluorescence results, which can be clearly distinguished. When the target mutates, the two-dimensional fluorescence results will add mutant droplet clusters on the basis of the wild-type results, and the mutant clusters will be located directly below the corresponding wild-type clusters.

[0073] Preferably, the method for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations for the purpose of non-disease diagnosis and / or treatment comprises the following steps:

[0074] (1) preparing the KN multiplex digital PCR system and the NBP multiplex digital PCR system;

[0075] (2) mixing the prepared amplification system, adding it to the sample well of the droplet generator, and adding droplet generating oil to the oil well to prepare droplets;

[0076] (3) Transferring the microdroplets to a PCR plate for amplification. The PCR reaction conditions are as follows: pre-denaturation at 95°C for 10 min; 94°C for 30 sec, 60°C for 30 sec, for a total of 50 cycles;

[0077] (4) After amplification is completed, droplet counting and fluorescence signal detection are performed to classify the droplets and determine whether mutations have occurred.

[0078] In a fifth aspect, the present invention provides a device for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, the device comprising a detection unit and an analysis unit;

[0079] The detection unit is used to perform the following steps:

[0080] Using the genome in the sample to be tested as a template, digital PCR is performed using the primer probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations described in the first aspect to perform fluorescence signal detection;

[0081] The analysis unit is used to perform the following steps:

[0082] The results were interpreted based on the digital PCR results.

[0083] Preferably, the reaction conditions of the digital PCR are:

[0084] Pre-denaturation at 93-98°C for 10 min;

[0085] 93-96°C, 25-35 seconds, 55-65°C, 25-35 seconds, for a total of 45-55 cycles.

[0086] Preferably, the result interpretation includes classifying the droplets and judging whether a mutation occurs.

[0087] Preferably, the detection unit is used to perform the following steps:

[0088] (1') preparing the KN multiplex digital PCR system and the NBP multiplex digital PCR system;

[0089] (2′) mixing the prepared amplification system, adding it to the sample well of the droplet generator, and adding droplet generation oil to the oil well to prepare droplets;

[0090] (3') Transferring the microdroplets to a PCR plate for amplification. The PCR reaction conditions are as follows: pre-denaturation at 95°C for 10 min; 94°C for 30 sec, 60°C for 30 sec, for a total of 50 cycles;

[0091] (4') After amplification, fluorescence signal detection is performed.

[0092] Compared with the prior art, the present invention has the following beneficial effects:

[0093] In the present invention, primers, wild-type probes and reference probes are designed for different exons of the KRAS gene (Exon2, Exon3, Exon4), two exons of the NRAS gene (Exon2, Exon3), the BRAF gene and the PIK3CA gene, which cooperate with each other and can simultaneously detect 14 hotspot mutations of the KRAS, NRAS, BRAF and PIK3CA genes based on digital PCR, including KRAS mutant genes p.G12S, p.G12D, p.G12C, p.G12V, p.G12A, p.G13D, p.Q61H, p.K117N (19940), p.K117N (28519); NRAS mutant genes p.G12D, p.Q61R, p.Q61K; BRAF mutant gene p.V600E; PIK3CA mutant gene p.H1047R. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1A This is a graph showing the results of ddPCR detection using a mixture of KRAS-E2, KRAS-E3, KRAS-E4, and NRAS-E2 wild-type plasmids as targets;

[0095] Figure 1B The results of ddPCR detection using a mixture of KRAS-E2, KRAS-E3, KRAS-E4, NRAS-E2 wild-type plasmids and their corresponding mutant plasmids (p.G12D, p.Q61H, p.K117N (28519), p.G12D (564));

[0096] Figure 2A This is the result of ddPCR detection using a mixture of NRAS-E3, BRAF, and PIK3CA wild-type plasmids as targets;

[0097] Figure 2B The results of ddPCR detection using a mixture of NRAS-E3, BRAF, PIK3CA wild-type plasmids and their corresponding mutant plasmids (p.Q61R, p.V600H, p.H1047R);

[0098] Figure 3 The figure shows the results of KN system ddPCR detection of wild-type samples, where (1) is the KRAS-E2 wild-type droplet cluster, (2) is the KRAS-E4 wild-type droplet cluster, (3) is the NRAS-E2 wild-type droplet cluster, (4) is the KRAS-E3 wild-type droplet cluster, and (5) is the double-positive droplet cluster;

