WO2021135072A1 - Probe and detection kit for detecting lung cancer driving gene mutation - Google Patents

Abstract

Disclosed in the present invention are a probe and a detection kit for detecting a lung cancer driving gene mutation. The probe comprises probes for BRAF, EGFR, KRAS, MET, NRAS and PIK3CA driving gene mutation, and probes for TP53 and UGT1A1 chemotherapy drug-related gene, and the total number of the probes is 276. The minimum detection limit of the probe provided by the present invention can reach 0.1%. The present invention can provide medication basis for lung cancer patients looking for targeted therapeutic drugs; and can also change therapeutic regimen for lung cancer patients experiencing severe toxic and side effects, drug resistance, and the like in the therapeutic process.

Description

Probe and detection kit for detecting lung cancer driving gene mutation Technical field

The invention relates to a cancer gene detection method, in particular to a probe and a detection kit for detecting lung cancer driving gene mutations.

Background technique

According to the statistics of the 2018 World Cancer Report, the global incidence and mortality of lung cancer ranks first among all cancer types, and it is increasing year by year. Among them, the global incidence of lung cancer accounts for 11.6% of the total cancer population, and the mortality rate accounts for 11.6% of the total cancer population. 18.4% of the total cancer population. In China, the incidence of lung cancer ranks first among all cancers, with an incidence of 787,000. Among men, the incidence is relatively high; among women, the incidence of lung cancer is second only to breast cancer, ranking second. However, the mortality rate of lung cancer in both men and women ranks first among cancer deaths, causing a serious burden on patients and society. In the process of clinical tumor treatment, it has been discovered that human tumors are very different. Even if it is a tumor at the same site, the treatment effect and method should be different from person to person. In addition, the heterogeneity of tumors is high. Even tumors in the same part of the same person may contain tumor cells of multiple mutation types, which brings great challenges to clinical treatment. The term "driver gene" was introduced by Professor Wu Yilong, deputy dean of Guangdong Provincial People's Hospital at the Asian Cancer Conference in 2012. He pointed out that genes related to the occurrence and development of cancer can be called driver genes, which determine the main cause of this cancer. When we know the driver gene, we know which drugs can be used to combat it, and then we can carry out individualized treatment. It is precisely because of this huge change in concept that genetic testing and individualized treatment of tumors have attracted more and more attention, and they have developed rapidly in just a few years.

Gene detection technology mainly relies on high-throughput sequencers. At present, Next Generation Sequencing (NGS) platforms are mainly represented by Illumina's Hiseq, Miseq, Nextseq series and Life's Ion Torrent ^TM series sequencers. It is widely used in biological research, prenatal diagnosis, genetic diagnosis and treatment. However, cancer patients often carry a small piece of gene change or a single base change. If you choose whole genome sequencing, it will cause a waste of cost and bring a heavier economic burden to the patient. The application of target sequence capture sequencing technology solves the above problems. This technology can customize the genomic region of interest into specific probes to hybridize with genomic DNA, enrich the DNA fragments of the target genomic region and then use second-generation sequencing Technology for sequencing. Compared with traditional polymerase chain reaction (PCR) and gene chips, this method has high throughput and high accuracy, and can detect known and unknown genes at one time, and can effectively detect high-frequency mutations and low-frequency or even low-frequency mutations in samples. Ultra-low frequency mutation.

With in-depth research on lung cancer genes, more and more driver genes related to lung cancer have been discovered, and a variety of drugs targeting specific gene mutations have been developed. Currently known driver genes such as EGFR, KRAS, BRAF, MET, NRAS, PIK3CA, etc., as well as chemotherapeutic-related genes such as TP53 and UGT1A1. Common targeted drugs are: EGFR target drug-gefitinib, erlotinib, icotinib, afatinib, osimertinib, AZD9291; MET target drug-Secure; BRAF target drug-Da Rafinis. Chemotherapy drugs such as: irinotecan, 5-FU, capecitabine, platinum drugs, etc.

