CN116004827A - Method and kit for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR - Google Patents
Method and kit for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR, comprising the following components: 5 xRT-qPCRMastermix, enzyme cocktail, 8-PCR tube reaction strips; the 5×RT-qPCR Master mix contains: dNTP, magnesium ion, salt required by reaction and buffer ion; the enzyme mixture comprises: reverse transcriptase, fastTaq enzyme, UDG enzyme; the 8-linkage PCR tube reaction strip contains primer probe mixed liquor, and specifically consists of 6 groups of fusion gene primer probe mixed liquor and human internal reference gene (GAPDH gene) primer probe mixed liquor. The invention detects the fusion of the ROS1, RET and ALK genes of lung cancer, can specifically help to distinguish the fusion into the ROS1/RET/ALK and other types of kits, and has certain significance for treatment selection. The kit adopts a real-time fluorescence quantitative PCR technology, judges whether fusion occurs or not by analyzing the amplified fluorescence curve, has simpler and more convenient operation, simpler required instruments and equipment and easier clinical popularization and use.
Description
Technical Field
The invention relates to the technical field of ocean monitoring, in particular to a method and a kit for detecting lung cancer ROS1, RET and ALK fusion genes by using one-step RT-qPCR.
Background
Lung cancer is one of the common malignant tumors seriously jeopardizing human health, and the incidence and mortality of lung cancer are rapidly rising in recent years, wherein non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer. In recent years, in the field of molecular targeted therapy research of lung cancer, research hotspots are mainly focused on EGFR, ALK, ROS, RET, KRAS and other sites, mutation or fusion of genes is related to targeted drug therapy, wherein ALK, ROS1 and RET gene fusion patients can benefit from tyrosine kinase inhibitor therapy. The NCCN guideline for non-small cell lung cancer clearly states that the status of a gene mutation should be detected before targeted therapy. Therefore, the combined detection of the polygenic mutation sites of NSCLC patients can provide more accurate treatment for the patients,
the fusion genes can be detected by the existing second generation sequencing, but the NGS is high in price, complex in experimental operation, complex in data analysis and processing and long in detection period. Meanwhile, the lung cancer patients are difficult to sample, the sample size is small, and the screening one by one can not be generally satisfied;
therefore, we propose a one-step RT-qPCR method for detecting lung cancer ROS1, RET and ALK gene fusion, and combine 13 lung cancer gene fusion types into 6 reaction systems, and simultaneously detect, directly perform result interpretation through Ct value, and the detection flow is simple, the result is easy to interpret, sample is saved, detection time is shortened, and clinical reference is provided for targeted treatment of lung cancer patients.
Disclosure of Invention
The invention aims to provide a method and a kit for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR, which solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions: a kit for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR comprises the following components: 5 xRT-qPCRMastermix, enzyme cocktail, 8-PCR tube reaction strips;
the 5 XRT-qPCRMastermix contains: dNTP, magnesium ion, salt required by reaction and buffer ion;
the enzyme mixture comprises: reverse transcriptase, fastTaq enzyme, UDG enzyme;
the 8-linkage PCR tube reaction strip contains primer probe mixed liquor, and specifically consists of 6 groups of fusion gene primer probe mixed liquor and human internal reference gene (GAPDH gene) primer probe mixed liquor.