[0099] Figure 4 This is the result of KN system ddPCR detection of KRAS-E3 mutation samples;

[0100] Figure 5 This is the result of KRAS-E2 mutation sample KN system ddPCR detection;

[0101] Figure 6 This is the result of KN system ddPCR test for NRAS-E2 mutation sample;

[0102] Figure 7 The results of the wild-type sample NBP system ddPCR test are shown in Figure 1, where (1) is a PIK3CA wild-type droplet cluster, (2) is a NRAS-E3 wild-type droplet cluster, (3) is a BRAF wild-type droplet cluster, and (4) is a double-positive droplet cluster;

[0103] Figure 8 This is the result of NBP system ddPCR test for PIK3CA mutation samples;

[0104] Fig. 9 This is the result of NBP system ddPCR detection of BRAF mutation samples. DETAILED DESCRIPTION

[0105] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0106] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0107] Example 1

[0108] In this embodiment, primer-probe combinations for detecting KRAS, NRAS, BRAF and PIK3CA genes are designed.

[0109] The sequences and concentrations of the primers are as follows:

[0110] KRAS-E2 forward primer (900 nM): 5′-AAGGCCTGCTGAAAATGACT-3′;

[0111] KRAS-E2 reverse primer (900 nM): 5′-GTCCTGCACCAGTAATATGC-3′;

[0112] KRAS-E3 forward primer (900 nM): 5′-TCTCCCTTCTCAGGATTCCT-3′;

[0113] KRAS-E3 reverse primer (900 nM): 5′-TACACAAAGAAAGCCCTCCC-3′;

[0114] KRAS-E4 forward primer (900 nM): 5′-GGACTCTGAAGATGTACCTATGG-3′;

[0115] KRAS-E4 reverse primer (900 nM): 5′-TCAGTGTTACTTACCTGTCTTGT-3′;

[0116] NRAS-E2 forward primer (900 nM): 5′-GGTTTCCAACAGGTTCTTGC-3′;

[0117] NRAS-E2 reverse primer (900 nM): 5′-ACTGGGCCTCACCTCTATG-3′;

[0118] NRAS-E3 forward primer (900 nM): 5′-CACACCCCCAGGATTCTTAC-3′;

[0119] NRAS-E3 reverse primer (900 nM): 5′-ATTGGTCTCTCATGGCACTG-3′;

[0120] BRAF forward primer (900 nM): 5′-TCTTCATGAAGACCTCACAGTAAA-3′;

[0121] BRAF reverse primer (900 nM): 5′-AGCCTCAATTCTTACCATCCA-3′;

[0122] PIK3CA forward primer (900 nM): 5′-AGAGGCTTTGGAGTATTTCATGA-3′;

[0123] PIK3CA reverse primer (900 nM): 5'-GTGGAATCCAGAGTGAGCTT-3'.

[0124] The sequences and concentrations of the probes are as follows:

[0125] (1)KN multiplex ddPCR system:

[0126] KRAS-E2 wild-type probe (250nM):

[0127] 5'-FAM-TGGAGCTGGTGGCGT-MGB-3';

[0128] KRAS-E3 wild-type probe (100nM):

[0129] 5'-FAM-CACAGCAGGTCAAGAGGA-MGB-3';

[0130] KRAS-E3 Reference Probe (200nM):

[0131] 5'-HEX-CCTCATGTACTGGTCCCTCATTGCACTG-Blackhole Quencher 1-3';

[0132] KRAS-E4 wild-type probe (80nM):

[0133] 5'-FAM-AGGAAATAAATGTGATTTGCCTTC-MGB-3';

[0134] KRAS-E4 Reference Probe (150nM):

[0135] 5'-HEX-TGCTAAGTCCT / iBHQ1dT / GAGCCTGTTTTGTGTCT-Blackhole Quencher 1-3' (this is a double-quenched probe, / iBHQ1dT / means that the quencher group BHQ1 is labeled on the middle base T of the sequence);

[0136] NRAS-E2 wild-type probe (150nM):

[0137] 5'-FAM-TGGTTGGAGCAGGTGG-MGB-3';

[0138] NRAS-E2 Reference Probe (120nM):