At present, the most commonly used clinical gene mutation detection methods include Sanger sequencing technology, fluorescence in situ hybridization (FISH), amplification hindered mutation system technology (ARMS-PCR), fluorescent PCR technology, etc. Sanger sequencing technology, as the gold standard for genetic testing, is mainly used to find specific gene mutations related to diseases. It is difficult to complete the screening of large sample cases without clear candidate genes or with a large number of candidate genes. Fluorescence quantitative PCR detection of the target gene only needs to detect whether the sample has an amplification signal, which can detect small mutations, but the throughput is low. ARMS-PCR technology can be used to detect known mutant genes, but it is not feasible for unknown mutant genes. Although FISH technology uses several different colors of fluorescein alone or mixed-labeled probes for in situ hybridization, it can detect multiple genes at the same time, but it is still limited in the detection of a large number of sites.

The invention provides a probe and a detection kit for detecting lung cancer driving gene mutations. It can be combined to detect multiple mutations of lung cancer genes, which greatly saves patients' samples and waiting time for testing, provides a basis and guidance for individualized treatment of lung cancer patients, and can also monitor the efficacy and predict prognosis after treatment.

Summary of the invention

The purpose of the present invention is to provide a probe and a detection kit for detecting lung cancer driving gene mutations, including probes for BRAF, EGFR, KRAS, MET, NRAS and PIK3CA driving gene mutations and TP53 and UGT1A1 chemotherapeutic drugs related Gene probes, through the detection of these genes to achieve individualized treatment of lung cancer and predict prognosis,

The present invention provides a probe for detecting lung cancer driving gene mutations, including the following 6 groups of 248 probes:

BRAF driver mutation specific probe sequence: its sequence is SEQ ID NO.1 to SEQ ID NO.3;

EGFR driver mutation specific probe sequence: its sequence is SEQ ID NO.4 to SEQ ID NO.98;

KRAS driver mutation specific probe sequence: its sequence is SEQ ID NO.99 to SEQ ID NO.148;

MET driver mutation specific probe sequence: its sequence is SEQ ID NO.149 to SEQ ID NO.150;

NRAS driver mutation specific probe sequence: its sequence is SEQ ID NO.151 to SEQ ID NO.193;

PIK3CA driver mutation specific probe sequence: its sequence is SEQ ID NO.194 to SEQ ID NO.248;

It also includes 28 probes in the following 2 groups:

TP53 specific probe nucleotide sequence: its sequence is SEQ ID NO.249 to SEQ ID NO.272;

UGT1A1 specific probe sequence: its sequence is SEQ ID NO.273 to SEQ ID NO.276.

The sequence of the above specific probe is as follows:

1. BRAF driver mutation probe sequence

2. EGFR driver mutation probe sequence

3. KRAS driver mutation probe sequence

4. MET driver mutation probe sequence

5. NRAS driver mutation probe sequence

6. PIK3CA driver mutation probe sequence

7. TP53 chemotherapeutic drug gene probe

8. UGT1A1 chemotherapeutic drug gene probe

The medication guidance program of the detection kit includes: BRAF, EGFR, KRAS, MET, NRAS, PIK3CA driving gene mutations and TP53 and UGT1A1 chemotherapy drug-related gene mutations. (1) Non-small cell lung cancer with BRAF V600E mutation is sensitive to BRAF inhibitor dabrafenib combined with MEK inhibitor trametinib. However, non-small cell lung cancer carrying BRAF G466V, G469A, Y472C and other non-V600E mutations may not be sensitive to current selective BRAF inhibitors. (2) Non-small cell lung cancer carrying EGFR activating mutations (such as exons 18, 19, 20 and 21) are sensitive to the first and second generation EGFR-TKIs. Non-small cell lung cancer with EGFR T790M mutation may be resistant to the first and second generation EGFR-TKI, but sensitive to the third generation EGFR-TKI. Non-small cell lung cancer carrying EGFR 20 exon insertion mutation may not be sensitive to existing EGFR-TKI. Lung squamous cell carcinoma carrying EGFR amplification may be more sensitive to anti-EGFR antibody combined with chemotherapy vs. chemotherapy alone. (3) Non-small cell lung cancer with KRAS mutation may be resistant to current targeted therapies such as EGFR-TKI. (4) Advanced non-small cell lung cancer carrying variable splicing mutations in exon 14 of MET is sensitive to MET inhibitors. Non-small cell lung cancer with high-level amplification of MET copy number may be sensitive to MET inhibitors. (5) Those with NRAS mutations may be sensitive to MEK inhibitors. (6) Carrying PIK3CA gene mutations (such as exons 9 and 20), the main target drug is brparlisib (BKM120). If PIK3CA has an activating mutation, it may be resistant to EGFR and HER2 targets, and Herceptin, Iressa, and Tarceva are not effective. (7) The rs1042522 locus of TP53 can predict the sensitivity to cisplatin, capecitabine, paclitaxel and oxaliplatin. (8) rs8175347 and rs4148323 of UGT1A1 can predict the risk of side effects to irinotecan.

