CN110904239A - Detection kit and detection method for lung cancer related molecular marker gene mutation - Google Patents
Detection kit and detection method for lung cancer related molecular marker gene mutation Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention provides a lung cancer related molecular marker gene mutation crRNA, a detection kit and a detection method, wherein the crRNA comprises: crRNA at the EGFR-T790M mutation site: as shown in any one of SEQ ID NO. 16-17; crRNA at EGFR-L858R mutation site: as shown in any one of SEQ ID NO. 18-19; crRNA at mutation site of BRAF gene: as shown in any one of SEQ ID NO. 20-21; crRNA at the mutation site of ALK gene: as shown in any one of SEQ ID NO. 22-23; crRNA at mutation site of ROS1 gene: as shown in any one of SEQ ID NO. 24-25. The invention designs 5 crRNAs related to the lung cancer related molecular marker gene mutation target, and performs mutation detection by combining the crRNAs with a CRISPR-cpf1 system.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a detection kit and a detection method for lung cancer related molecular marker gene mutation.
Background
The tumor is a new organism formed by abnormal proliferation of cells in certain or some parts of tissues in a human body under the action of various adverse factors, and the cells with abnormal proliferation are tumor cells.
Primary lung cancer is the first of various malignant tumors with high morbidity and mortality in China, and seriously threatens human health. Incidence and mortality of lung cancer worldwide between about 80% and 85% of lung cancer patients are non-small cell lung cancer (NSCL), and about 50% of patients have been diagnosed with advanced stage and poor prognosis. The biological information of the tumor is accurately obtained, so that the individual treatment under the guidance of the genotyping is important for guiding the clinical medication. The incidence of lung cancer is most common to non-small cell lung adenocarcinoma, and the mutation of partial genes can cause the generation of lung cancer, for example, the mutation of genes such as EGFR, BRAF, KARS and the like can be detected in partial patients. Wherein, the EGFR is a transmembrane receptor protein and is related to various signal transduction pathways such as cell proliferation, metastasis, apoptosis and the like. EGFRP.L858R mutation, exon 19 insertion or deletion, and increased sensitivity to EGFR TKIs drugs; and the EGFR gene p.T790M mutation has reduced sensitivity to first-generation EGFR TKIs (erlotinib, erlotinib and gefitinib); the sensitivity to the third-generation EGFR TKis medicament (Osimetinib, Rociletinib) is increased. Therefore, for the observation of the curative effect of a patient with an early p.L858R mutation, exon 19 insertion or deletion mutation on a targeted drug, whether the mutation occurs at the p.T790M site can be determined, and the detection of the mutation state of the EGFR gene is a prerequisite for determining whether the patient can apply EGFR-TKI treatment.
However, at present, in clinical practice, gene mutation detection of relevant sites of patients can be performed by means of Quantitative real-time polymerase chain reaction (Q-PCR), nonradioactive in situ hybridization (Fish), multiplex PCR (multiplex PCR) and the like, and these techniques have the problems of only one gene being detected at a time or high detection cost, and cannot be applied to large-scale clinical sample research, so there is an urgent need to provide a specific fragment detection method with simple temperature control, high sensitivity and high specificity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a detection kit and a detection method for lung cancer related molecular marker gene mutation, which have strong specificity and high sensitivity, and the lowest detection limit reaches 10 copies/uL.
The invention is realized by the following steps:
one of the objects of the present invention is to provide a crDNA for detecting a mutation in a lung cancer-associated molecular marker gene, the crDNA comprising:
crDNA of EGFR-T790M mutation site: the sequence is shown in any one of SEQ ID NO. 6-7;
crDNA at EGFR-L858R mutation site: the sequence is shown in any one of SEQ ID NO. 8-9;
crDNA of mutation site of BRAF gene: the sequence is shown in any one of SEQ ID NO. 10-11;
crDNA of ALK gene mutation site: the sequence is shown in any one of SEQ ID NO. 12-13;
crDNA of mutant site of ROS1 gene: the sequence of the polypeptide is shown in any one of SEQ ID NO. 14-15.