[0139] 5'-HEX-CTGGATTAGCTGGATTGTCAGTGCGCT-Blackhole Quencher 1-3';

[0140] (2)NBP multiplex ddPCR system:

[0141] NRAS-E3 wild-type probe (100nM):

[0142] 5'-FAM-ATACAGCTGGACAAGAAGAGT-MGB-3';

[0143] NRAS-E3 Reference Probe (100nM):

[0144] 5'-HEX-CCAGTATGTCCAACAAACAGGTTTCACCA-Blackhole Quencher 1-3';

[0145] BRAF wild-type probe (80nM):

[0146] 5'-FAM-AGCTACAGTGAAATCTCGATGG-MGB-3';

[0147] BRAF Reference Probe (200nM):

[0148] 5'-HEX-ACTGTTCAAACTGATGGGACCCACTCCA-Blackhole Quencher 1-3';

[0149] PIK3CA wild-type probe (250nM):

[0150] 5'-FAM-TGCACATCATGGTGGCT-MGB-3';

[0151] PIK3CA Reference Probe (80nM):

[0152] 5'-HEX-TGCATGCTGTTTAATTGTGTGGAAGATCCA-Blackhole Quencher 1-3'.

[0153] Example 2

[0154] The KN multiplex ddPCR system was used to detect the wild-type plasmid mixture of KRAS-E2, KRAS-E3, KRAS-E4, NRAS-E2 and the mixture of the four wild-type plasmids and the corresponding mutant plasmids.

[0155] Primers were designed for KRAS-E2, KRAS-E3, KRAS-E4, and NRAS-E2 genes, and the corresponding gene fragments were amplified from clinical gDNA using the primers. Cloning and transformation were completed using the pEASY-Blunt Cloning Kit; plasmids were extracted from E. coli using the TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0 (TaKaRa). After preparing the site-directed mutation of the wild-type plasmid, the mutant plasmid was obtained. The four wild-type plasmids of KRAS-E2, KRAS-E3, KRAS-E4, and NRAS-E2 were mixed to obtain a wild-type mixed sample, and the four wild-type plasmids were mixed with their corresponding four mutant plasmids to obtain a mutant mixed sample. The 20 μL reaction system of ddPCR was: 10 μL of ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 1.8 μL of primer mixture, 0.5 μL of KRAS-E2 wild-type probe, 0.2 μL of KRAS-E3 wild-type probe, 0.4 μL of KRAS-E3 reference probe, 0.3 μL of KRAS-E4 wild-type probe, 0.16 μL of KRAS-E4 reference probe, 0.3 μL of NRAS-E2 wild-type probe, 0.24 μL of NRAS-E2 reference probe, 2 μL of sample to be tested, and 4.2 μL of sterile water.

[0156] After mixing, the prepared PCR amplification system was added to the sample well of the droplet generator, 70 μL of droplet generation oil was added to the oil well, covered with a gasket, and placed in the QX200 Droplet Generator of the Bio-rad ddPCR system to prepare droplets. Transfer 40 μL of droplets to a 96-well PCR plate, seal the plate and amplify in a Bio-rad T100 PCR instrument (PCR reaction conditions were 95°C pre-denaturation for 10 min; 94°C 30 sec, 60°C 30 sec for a total of 50 cycles). After amplification, transfer the PCR reaction plate to the QX200 Droplet Reader of the Bio-rad ddPCR system, and perform droplet counting and fluorescence signal detection according to the instrument and software operating instructions; use QuantaSoft Analysis Pro version 1.0.596 software to classify droplets and detect mutants. The ddPCR results are as follows: Figure 1A and Figure 1BWhen the system detects four wild-type plasmids, the results are as follows Figure 1A As shown in the figure, single positive droplets (serial numbers 1-4) and double positive droplets (serial numbers 5-10) of different targets show different clusters in the scatter plot and can be clearly distinguished. (11) is the droplet scatter plot containing three targets. When the system detects four wild-type plasmids and the corresponding four mutant plasmids, the results are as follows Figure 1B As shown, the four wild-type droplet clusters (serial numbers 1-4) and the four mutant droplet clusters (serial numbers 5-8) can be clearly distinguished, and the mutant clusters are located directly below the wild-type clusters (serial numbers 1 and 5, 2 and 6, 3 and 7, 4 and 8). Serial number (9) is a droplet scatter point containing two or more targets.