The present invention also provides a detection kit for detecting lung cancer driving gene mutations, which includes the above-mentioned 6 groups of 248 probes and 2 groups of 28 probes.

The beneficial effects of the present invention are: the probe provided by the present invention has high specificity, good accuracy, and the minimum detection limit can reach 0.1%. It can provide medication basis for lung cancer patients who are looking for targeted therapy drugs; it can also change the treatment plan for lung cancer patients who have severe toxic side effects and drug resistance during the treatment process.

Detailed ways

The present invention is described below through specific examples. It should be noted that the following examples are only used to further illustrate the present invention, but cannot be used to limit the scope of protection of the present invention. Those skilled in the art should understand that based on the present invention Under the technical premise, improvements and modifications made are also regarded as the protection scope of the present invention.

In this example, fresh whole blood samples from 2 patients with lung cancer and 3 standard products with different mutation frequencies (0.1%, 1%, and 5%) were tested. The form of the sample in the present invention is any form of tumor sample from which nucleic acid can be extracted. Among them, whole blood samples are preferred, including but not limited to tissue samples, puncture samples, serum and plasma; and tissue samples include but not limited to paraffin-embedded tissue, Fresh tissue and frozen sectioned tissue.

In this example, genes related to lung cancer were screened from NCCN guidelines, COSMIC database, and drug-related databases. The specific genes are as follows: BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, TP53 and UGT1A1. The probe sequence refers to SEQ ID NO.1-SEQ ID NO.276.

In the present invention, the method of constructing a library and hybridizing and capturing is adopted to verify the probes involved in the present invention, and the specific steps are as follows:

1 Sample preparation

1.1 Separation of plasma and blood cells

Centrifuge the fresh whole blood samples of 2 patients with lung cancer at 3000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. Centrifuge the supernatant again at 3000 rpm for 5 minutes, and save the supernatant (plasma) in a new centrifuge tube for later use. Make a mark (if not used immediately, it can be stored at -20°C).

1.2 Extraction of cfDNA

Strictly follow the instructions of the magnetic bead method large-volume free nucleic acid extraction kit (Tiangen, item number: DP710-01) to extract cfDNA from plasma, and Qubit3.0 to detect the concentration, without further fragmentation.

1.3 Extraction and fragmentation of gDNA

1.3.1 Extraction of gDNA

Strictly follow QIAamp DNA Blood Mini Kit (QIAGEN, article number: 163026054) to extract gDNA from blood cells, check the concentration by Qubit3.0, and check DNA integrity by agarose gel electrophoresis.

1.3.2 Fragmentation of gDNA

1.3.2.1 Turn on the interrupter in advance to reduce its temperature to 4°C.

1.3.2.2 Take 300ng of gDNA and add it to the interrupted tube, use 1x IDTE Buffer to make up to 80μL, vortex to mix, and centrifuge for 1-3s.

1.3.2.3 Set a program of 5 (run 30s, stop 30s) × 3 times to interrupt.

1.3.2.4 Agilent 2200 detects the fragment size of the broken DNA (generally 200-300bp).

1.3.2.5 After equilibrating at room temperature for 30 minutes

AMPure XP Beads (magnetic beads) vortex and mix well, transfer 144μL to a new 1.5mL centrifuge tube, and mark it.

1.3.2.6 Transfer the fragmented product to the 1.5 mL centrifuge tube in step 1.3.2.5, vortex to mix, and incubate at room temperature for 5 min.

1.3.2.7 Place the 1.5 mL centrifuge tube from step 1.3.2.6 on the magnetic stand and let it stand until the solution is completely clear. Aspirate and discard the supernatant, taking care to avoid attracting the magnetic beads.

1.3.2.8 Add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for 30 sec, and aspirate and discard the supernatant.

1.3.2.9 repeat 1.3.2.8.

1.3.2.10 Microcentrifuge the 1.5 mL centrifuge tube from step 1.3.2.9 and place it on a magnetic stand, aspirate the remaining solution, open the lid and dry at room temperature until the ethanol is completely evaporated.