As a preferred embodiment of the invention, the specific fusion sites of the ROS1 fusion gene, RET fusion gene and ALK fusion gene are optimized, and the grouping and primer probe sequences are as follows:
the upstream exon of ROS1 fusion gene combination 1 is CD74 exon and the downstream exon of ROS1 is E32 exon
The upstream exogene of ROS1 fusion gene combination 1 is SLC34A2 exon and the downstream exogene of ROS1 exon is E32
The upstream exon of ROS1 fusion gene combination 1 is SLC34A2 exon and the downstream exon of ROS1 exon is E32
The upstream exogene of the ROS1 fusion gene combination 2 is CD74 exon and the downstream exogene is ROS1 exon and E34
The upstream exon of the ROS1 fusion gene combination 2 is EZR exon E10, the downstream exon of ROS1 is ROS1 exon E34
RET3 fusion gene combination 3 upstream external gene is CCDC6 exon and E1 downstream external gene is RET exon and E12
RET3 fusion gene combination 3 upstream external gene is CCDC6 exon and E2 downstream external gene is RET exon and E12
RET3 fusion gene combination 3 upstream external gene is NCOA4 exon and E8 downstream external gene is RET exon and E12
The RET4 fusion gene combination 1 has the upstream external gene of CCDC6 as E8 and the downstream external gene of RET as E11;
the ALK fusion gene combination 5 has the upstream exon of NPM1 as E5 and the downstream exon of ALK as E20;
the ALK fusion gene combination 5 has the upstream exon of NPM4 as E6 and the downstream exon of ALK as E20;
the upstream exon of ALK fusion gene combination 6 is NPM4 exon and the downstream exon of E13 is ALK exon and E20;
the upstream exon of ALK fusion gene combination 6 is EML4 exon and the downstream exon of E20 exon is ALK exon and E20.
As a preferred embodiment of the invention, 1) GAPDH reference gene: CY5 fluorescent upstream primer: 5'-CAGGTCAACTGGTATCGTGAC-3'
A downstream primer: 5'-CCACTAGAGGGGGATTGTGA-3'
Fluorescent probe: 5'-CY5-CTACGCAGTAGCACTGCCATCG- -BHQ1-3'
2) ROS1 fusion gene combination 1: FAM fluorescence
An upstream primer: 5'-GTTTCAACTGTGCAGGCACTC-3'
An upstream primer: 5'-TCTAGTTTTTCTTGTGCTGCC-3'
An upstream primer: 5'-GGCTCCTGATACCTCTGTTAAC-3'
A downstream primer: 5'-TCAGCTTTCACCACTGTATTG-3'
Fluorescent probe: 5'-FAM-AGTCCCAAAACCAGGCATTCCCA-BHQ1-3'
3) ROS1 fusion gene combination 2: FAM fluorescence
An upstream primer: 5'-GTTTGCATGCGCCCGCACTC-3'
An upstream primer: 5'-AAGGAGACTTCATCCTGC-3'
A downstream primer: 5'-TCAGCTTTCACCACTGTATTG-3'
Fluorescent probe: 5'-FAM-AGTCCCAAAACCAGGCATTCCCA-BHQ1-3'
4) RET fusion gene combination 3: FAM fluorescence
An upstream primer: 5'-AAGAAACCCTTGCTGTAAATTATGAG-3'
An upstream primer: 5'-TGAGCTCTCCAGAAAATTGATGC-3'
An upstream primer: 5'-CTTACATACCGAGCACGGAC-3'
A downstream primer: 5'-GACCACCATTCGAAATCGCC-3'
Fluorescent probe: 5'-FAM-AATTCCTCGGAAGAACTTGTTC-BHQ1-3'
5) RET fusion gene combination 4: FAM fluorescence
An upstream primer: 5'-CCCATAGGTTTCACAACCCACTG-3'
A downstream primer: 5'-TGTGGCCAAACTTCTGGAAG-3'
Fluorescent probe: 5'-FAM-TCTCCTTCTTCGTCACCGTGCTG-BHQ1-3'
6) ALK fusion gene combination 5: FAM fluorescence
An upstream primer: 5'-TTCAGGCCCAGTCCATAATAG-3'
An upstream primer: 5'-GTTACCAAAACTGCAGACAAGC-3'
A downstream primer: 5'-GGAGCCTGCTCAGCTTGTACT-3'
Fluorescent probe: 5'-FAM-CCGCGGAAGCAAGCAGGAG-BHQ1-3'
7) ALK fusion gene combination 6: FAM fluorescence
An upstream primer: 5'-GGTGGAGTCATGCTTATATGGAG-3'
An upstream primer: 5'-TCTAACTCGGGAGACTATGAAATATTG-3'
A downstream primer: 5'-GGAGCCTGCTCAGCTTGTACT-3'
Fluorescent probe: 5'-FAM-CCGCGGAAGCAAGCAGGAG-BHQ1-3'.