[0157] Example 3

[0158] The NBP multiplex ddPCR system was used to detect the NRAS-E3, BRAF, PIK3CA wild-type plasmid mixture and the mixture of three wild-type plasmids and corresponding mutant plasmids.

[0159] Primers were designed for NRAS-E3, BRAF, and PIK3CA genes, and the corresponding gene fragments were amplified from clinical gDNA using the primers. Cloning and transformation were completed using the pEASY-Blunt Cloning Kit. Plasmids were extracted from E. coli using the TaKaRa MiniBESTPlasmid Purification Kit Ver.4.0 (TaKaRa). After preparing the wild-type plasmid site-directed mutagenesis, mutant plasmids were obtained. The three wild-type plasmids of NRAS-E3, BRAF, and PIK3CA were mixed to obtain a wild-type mixed sample. The three wild-type plasmids were mixed with their corresponding three mutant plasmids to obtain a mutant mixed sample. The 20 μL reaction system of ddPCR was: 10 μL of ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 1.8 μL of primer mixture, 0.2 μL each of NRAS-E3 wild-type and NRAS-E3 reference probes, 0.16 μL of BRAF wild-type probe, 0.4 μL of BRAF reference probe, 0.5 μL of PIK3CA wild-type probe, 0.16 μL of PIK3CA reference probe, 2 μL of sample to be tested, and 4.58 μL of sterile water.

[0160] After mixing, the prepared PCR amplification system was added to the sample well of the droplet generator, 70 μL of droplet generation oil was added to the oil well, covered with a gasket, and placed in the QX200 Droplet Generator of the Bio-rad ddPCR system to prepare droplets. Transfer 40 μL of droplets to a 96-well PCR plate, seal the plate, and amplify in a Bio-rad T100 PCR instrument (PCR reaction conditions were 95°C pre-denaturation for 10 min; 94°C, 30 sec, 60°C, 30 sec for a total of 50 cycles). After amplification, transfer the PCR reaction plate to the QX200 Droplet Reader of the Bio-rad ddPCR system, and perform droplet counting and fluorescence signal detection according to the instrument and software operating instructions; use QuantaSoft Analysis Pro version 1.0.596 software to classify droplets and detect mutants. The ddPCR results are as follows: Figure 2A and Figure 2B When the system detects NRAS-E3, BRAF, and PIK3CA wild-type plasmids, the results are as follows Figure 2A As shown in the figure, the single positive droplets (serial numbers 1-3) and double positive droplets (serial numbers 4-6) of different targets show different clusters in the scatter plot and can be clearly distinguished. (7) is the droplet scatter plot containing three targets. When the system detects three wild-type plasmids and the corresponding three mutant plasmids, the results are as follows Figure 2B As shown, the three wild-type droplet clusters (serial numbers 1-3) and the three mutant droplet clusters (serial numbers 4-6) can be clearly distinguished, and the mutant clusters are located directly below the wild-type clusters (serial numbers 1 and 4, 2 and 5, 3 and 6). Numbers (7-18) are double-positive droplet clusters containing two targets, and (19) are droplet scatter points containing three targets.

[0161] Example 4

[0162] This example performs multi-site mutation detection on clinical tumor FFPE samples.