1.3.2.11 Add 52μL of NFW, vortex to mix, incubate at room temperature for 2min, place on a magnetic stand, after the solution is completely clear, transfer 50μL of supernatant to a new labeled 0.2mL PCR tube to enter the next reaction.

1.3.2.12 Take 1μL and use Qubit3.0 to detect the concentration.

2 Library construction

2.1 End repair and 3'end with "A"

2.1.1 Take 30ng cfDNA/40ng standard, make up to 50μL with NFW, vortex to mix and centrifuge.

2.1.2 Prepare the following reaction system in 0.2mL PCR Tube:

Component	Volume (μL)
cfDNA/gDNA	50
End Repair&A-Tailing Buffer	7
End Repair&A-Tailing Enzyme Mix	3
total capacity	60

2.1.3 Vortex to mix well, and perform the following reactions after centrifugation:

2.2 add Adapter

2.2.1 Centrifuge the reaction product of the previous step, and then prepare the following reaction system:

Component	Volume (μL)
End Repair and A-Tailing Reaction Product	60
NFW	5
Ligation Buffer	30
Duplex Seq Adapters(3μM)	5
DNA Ligase	10
total capacity	110

2.2.2 Vortex to mix well, and perform the following reactions after centrifugation:

step	temperature reflex	Reaction time
Adapter Ligation	20℃	15min
Hold	4℃	∞

2.3 Purification after connection

2.3.1 After equilibrating at room temperature for 30 minutes

AMPure XP Beads (magnetic beads) are vortexed and mixed, and 88μL is transferred to a new 1.5mL centrifuge tube and marked.

2.3.2 Transfer the connected product to the 1.5 mL centrifuge tube in step 2.3.1, vortex to mix, and incubate at room temperature for 5 min.

2.3.3 Place the 1.5mL centrifuge tube of step 2.3.2 on the magnetic stand and let it stand until the solution is completely clear. Aspirate and discard the supernatant, taking care to avoid attracting the magnetic beads.

2.3.4 Add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for 30 sec, and aspirate the supernatant.

2.3.5 Repeat 2.3.4.

2.3.6 Microcentrifuge the 1.5mL centrifuge tube in step 2.3.5 and place it on the magnetic stand, aspirate the remaining solution, open the lid and dry at room temperature until the ethanol is completely evaporated.

2.3.7 Add 22μL of NFW, vortex to mix, incubate at room temperature for 2min, place on a magnetic stand, after the solution is completely clear, transfer 20μL of supernatant to a new labeled 0.2mL PCR tube.

2.4 PCR amplification

2.4.1 Prepare the following reaction system in 0.2mL PCR Tube:

Component	Volume (μL)
2X KAPA HiFi HotStart ReadyMix	25
UDI Primer Mix (5μM/Primer)	5
Adapter-ligated Library	20
total capacity	50

2.4.2 Vortex centrifugation, perform the following reactions after centrifugation

2.5 PCR product purification

2.5.1 After equilibrating at room temperature for 30 minutes

AMPure XP Beads (magnetic beads) vortex and mix well, take 50μL into a new 1.5mL centrifuge tube, and mark it.

2.5.2 Transfer the PCR product to the 1.5 mL centrifuge tube in step 2.5.1, vortex to mix, and incubate at room temperature for 5 min.

2.5.3 Place the 1.5mL centrifuge tube of step 2.5.2 on the magnetic stand, and let it stand until the solution is completely clear. Discard the supernatant, taking care to avoid attracting the magnetic beads.

2.5.4 Add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for 30 sec, and aspirate the supernatant.

2.5.5 Repeat 2.5.4.

2.5.6 Microcentrifuge the 1.5mL centrifuge tube in step 2.5.5 and place it on the magnetic stand, aspirate the remaining solution, open the lid and dry at room temperature until the ethanol is completely evaporated.

2.5.7 Add 22μL of NFW, vortex to mix, incubate at room temperature for 2min, place on a magnetic stand, after the solution is completely clear, transfer 20μL of supernatant to a new labeled 1.5mL centrifuge tube.

2.5.8 Quality Inspection

Take 1μL of DNA library solution and use Qubit Fluorometer to detect the concentration.