The invention also aims to provide a crRNA for detecting a lung cancer related molecular marker gene mutation, wherein the crRNA comprises:
crRNA at the EGFR-T790M mutation site: the sequence is shown in any one of SEQ ID NO. 16-17;
crRNA at EGFR-L858R mutation site: the sequence is shown in any one of SEQ ID NO. 18-19;
crRNA at mutation site of BRAF gene: the sequence is shown in any one of SEQ ID NO. 20-21;
crRNA at the mutation site of ALK gene: the sequence is shown in any one of SEQ ID NO. 22-23;
crRNA at mutation site of ROS1 gene: the sequence of the polypeptide is shown in any one of SEQ ID NO. 24-25.
The invention also aims to provide a detection kit for the gene mutation of the lung cancer related molecular marker, which contains the crDNA or the crRNA.
Preferably, the kit further comprises cpf1 protein and a fluorescent probe.
The fluorescent probe is selected from one of the following probes:
fluorescent probe 1: the sequence is shown as SEQ ID NO. 26;
and (3) fluorescent probe 2: the sequence is shown as SEQ ID NO. 27;
fluorescent probe 3: the sequence is shown in SEQ ID NO. 28;
the 5 'end of the sequence of the fluorescent probe is marked with a fluorescent group, and the 3' end is marked with a quenching group.
Preferably, the fluorophore comprises one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the quencher comprises one of TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3. The 5 'end of the sequence of the fluorescent probe in the invention is marked by HEX group, and the 3' end is marked by BHQ-1 group.
Preferably, the kit further comprises a dnase inhibitor.
Preferably, the detection kit is a colloidal gold detection kit or a real-time fluorescent quantitative detection kit.
The fourth purpose of the invention is to provide the crRNA and the application of the kit in detecting the lung cancer related molecular marker gene mutation.
The fifth purpose of the invention is to provide a detection method of lung cancer related molecular marker gene mutation, which comprises the following steps:
s1, amplifying the nucleic acid of the sample to be detected to obtain an amplification product;
s2, detecting the detection system consisting of the amplification product, crRNA, cpf1 protein, the fluorescent probe shown in any one of SEQ ID NO.26-28 and enzyme-free water.
Preferably, the detection system is added to a colloidal gold test strip sample detection area for detection after incubation, or a fluorescence detector measures a fluorescence value after the detection system is incubated.
As one of the above embodiments, the amplified product, cpf1 protein/crRNA complex solution, added with fluorescent probe and no enzyme water after incubation, is added to the sample detection zone of the colloidal gold test strip. The invention provides a CRISPR-cpf 1-based system for visually detecting a fluorescent group by using colloidal gold, presents a detection result in a test strip mode, is simple and convenient to operate, does not need an instrument, has a very strong application range and place, and is extremely high in practicability.
As one of the other preferable modes, the fluorescence value is measured by a fluorescence detector after adding a fluorescent probe and incubating the amplification product and cpf1/crRNA complex solution in the absence of enzyme water. The kit can be made into a portable detector which can detect the sample at any time and any place in time, and the application and the method provide a platform for the development of subsequent kits and the application of clinical diagnosis, and are suitable for large-scale clinical sample detection.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides a crRNA, a kit and a detection method for detecting lung cancer related molecular marker gene mutation, designs a crRNA related to a human gene mutation target, and uses the crRNA to combine with a CRISPR-cpf1 system to detect mutation, and the method has the advantages of low cost, repeated detection for many times, simple method, high detection speed, sensitivity (the lowest detection limit reaches 10 copies/uL), specificity, and detection of target nucleic acid containing corresponding mutation by the change of a fluorescence signal in a short time:
(1) in the detection of EGFR-T790M, the fluorescence signal of the mutant gene was significantly higher than that of the wild-type gene by 20-30 minutes. This method can distinguish between 10 copies of the mutant and 104The sensitivity of the copied wild strain is higher than that of the common PCR.