[0163] Tumor FFPE samples were collected, and the tumor area was divided according to the HE staining results. Four samples with a thickness of 10 μm and a tumor area greater than 60% were cut using a microtome. The gDNA was extracted using the DNA FFPE Tissue Kit (Qiagen), and finally eluted with 50 μL Buffer AE. The KN multiplex ddPCR system and the NBP multiplex ddPCR system were used to perform ddPCR amplification and detection on the clinical sample gDNA. The reaction process was referred to Example 2 and Example 3. Finally, the droplets were classified using QuantaSoft Analysis Pro version 1.0.596 software to determine whether mutations occurred. The ddPCR results are shown in Figure 2. Figure 3 to Figure 9 As shown, in the KN system, the wild-type sample test results are as follows Figure 3 As shown, (1) is a KRAS-E2 wild-type droplet cluster, (2) is a KRAS-E4 wild-type droplet cluster, (3) is a NRAS-E2 wild-type droplet cluster, (4) is a KRAS-E3 wild-type droplet cluster, and (5) is a double-positive droplet cluster. Figures 4 to 6 As shown, Figure 4 For KRAS-E3 mutation samples, Figure 5 For KRAS-E2 mutation samples, Figure 6 is a NRAS-E2 mutant sample, numbers (1-4) are four wild-type droplet clusters, (5) is a mutant droplet cluster, and (6) is a double-positive droplet cluster. In the NBP system, the wild-type sample detection results are as follows Figure 7 As shown, (1) is a PIK3CA wild-type droplet cluster, (2) is a NRAS-E3 wild-type droplet cluster, (3) is a BRAF wild-type droplet cluster, and (4) is a double-positive droplet cluster. Figure 8 The results of BRAF mutant samples are shown. Numbers (1-3) are three wild-type droplet clusters, (4) is a mutant droplet cluster, and (5) is a double-positive droplet cluster. Fig. 9 The results of PIK3CA mutant samples are shown. Numbers (1-3) are three wild-type droplet clusters, (4) is a mutant droplet cluster, and (5) is a double-positive droplet cluster.

[0164] In summary, the present invention designs a primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA genes, and uses digital PCR to qualitatively detect 14 hotspot mutations of KRAS, NRAS, BRAF and PIK3CA genes in tumors, including KRAS mutant genes p.G12S, p.G12D, p.G12C, p.G12V, p.G12A, p.G13D, p.Q61H, p.K117N (19940), p.K117N (28519); NRAS mutant genes p.G12D, p.Q61R, p.Q61K; BRAF mutant gene p.V600E; PIK3CA mutant gene p.H1047R, which has good specificity and sensitivity, is easy to operate and has low cost.

[0165] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention. Sequence Listing <110> Shenzhen University <120> A primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations and its application <130> 2022-05-28 <160> 27 <170> PatentIn version 3.3 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 aaggcctgct gaaaatgact 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 gtcctgcacc agtaatatgc 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 tctcccttct caggattcct 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 tacacaaaga aagccctccc 20 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> 5 ggactctgaa gatgtaccta tgg 23 <210> 6 <211> twenty three <212> DNA <213> Artificial sequence <400> 6 tcagtgttac ttacctgtct tgt 23 <210> 7 <211> 15 <212> DNA <213> Artificial sequence <400> 7 tggagctggt ggcgt 15 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <400> 8 cacagcaggt caagagga 18 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <400> 9 aggaaataaa tgtgatttgc cttc 24 <210> 10 <211> 28 <212> DNA <213> Artificial sequence <400> 10 cctcatgtac tggtccctca ttgcactg 28 <210> 11 <211> 28 <212> DNA <213> Artificial sequence <400> 11 tgctaagtcc tgagcctgtt ttgtgtct 28 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <400> 12 ggtttccaac aggttcttgc 20 <210> 13 <211> 19 <212> DNA <213> Artificial sequence <400> 13 actgggcctc acctctatg 19 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 cacaccccca ggattcttac 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <400> 15 attggtctct catggcactg 20 <210> 16 <211> 16 <212> DNA <213> Artificial sequence <400> 16 tggttggagc aggtgg 16 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <400> 17 atacagctgg acaagaagag t 21 <210> 18 <211> 27 <212> DNA <213> Artificial sequence <400> 18 ctggattagc tggattgtca gtgcgct 27 <210> 19 <211> 29 <212> DNA <213> Artificial sequence <400> 19 ccagtatgtc caacaaacag gtttcacca 29 <210> 20 <211> twenty four <212> DNA <213> Artificial sequence <400> 20 tcttcatgaa gacctcacag taaa 24 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <400> twenty one agcctcaatt cttaccatcc a 21 <210> twenty two <211> twenty two <212> DNA <213> Artificial sequence <400> twenty two agctacagtg aaatctcgat gg 22 <210> twenty three <211> 28 <212> DNA <213> Artificial sequence <400> twenty three actgttcaaa ctgatgggacccactcca 28 <210> twenty four <211> twenty three <212> DNA <213> Artificial sequence <400> twenty four agaggctttg gagtatttca tga 23 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <400> 25 gtggaatcca gagtgagctt 20 <210> 26 <211> 17 <212> DNA <213> Artificial sequence <400> 26 tgcacatcat ggtggct 17 <210> 27 <211> 30 <212> DNA <213> Artificial sequence <400> 27 tgcatgctgt ttaattgtgt ggaagatcca 30