3 hybrids

3.1 Configure the following reaction in the 1.5ml EP tube:

Blocker component	Volume per reaction(μL)
Human Cot DNA	5
xGen Blocking Oligos based on your library adapters	2

3.2 Mix 2ug of the library and add it to the mixed solution of step 3.1.

3.3 Vacuum dry.

3.4 Configure the following systems in the vacuum-dried sample EP tube

Hybridization Master Mix component	Volume per reaction(μL)
xGen 2X Hybridization Buffer	8.5
xGen Hybridization Buffer Enhancer	2.7
xGen Lockdown Panel or custom probes	4
Nuclease-Free Water	1.8
Total	17

3.5 Shake, centrifuge, and repeat twice.

3.6 Carry out the following PCR reaction procedures:

4 capture.

Configure the cleaning buffer according to the following table:

Configure Bead Resuspension Mix according to the following table

Bead Resuspension Mix component	Volume per reaction(μL)
xGen 2X Hybridization Buffer	8.5
xGen Hybridization Buffer Enhancer	2.7
Nuclease-Free Water	5.8
Total	17

4.1 Cleaning streptavidin magnetic beads

4.1.1 Shake and mix the magnetic beads, and draw 50μL of each captured magnetic beads into the same 1.5mL EP tube.

4.1.2 Add 100μL of Bead Wash Buffer for each capture, blow with a gun more than 10 times, put it on the magnetic stand and let it stand until it is completely clarified, and discard the supernatant.

4.1.3 Repeat 4.1.2 operation twice.

4.1.4 Add the configured Bead Resuspension Mix, capture 17 μL of each, pipette to mix, and transfer to a 0.2 mL PCR tube.

4.2 Capture

4.2.1 After the probe hybridization is over, quickly adjust the gradient PCR instrument program to the following table

4.2.2 Quickly transfer all the hybridized samples to the 0.2mL PCR tube containing Bead Resuspension Mix in step 4.1.4, pipette to mix and centrifuge.

4.2.3 Put the sample into the PCR machine with the program set in step 4.2.1, place it for 45 minutes, and take out the rapid shaking and mix every 10-12 minutes to ensure that the magnetic beads are in a resuspended state.

4.3 Hot wash

4.3.1 The thermostat warms up the 1X Wash Buffer 1 and 1X Stringent Wash Buffer at 65°C for at least 15 minutes.

4.3.2 Add 100μL of preheated 1X Wash Buffer 1 to 4.2.3 samples, and quickly pipette and mix with a gun to avoid air bubbles.

4.3.3 Place on the magnetic stand until clarified, discard the supernatant, and remove the magnetic stand.

4.3.4 Add 150μL of pre-heated 1X Stringent Wash Buffer to the sample, and quickly mix with a gun to avoid bubbles. Incubate at 65°C for 5 min, place on a magnetic stand until clear, and discard the supernatant.

4.3.5 Repeat step 4.3.4.

4.4 Wash at room temperature

4.4.1 Add 150μL Wash Buffer 1 to the sample in the hot wash step 4.3.5, and centrifuge with shaking. Incubate for 2min at room temperature, shake once every 30s (not too violently) to ensure that the magnetic beads are in a resuspended state.

4.4.2 After the incubation, microcentrifuge. Place on the magnetic stand for 1 min, until clear, discard the supernatant.

4.4.3 Add 150μL Wash Buffer 2, shake and mix. Incubate for 2min at room temperature, shake once every 30s (not too violently) to ensure that the magnetic beads are in a resuspended state.

4.4.4 After the incubation is over, microcentrifuge. Place on the magnetic stand for 1 min, until clear, discard the supernatant.

4.4.5 Add 150μL Wash Buffer 3, shake and mix. Incubate for 2 minutes at room temperature, shake once for 30 seconds (not too vigorously) to ensure that the magnetic beads are in a resuspended state.

4.4.6 After the incubation, microcentrifuge. Place on the magnetic stand for 1 min, until clear, discard the supernatant.

4.4.7 Use a gun to absorb the remaining Wash Buffer 3 and remove the magnetic stand.

4.4.8 Add 20μL NFW to each capture, mix well by pipetting with a gun, and transfer to a 0.2mL PCR tube.

5 PCR amplification and purification

5.1 PCR amplification

5.1.1 Add the following system to each capture

Amplification Reaction Mix component	Volume per reaction(μL)
2X KAPA HiFi HotStart ReadyMix*	25

KAPA Primer mix

5

5.1.2 Carry out the following PCR amplification

5.2 PCR product purification

5.2.1 Configure 80% ethanol in advance, 250μL is required for each capture.

5.2.2 Add 75μL to each capture

AMPure XP beads (AMP), mix well by pipetting and incubate at room temperature for 5-10min. Set the magnetic stand for 2-5 minutes until it is clear, and discard the supernatant.