(2) In the detection of EGFR-L858R, the fluorescence signal of the mutant gene is obviously higher than that of the wild-type gene when the reaction is carried out for 20-30 minutes. This method can distinguish between 10 copies of the mutant and 104The sensitivity of the copied wild strain is higher than that of the common PCR.
(3) In the detection of BRAF, the fluorescence signal of the mutant gene is obviously higher than that of the wild gene when the reaction is carried out for 20-30 minutes. This method can distinguish between 10 copies of the mutant and 104The sensitivity of the copied wild strain is higher than that of the common PCR.
(4) In ALK detection, the fluorescence signal of the mutant gene is obviously higher than that of the wild gene when the reaction is carried out for 20-30 minutes. The method can distinguish 10Copy mutant and 104The sensitivity of the copied wild strain is higher than that of the common CR.
(5) In the detection of ROS1, the fluorescence signal of the mutant gene is obviously higher than that of the wild gene when the reaction is carried out for 20-30 minutes. This method can distinguish between 10 copies of the mutant and 104The sensitivity of the copied wild strain is higher than that of the common PCR.
Drawings
FIG. 1 shows the result of specific detection of EGFR-T790M;
FIG. 2 shows the result of specific detection of EGFR-L858R;
FIG. 3 shows the result of specific detection of BRAF;
FIG. 4 shows the results of specific detection of ALK;
FIG. 5 shows the results of specific detection of ROS 1;
FIG. 6 shows the results of sensitivity detection of EGFR-T790M and EGFR-L858R mutations;
FIG. 7 shows the results of sensitive detection of BRAF mutation;
FIG. 8 shows the results of the sensitivity detection of ALK mutation;
FIG. 9 shows the results of sensitive detection of the mutation of ROS 1.
Detailed Description
Example 1 design and acquisition of crRNA targeting Gene mutation site
1. Discovery of lung cancer detection site based on CRISPR-cpf1 system
According to the newly issued NCCN guideline, the molecular marker gene for detecting the common mutation of the lung cancer is obtained. Determining the sequence of the common mutation site of the gene according to the gene sequence and a large amount of clinical detection data, designing crRNA aiming at different regions and constructing a CRISPR/cpf1 system for research. The result shows that the region sequence shown in SEQ ID NO.1-5 is used as the lung cancer mutation detection site based on the CRISPR/cpf1 system (the thickened part is the mutation region), and the detection effect is good.
TABLE 1
2. Design of crRNA targeting gene mutation site
(1) Design principle of targeted gene mutation site crRNA
Since the CRISPR-cpf1 system is a novel targeted DNA gene editing system in which cpf1 binds to crRNA to form a monitoring complex, the guide region of the crRNA recognizes the target DNA with complementary sequences, and cpf1 degrades the target DNA strand, the crRNA design requires: the crRNA includes a protein anchor sequence and a guide sequence in the sequence format: 5 '-anchor sequence binding to cpf1 protein-crRNA guide sequence-3', protein anchor sequence needs to be determined according to cpf1 protein, so that it can match and bind to selected cpf1 protein; the guide sequence is matched to a fragment in the targeting DNA. The crRNA guide sequence cannot be too close to the initiation codon (ATG); the length is 22-24 nucleotides.