As a preferable implementation mode of the invention, the concentration of the primer probe of the mixed liquid primer probe in the 8-joint PCR tube reaction strip is 0.5-3 mu M.
The invention also relates to a method for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR, which comprises an amplification reaction system and a reaction amplification program shown in tables 1-2;
TABLE 1
After preparation, uniformly mixing on a vortex device, centrifuging for 15 seconds, lightly uncovering the strip cover of the 8-link PCR tube reaction strip, dividing an amplification reaction system into the 8-link PCR tube reaction strip, uniformly mixing, centrifuging, and putting into a real-time PCR instrument for detection, wherein the detection is shown in the following table;
table 2.
As a preferred embodiment of the present invention.
As a preferred embodiment of the present invention.
Compared with the prior art, the invention has the following beneficial effects:
the detection of the fusion of the ROS1, RET and ALK genes of lung cancer can be particularly helpful for distinguishing the fusion into the ROS1/RET/ALK and other types of kits, and the detection method has a certain significance for treatment selection. The kit adopts a real-time fluorescence quantitative PCR technology, judges whether fusion occurs or not by analyzing the amplified fluorescence curve, has simpler and more convenient operation, simpler required instruments and equipment and easier clinical popularization and use;
13 fusion types are detected simultaneously in one 8-joint tube by adopting a one-step RT-qPCR method, the sample use amount is less, the cost is saved, the pollution caused by repeated uncovering is avoided by adopting the one-step RT-qPCR reaction, the experimental steps are reduced, and the experimental time is shortened.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1CD74-ROS1E6:E32 fusion gene amplification results;
FIG. 2SLC34A2-ROS1E4:E32 fusion gene amplification results;
FIG. 3 results of amplification of the SLC34A2-ROS1E13:E32 fusion gene;
FIG. 4CD74-ROS1E6:E34 fusion gene amplification results;
FIG. 5EZR-ROS1E10:E34 fusion gene amplification results;
FIG. 6CCDC6-RETE1: E12 fusion gene amplification results;
FIG. 7CCDC6-RETE2:E12 fusion gene amplification results;
FIG. 8NCOA4-RETE8:E12 fusion gene amplification results;
FIG. 9CCDC6-RETE8:E11 fusion gene amplification results;
FIG. 10NPM1-ALKE5:E20 fusion gene amplification results;
FIG. 11EML4-ALKE6:E20 fusion gene amplification results;
FIG. 12EML4-ALKE13:E20 fusion gene amplification results;
FIG. 13EML4-ALKE20:E20 fusion gene amplification results;
FIG. 14 results of clinical sample detection of CD74-ROS1E6:E32 fusion gene;
FIG. 15 detection results of clinical samples of CD74-ROS1E6:E34 fusion gene;
FIG. 16CCDC6-RETE1: E12 fusion gene clinical sample detection results;
FIG. 17CCDC6-RETE8:E11 fusion gene clinical sample detection results;
FIG. 18EML4-ALKE20:E20 fusion gene clinical sample detection results.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.
Example 1
Verifying amplification effect of screened primer probe by concentration gradient dilution standard
1. Instrument, reagent and sample
1) The main instrument is as follows: BSC-1300-IIA2 biosafety cabinet; OSE-MCBmini centrifuge; SW-CJ-1FD single use ultra clean bench; ABI7500.
2) The main reagent comprises: vazymeT7HighYieldRNATranscription Kit kit, 5 XRT-qPCRMastermix, enzyme cocktail, 8-up PCR tube reaction strips.
3) Sample: the fusion gene transcribes RNA in vitro.
2. The experimental steps are as follows:
1) Sample processing: the synthesized plasmid DNA of the fusion gene in 13 is subjected to in vitro transcription by using a Vazyme T7HighYIeldRNATranscriptionkit kit, agarose gel electrophoresis is carried out after amplification is finished, in vitro transcription RNA is recovered by cutting gel, and the yield is quantitatively confirmed by Qubit for subsequent use.