5.2.3 Add 125 μL of 80% ethanol, let stand for 1 min at room temperature, and discard the supernatant.

5.2.4 Repeat step 5.2.3.

5.2.5 Dry the magnetic beads at room temperature for about 1-3 minutes.

5.2.6 After the magnetic beads are dried, remove the magnetic stand, add 22μL TE, and mix by pipetting.

5.2.7 Incubate at room temperature for 5 minutes. Set the magnetic stand until it is clear, and transfer 20 μL to a new 1.5 mL EP tube for storage.

5.2.8 Library quality inspection

Use Qubit 3.0 and Agilent 2200 for detection.

6. Nextseq on-machine sequencing.

7. Data analysis results

The main quality control of the two patient samples is shown in Table 1: DNA quality evaluation and sequencing quality evaluation are both qualified, and the next step data analysis can be carried out. The data analysis results of lung cancer patients and standard products are as follows:

(1) The results of sequencing data analysis of a lung cancer patient showed that: (1) The c.2294T>G mutation in Exon 20 of the EGFR gene was detected, which caused the valine (V) at position 765 to be changed to glycine (G). ). At present, there are no drugs specifically targeting this mutation site, and there are gene-targeted drugs approved by the FDA or cFDA. (2) The genotype at the rs8175347 locus of the UGT1A1 gene is heterozygous, indicating that the use of irinotecan has a greater risk of side effects (neutropenia, diarrhea, weakness).

The results of sequencing data analysis of another lung cancer patient showed that the genotype at the rs8175347 locus (*28) of the UGT1A1 gene was heterozygous, indicating that the use of irinotecan had a greater risk of side effects; the genotype at the rs1042522 locus of the TP53 gene was heterozygous. The combined type suggests that the sensitivity of using cisplatin, capecitabine, paclitaxel, and oxaliplatin may be low.

(2) The test results of the three standard products are shown in Table 2: The known mutation sites with allele frequencies of 0.1%, 1%, and 5% were detected. It shows that the detection kit can detect the gene locus of ultra-low frequency mutation, and the minimum detection limit can reach 0.1%.

Table 1:

Table 2:

gene	Known mutation sites	Expected allele frequency (%)	Frequency of alleles detected (%)
EGFR	T790M	5.0	4.7
EGFR	delE746-A750	5.0	1.8
EGFR	L858R	5.0	3.9
EGFR	V769-D770insASV	5.0	3.0
PIK3CA	E545K	6.3	5.9
KRAS	G12D	6.3	5.0
EGFR	T790M	1.0	0.52
EGFR	delE746-A750	1.0	0.19
EGFR	L858R	1.0	0.58
EGFR	V769-D770insASV	1.0	0.39
PIK3CA	E545K	1.3	0.25
KRAS	G12D	1.3	0.25
EGFR	T790M	0.1	0.0021

Claims (2)

1. A probe for detecting lung cancer driving gene mutations, which is characterized in that it includes the following 6 sets of 248 probes:

   BRAF driver mutation specific probe sequence: its sequence is SEQ ID NO.1 to SEQ ID NO.3;

   EGFR driver mutation specific probe sequence: its sequence is SEQ ID NO.4 to SEQ ID NO.98;

   KRAS driver mutation specific probe sequence: its sequence is SEQ ID NO.99 to SEQ ID NO.148;

   MET driver mutation specific probe sequence: its sequence is SEQ ID NO.149 to SEQ ID NO.150;

   NRAS driver mutation specific probe sequence: its sequence is SEQ ID NO.151 to SEQ ID NO.193;

   PIK3CA driver mutation specific probe sequence: its sequence is SEQ ID NO.194 to SEQ ID NO.248;

   It also includes 28 probes in the following 2 groups:

   TP53 specific probe nucleotide sequence: its sequence is SEQ ID NO.249 to SEQ ID NO.272;

   UGT1A1 specific probe sequence: its sequence is SEQ ID NO.273 to SEQ ID NO.276.
2. A detection kit for detecting lung cancer driving gene mutations, comprising: 6 sets of 248 probes in total and 2 sets of 28 probes in total according to claim 1.