Selection of crDNA sequences
The anchoring sequence SEQ ID NO.14 combined with the cpf1 protein; the crRNA guide sequence is designed according to a mutation site region in a sequence shown by SEQID NO. 1-5; finally, the crDNA fragments of 5 mutation sites were obtained as shown below.
crDNA of EGFR-T790M mutation site: the sequence is shown in any one of SEQ ID NO. 6-7;
crDNA at EGFR-L858R mutation site: the sequence is shown in any one of SEQ ID NO. 8-9;
crDNA of mutation site of BRAF gene: the sequence is shown in any one of SEQ ID NO. 10-11;
crDNA of ALK gene mutation site: the sequence is shown in any one of SEQ ID NO. 12-13;
crDNA of mutant site of ROS1 gene: the sequence of the polypeptide is shown in any one of SEQ ID NO. 14-15.
3. Obtaining crRNA
The obtained crDNA fragments with 5 mutation sites are respectively subjected to the action of T7 RNA polymerase (the transcription reaction system is shown in Table 2) to generate RNA, and the RNA is recovered and purified to obtain the crRNA.
TABLE 2
The resulting crRNA includes:
crRNA at the EGFR-T790M mutation site: the sequence is shown in any one of SEQ ID NO. 16-17;
crRNA at EGFR-L858R mutation site: the sequence is shown in any one of SEQ ID NO. 18-19;
crRNA at mutation site of BRAF gene: the sequence is shown in any one of SEQ ID NO. 20-21;
crRNA at the mutation site of ALK gene: the sequence is shown in any one of SEQ ID NO. 22-23;
crRNA at mutation site of ROS1 gene: the sequence of the polypeptide is shown in any one of SEQ ID NO. 24-25.
Example 2 detection kit and detection method for Gene mutation of molecular marker related to Lung cancer
1. Composition of the kit
The kit comprises 5 crRNAs (the crRNAs at 5 mutation sites are obtained as shown in example 1) related to lung cancer gene mutation detection or 5 crDNAs at 5 mutation sites (when the crDNAs are in the kit, an operator needs to firstly generate the crDNAs under the action of T7 RNA polymerase respectively by a crDNA fragment, and the crRNAs are obtained by recovery and purification, specifically shown in example 1), a specific fluorescent probe (the sequence is shown in Table 3, and any probe is shown as SEQ ID NO. 26-28), cpf1 protein, enzyme-free water and a DNase inhibitor;
TABLE 3
Preferably, the kit also comprises an amplification system, wherein the amplification system comprises an isothermal amplification primer pair, and the sequence of the isothermal amplification primer pair at the EGFR-T790M mutation site is shown as SEQ ID NO. 29-30; the sequence of the isothermal amplification primer pair at the EGFR-L858R mutation site is shown as SEQ ID NO. 31-32; the sequence of the isothermal amplification primer pair of the BRAF gene mutation site is shown in SEQ ID NO. 33-34; the sequence of an isothermal amplification primer pair of the ALK gene mutation site is shown in SEQ ID NO. 35-36; the sequence of the isothermal amplification primer pair of the mutation site of the ROS1 gene is shown in SEQ ID NO. 37-38.
2. Detection method of lung cancer related molecular marker gene mutation
(1) Taking 50-100ng of DNA of a sample to be detected, and adding the DNA into an isothermal amplification system, wherein the amplification system is shown in a table 4;
TABLE 4
(2) Mixing the obtained amplification product with the detection reagent: crRNA, cpf1 protein, fluorescent probe, and enzyme-free water were made into detection systems, as shown in table 5.
TABLE 5
100-250nM purified cpf1, 250-500nM crRNA, 1-5. mu.l of synthetic fluorescent probe, 2. mu.l of DNase inhibitor, 5-10ul of target DNA amplification product were incubated in detection buffer (NEBuffer 3) at 37 ℃ for 1-3 hours. And a negative control group is set at the same time. And adding the incubated detection system into a colloidal gold test strip sample detection area for detection, or incubating the detection system and then determining the fluorescence value by a fluorescence detector. And (5) after the reaction is finished, counting and analyzing the change condition of the fluorescence value.