The in vitro transcription system and reaction procedure are shown in tables 3 and 4;
TABLE 3 Table 3
| Reaction temperature | Reaction time |
| 37℃ | 2H |
TABLE 4 Table 4
In vitro transcribed RNA recovery:
160uLRNasefree water is added into the in vitro transcription system respectively, and the product is diluted to 180uL;
adding 20uL of 3M sodium acetate (pH 5.2), fully mixing and centrifuging;
adding 200uL of chloroform, fully and uniformly vortex and uniformly mixing, centrifuging at 12000rpm for 5min at room temperature, and sucking 180uL of an upper water phase into a new centrifuge tube;
adding 360uL of absolute ethyl alcohol into a new centrifuge tube, mixing the materials evenly in a reverse way, standing at the temperature of minus 20 ℃ for 30min, and centrifuging at the temperature of 4 ℃ for 15min at 12000 rpm;
discarding the supernatant, adding 500uL of precooled 70% ethanol, washing the precipitate, centrifuging at 12000rpm at 4 ℃ for 15min, and discarding the supernatant;
uncapping and drying for 2min, adding 500uLRNasefree water to dissolve RNA precipitate, quantitatively confirming the yield by Qubit, and storing at-80 ℃ for later use;
3) Carrying out RT-qPCR amplification reaction on the sample:
the reaction system is as follows:
mixing the reaction systems uniformly, centrifuging, and respectively split charging the reaction systems into 8-joint PCR tube reaction strips, and performing ABI7500 fluorescence detection;
the reaction procedure was as follows:
experimental results: see FIGS. 1-13 of the drawings
Summarizing: the RNA transcribed from the outside of 13 groups of fusion genes is used as a sample, the optimized 6 groups of fusion gene primer probe grouping amplification curves show typical S-shaped curves, the concentration gradients are in good linear relation, the 10copies in-vitro transcription samples can be amplified, and the sensitivity can be completely used for clinical sample detection.
Example two
Fusion gene positive clinical sample detection
1. Instrument, reagent and sample
1) The main instrument is as follows: BSC-1300-IIA2 biosafety cabinet; OSE-MCBmini centrifuge; SW-CJ-1FD single use ultra clean bench; berle CFX96 fluorescent quantitative PCR instrument.
2) The main reagent comprises: total RNA extraction kit (Tiangen Biochemical technology Co., ltd., product number: DP 439), 5 XRT-qPCRMastermix, enzyme cocktail, 8-PCR tube reaction strip.
3) Sample: paraffin tissue sections of lung cancer patients;
2. the experimental steps are as follows:
taking paraffin tissue sections of the lung cancer patient with the determined fusion type, detecting, and verifying the reaction effect;
1) Sample processing:
cutting paraffin wax sample into 5-10 μm thick slices;
note that: if the sample surface is exposed to air, the first 2-3 sheets are discarded.
Rapidly placing 2-8 slices into a centrifuge tube of 1.5mLRNase-Free, adding 1mL of dimethylbenzene, and severely swirling for 10sec;
centrifuging at 12.000rpm for 2min at room temperature;
the supernatant was aspirated with the gun head, taking care not to aspirate the pellet;
adding 1mL of absolute ethyl alcohol into the sediment, and uniformly mixing by vortex;
centrifuging at 12000rpm for 2min at room temperature;
the supernatant was aspirated with the gun head, taking care not to aspirate sediment (carefully aspirate residual ethanol with a new gun head);
opening the tube cover, and standing at room temperature or 37 ℃ for 10min until the residual ethanol is completely volatilized;
it is important to note that complete removal of residual ethanol, which can have an effect on RNA.