Claims

1. A primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, characterized in that: The primer-probe combination includes primers and probes for detecting KRAS, NRAS, BRAF and PIK3CA genes; The primers for the KRAS gene include 3 pairs of primers, and the nucleic acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.6; The nucleic acid sequence of the wild-type probe of the KRAS gene is shown in SEQ ID NO.7 to SEQ ID NO.9; The nucleic acid sequence of the reference probe of the KRAS gene is shown in SEQ ID NO.10 to SEQ ID NO.11; The primers of the NRAS gene include 2 pairs of primers, and the nucleic acid sequences are shown in SEQ ID NO.12 to SEQ ID NO.15; The nucleic acid sequences of the wild-type probe of the NRAS gene are shown in SEQ ID NO.16 and SEQ ID NO.17; The nucleic acid sequences of the reference probes of the NRAS gene are shown in SEQ ID NO.18 and SEQ ID NO.19; The primers for the BRAF gene include a pair of primers, and the nucleic acid sequences are shown in SEQ ID NO.20 and SEQ ID NO.21; The nucleic acid sequence of the wild-type probe of the BRAF gene is shown in SEQ ID NO.22; The nucleic acid sequence of the reference probe of the BRAF gene is shown in SEQ ID NO.23; The primers for the PIK3CA gene include a pair of primers, and the nucleic acid sequences are shown in SEQ ID NO.24 and SEQ ID NO.25; The nucleic acid sequence of the wild-type probe of the PIK3CA gene is shown in SEQ ID NO.26; The nucleic acid sequence of the reference probe of the PIK3CA gene is shown in SEQ ID NO.27; The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

2. The primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to claim 1, characterized in that: The fluorescent group includes FAM, VIC, TET, JOE, HEX, TAMRA, ROX, CY5 or Texas Red.

3. The primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to claim 1, characterized in that: The quenching group includes MGB, TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 or Eclipse.

4. Use of the primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to any one of claims 1 to 3 in the preparation of products for detecting KRAS, NRAS, BRAF and PIK3CA genes.

5. A kit for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, characterized in that: The kit comprises the primer-probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to any one of claims 1 to 3.

6. The kit for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to claim 5, characterized in that: The kit also includes a PCR reaction solution.

7. The kit for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to claim 6, characterized in that: The PCR reaction solution includes DNA polymerase, Mg 2+ , dNTPs and water.

8. A method for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations for the purpose of non-disease diagnosis and / or treatment, characterized in that: The method comprises: Using the genome in the sample to be tested as a template, digital PCR is performed using the primer probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to any one of claims 1 to 3, and fluorescence signal detection is performed, and the results are interpreted according to the digital PCR results.

9. The method for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations for the purpose of non-disease diagnosis and / or treatment according to claim 8, characterized in that: The reaction conditions of the digital PCR are: Pre-denaturation at 93-98°C for 10 min; 93-96°C, 25-35 seconds, 55-65°C, 25-35 seconds, for a total of 45-55 cycles.

10. The method for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations for the purpose of non-disease diagnosis and / or treatment according to claim 8, characterized in that: The result interpretation includes classifying the droplets and judging whether a mutation occurs.

11. A device for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations, characterized in that: The device comprises a detection unit and an analysis unit; The detection unit is used to perform the following steps: Using the genome in the sample to be tested as a template, digital PCR is performed using the primer probe combination for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to any one of claims 1 to 3, and droplet counting and fluorescence signal detection are performed; The analysis unit is used to perform the following steps: The results were interpreted based on the digital PCR results.

12. The device for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to claim 11, characterized in that: The reaction conditions of the digital PCR are: Pre-denaturation at 93-98°C for 8-12 min; 93-96°C, 25-35 seconds, 55-65°C, 25-35 seconds, for a total of 45-55 cycles.

13. The device for detecting KRAS, NRAS, BRAF and PIK3CA gene mutations according to claim 12, characterized in that: The result interpretation includes classifying the droplets and judging whether a mutation occurs.

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