Example 3 specific detection of mutant crRNA on wild-type and mutant sequences
Target sequences of the wild strain and the mutant strain were synthesized, and their specificities were examined by using the above 5 mutant crRNAs, respectively. CRISPR-cpf1 systems constructed from the crRNAs at 5 mutation sites prepared in example 1 are used for verifying the cutting effectiveness in vitro.
100-250nM purified cpf1, 250-500nM crRNA, 1-5. mu.l of synthetic fluorescent probe, 2. mu.l of DNase inhibitor, target DNA at different dilution concentrations were incubated in detection buffer (NEBuffer 3) at 37 ℃ for 1-3 hours. A blank control was also set up. The blank control group is the signal group corresponding to each experimental group without adding crRNA and without adding cpf 1. Several sets of reaction mixtures were reacted simultaneously in a portable detector (temperature set at 37 ℃ C., kinetic measurements were performed every 10min for 1 hour). And detecting the change of the fluorescence signal of the system by using a fluorescence quantitative PCR instrument.
1. Specific detection of EGFR-T790M
We have tested the detection effect of EGFR-T790M crRNA on wild EGFR-T790M and mutant EGFR-T790M, and FIG. 1 shows the systematic fluorescence value result of EGFR-T790M mutant site. The result shows that the fluorescence intensity of the blank control is kept unchanged within the range of 4000-; the fluorescence signal is increased after the templates of wild EGFR-T790M and mutant EGFR-T790M are added, wherein the signal of the template of EGFR-T790M mutation with different concentrations which are completely matched with EGFR-T790M crRNA is generally higher than that of the wild-type control group.
2. Specific detection of EGFR-L858R
We have detected the detection effect of EGFR-L858R crRNA on wild EGFR-L858R and mutant EGFR-L858R, and FIG. 2 is the system fluorescence value result of EGFR-L858R mutation site. The result shows that the fluorescence intensity of the blank control is kept unchanged within the range of 4000-; the fluorescence signal is increased after the templates of wild EGFR-L858R and mutant EGFR-L858R are added, wherein the signal of EGFR-L858R mutant template which is completely matched with EGFR-L858R crRNA and the signal of wild template are generally higher than that of wild control group along with the time.
3. Specific detection of BRAF
We have tested the effect of BRAF crRNA on wild BRAF and mutant BRAF, and FIG. 3 shows the result of system fluorescence value of BRAF mutation site. The result shows that the fluorescence intensity of the blank control is kept unchanged within the range of 4000-; the increase of fluorescence signals appears after the templates of wild BRAF and mutant BRAF are added, wherein the signals of the BRAF mutant template completely matched with BRAFcrRNA and the wild template are generally higher than those of the wild control group along with the time.
4. Specific detection of ALK
We examined the effect of ALK crRNA on the detection of wild ALK and mutant ALK, and FIG. 4 shows the results of the system fluorescence value of ALK mutation sites. The result shows that the fluorescence intensity of the blank control is kept unchanged within the range of 4000-; the fluorescence signal is increased after the templates of the wild ALK and the ALK mutant are added, wherein the signal of the ALK mutant template which is completely matched with the ALKcrRNA and the signal of the wild template are generally higher than that of the wild control group along with the time.
5. Specific detection of ROS1
We tested the detection effect of ROS1crRNA on wild ROS1 and mutant ROS1, and FIG. 5 shows the result of the system fluorescence value of ROS1 mutant site. The result shows that the fluorescence intensity of the blank control is kept unchanged within the range of 4000-; the fluorescence signal increases after adding the templates of wild ROS1 and mutant ROS1, wherein the signal of ROS1 mutant template which is completely matched with ROS1crRNA is generally higher than that of the wild-type template along with the time.
In conclusion, the results all show that the trend of the change of the fluorescence value of the system for detecting the target DNA by the target crRNA is obviously higher than that of the negative control. The 5 crRNAs for detecting the lung cancer related molecular marker gene mutation can respectively and specifically detect corresponding mutant sequences.
Example 4 sensitivity test of mutant crRNA to wild-type and mutant sequences
1. Sensitive detection of EGFR-T790M, EGFR-L858R mutations
EGFR-T790M, EGFR-L858R mutant plasmid and wild type plasmid standard product are subjected to PCR amplification as a template after being diluted in a gradient way, and then are detected by 1.0% agarose gel electrophoresis, and the electrophoresis result is shown in FIG. 6. Negative Control (NC) no band, indicating no contamination during amplification; a single band with the same size as the target band (200bp and 306bp) appears between 100 and 500bp respectively; template concentration at 104Electrophoresis bands can be observed when the ratio of copies/mu L is more than that; and when the template concentration is less than 103No amplified band was observed at copies/. mu.L, indicating that the sensitivity of detecting PCR product by agarose gel electrophoresis was about 103About copies/. mu.L.
2. Sensitive detection of BRAF gene mutation
The BRAF mutant plasmid and the wild type plasmid standard product are subjected to PCR amplification as a template after being diluted in a gradient way, and then are detected by 1.0% agarose gel electrophoresis, and the electrophoresis result is shown in figure 7. Negative Controls (NC) corresponding to the four groups of templates have no bands, which indicates that no pollution exists in the amplification process; a single band with the same size (300bp) as the target band appears between 250 and 500bp respectively; the concentration of the three groups of templates is 105clear electrophoresis bands can be observed when the dosage is more than or equal to mu L; and when the template concentration is less than 102No amplified band was observed at copies/. mu.L, indicating that the sensitivity of detecting PCR product by agarose gel electrophoresis was about 102About copies/. mu.L.
3. Sensitive detection of ALK gene mutation
The ALK mutant plasmid and the wild type plasmid standard product are subjected to PCR amplification as a template after being diluted in a gradient manner, and then are detected by electrophoresis in 1.0% agarose gel, and the electrophoresis result is shown in FIG. 8. Negative Controls (NC) corresponding to the four groups of templates have no bands, which indicates that no pollution exists in the amplification process; a single band with the same size as the target band (86bp and 109bp) appears between 80 bp and 120bp respectively; the concentration of the three groups of templates is 105clear electrophoresis bands can be observed when the dosage is more than or equal to mu L; and when the template concentration is less than 102No amplified band was observed at copies/. mu.L, indicating that the sensitivity of detecting PCR product by agarose gel electrophoresis was about 102About copies/. mu.L.
4. Sensitive detection of ROS1 gene mutation
ROS1 mutant plasmid and wild type plasmid standard were subjected to gradient dilution and PCR amplification as template, followed by detection by 1.0% agarose gel electrophoresis, the results of which are shown in FIG. 9. Negative Controls (NC) corresponding to the four groups of templates have no bands, which indicates that no pollution exists in the amplification process; a single band with the same size as the target band (385bp and 346bp) appears between 100-300bp respectively; the concentration of the three groups of templates is 105clear electrophoresis bands can be observed when the ratio of copies/mu L is higher than(ii) a And when the template concentration is less than 102No amplified band was observed at copies/. mu.L, indicating that the sensitivity of detecting PCR product by agarose gel electrophoresis was about 102About copies/. mu.L.
The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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uaauuucuac ucuuguagau cggcgguaca cuuuaggucc uuu 43
<210>23
<211>41
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
uaauuucuac ucuuguagau cggcgguaca cuuuaggucc u 41
<210>24
<211>41
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
uaauuucuac ucuuguagau caaaaaucau cuccaaccag c 41
<210>25
<211>43
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
uaauuucuac ucuuguagau caaaaaucau cuccaaccag cug 43
<210>26
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
ctcactacag acgcacgcta 20
<210>27
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
ctcactacag acgcacgcta 20
<210>28
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
cacatcagca gcctacagca 20
<210>29
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
tccttctggc caccatgcga agccacactg a 31
<210>30
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
cgatctgcac acaccagttg agcaggtact gg 32
<210>31
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
tccctcacag cagggtcttc tctgtttca 29
<210>32
<211>34
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
ccctggtgtc aggaaaatgc tggctgacct aaag 34
<210>33
<211>33
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
cacctcagat atatttcttc atgaagacct cac 33
<210>34
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
cctcaattct taccatccac aaaatggatc 30
<210>35
<211>32
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
ctgcaaataa tctagggttt ggtgaatata gt 32
<210>36
<211>33
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
ccaactataa tagtaagtat gaaacttgtt tct 33
<210>37
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
ccacacctgc cactctcgct gatcctctc 29
<210>38
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
ctctgcagct ccatctgcat ggcttgca 28
Claims (10)
1. A crDNA for detecting a lung cancer-associated molecular marker gene mutation, the crDNA comprising:
crDNA of EGFR-T790M mutation site: the sequence is shown in any one of SEQ ID NO. 6-7;
crDNA at EGFR-L858R mutation site: the sequence is shown in any one of SEQ ID NO. 8-9;
crDNA of mutation site of BRAF gene: the sequence is shown in any one of SEQ ID NO. 10-11;
crDNA of ALK gene mutation site: the sequence is shown in any one of SEQ ID NO. 12-13;
crDNA of mutant site of ROS1 gene: the sequence of the polypeptide is shown in any one of SEQ ID NO. 14-15.
2. A crRNA for detecting a lung cancer-associated molecular marker gene mutation, the crRNA comprising:
crRNA at the EGFR-T790M mutation site: the sequence is shown in any one of SEQ ID NO. 16-17;
crRNA at EGFR-L858R mutation site: the sequence is shown in any one of SEQ ID NO. 18-19;
crRNA at mutation site of BRAF gene: the sequence is shown in any one of SEQ ID NO. 20-21;
crRNA at the mutation site of ALK gene: the sequence is shown in any one of SEQ ID NO. 22-23;
crRNA at mutation site of ROS1 gene: the sequence of the polypeptide is shown in any one of SEQ ID NO. 24-25.
3. A kit for detecting a gene mutation in a molecular marker related to lung cancer, comprising the crDNA according to claim 1 or the crRNA according to claim 2.
4. The test kit of claim 3, further comprising cpf1 protein and a fluorescent probe.
5. The test kit of claim 4, wherein the fluorescent probe is selected from one of the following probes:
fluorescent probe 1: the sequence is shown as SEQ ID NO. 26;
and (3) fluorescent probe 2: the sequence is shown as SEQ ID NO. 27;
fluorescent probe 3: the sequence is shown in SEQ ID NO. 28;
the 5 'end of the sequence of the fluorescent probe is marked with a fluorescent group, and the 3' end is marked with a quenching group.
6. The assay kit of claim 5, wherein the fluorescent moiety comprises one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE, and Texas Red and the quenching moiety comprises one of TAMRA, DABCYL, MGB, BHQ-1, BHQ-2, and BHQ-3.
7. The test kit of claim 3, wherein the kit further comprises a DNase inhibitor.
8. Use of the crDNA of claim 1, the crRNA of claim 2, and the kit of any one of claims 2 to 7 for detecting a mutation in a lung cancer-associated molecular marker gene.
9. A detection method for lung cancer related molecular marker gene mutation is characterized by comprising the following steps:
s1, amplifying the nucleic acid of the sample to be detected to obtain an amplification product;
s2, detecting the amplification product, the crRNA, the cpf1 protein, the fluorescent probe shown in any one of SEQ ID NO.26-28 and a detection system consisting of enzyme-free water, wherein the crRNA, the cpf1 protein and the detection system consist of the enzyme-free water.
10. The assay of claim 9 wherein the assay is incubated and added to a colloidal gold dipstick sample detection zone for detection or the assay is incubated and a fluorescence detector measures fluorescence.
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