200. Mu.L of lysate RF and 10. Mu.L of LProteinaseK are added to the pellet and thoroughly vortexed;
incubating at 55 ℃ for 15min and then incubating at 80 ℃ for 15min;
centrifuging at 12,000rpm for 5min at room temperature, and transferring supernatant into a new RNase-Free centrifuge tube;
adding 220 mu L of buffer RB, and uniformly mixing by vortex;
660. Mu.L of absolute ethanol is added, and the mixture is stirred and mixed uniformly (precipitation can occur);
transferring 700 μl of the solution and precipitating into an adsorption column CR3 (the adsorption column is placed in a collection tube), centrifuging at 12,000rpm for 1min, discarding the waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
step n is repeated until all the solution and precipitate passes completely through the column CR3, the waste liquid is discarded, and the column CR3 is returned to the collection tube.
Preparing DNaseI working solution, namely placing 10 mu LDNaseI storage solution into a new RNase-Free centrifuge tube, adding 70 mu LRDD buffer solution, and gently mixing;
adding 80 mu L of DNaseI working solution into the center of an adsorption column CR3, and standing at room temperature for 15min;
adding 500 μl deproteinized liquid RW1 into the adsorption column CR3, centrifuging at 12,000rpm at room temperature for 30-60sec, discarding the waste liquid, and placing the adsorption column back into the collection tube;
adding 500 μl of rinsing liquid RW (before use, checking whether ethanol has been added) into the adsorption column CR3, standing at room temperature for 2min, centrifuging at 12,000rpm for 30-60sec, discarding the waste liquid, and placing the adsorption column CR3 back into the collection tube;
repeating step s;
centrifuge at 12.000rpm for 2min at room temperature and discard the waste. Placing the adsorption column CR3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
note that this step aims at removing the residual rinse liquid from the adsorption column CR3, which may affect the subsequent experiments such as RT.
Transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, suspending and dripping 50 mu LRNase-Free dHO into the middle part of the adsorption film, standing at room temperature for 2min, centrifuging at 12,000rpm for 2min to obtain RNA solution, and preserving at-70deg.C for use;
2) Carrying out RT-qPCR amplification reaction on the sample;
the reaction system is as follows:
mixing and centrifuging the reaction system, and respectively split charging the mixture into 8-joint PCR tube reaction strips, and performing fluorescence detection on Bio-RadFX 96;
the reaction procedure was as follows:
3) Experimental results: see fig. 14-18;
summarizing: the experimental result of the embodiment shows that the kit for detecting ROS1, RET and ALK lung cancer gene fusion can be used for detecting clinical samples well.
Claims (5)
1. A kit for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR is characterized in that: comprises the following components: 5 xRT-qPCR Master mix, enzyme mix, 8-PCR tube reaction strips;
the 5 XRT-qPCRMastermix contains: dNTP, magnesium ion, salt required by reaction and buffer ion;
the enzyme mixture comprises: reverse transcriptase, fastTaq enzyme, UDG enzyme;
the 8-linkage PCR tube reaction strip contains primer probe mixed liquor, and specifically consists of 6 groups of fusion gene primer probe mixed liquor and human internal reference gene (GAPDH gene) primer probe mixed liquor.
2. The kit for detecting lung cancer ROS1, RET and ALK fusion genes by using one-step RT-qPCR according to claim 1, wherein the kit is characterized in that: the specific fusion sites of the ROS1 fusion gene, RET fusion gene and ALK fusion gene are optimized, and the grouping and primer probe sequences are as follows:
the upstream exon of ROS1 fusion gene combination 1 is CD74 exon and the downstream exon of ROS1 is E32 exon
The upstream exogene of ROS1 fusion gene combination 1 is SLC34A2 exon and the downstream exogene of ROS1 exon is E32
The upstream exon of ROS1 fusion gene combination 1 is SLC34A2 exon and the downstream exon of ROS1 exon is E32
The upstream exogene of the ROS1 fusion gene combination 2 is CD74 exon and the downstream exogene is ROS1 exon and E34
The upstream exon of the ROS1 fusion gene combination 2 is EZR exon E10, the downstream exon of ROS1 is ROS1 exon E34
RET3 fusion gene combination 3 upstream external gene is CCDC6 exon and E1 downstream external gene is RET exon and E12
RET3 fusion gene combination 3 upstream external gene is CCDC6 exon and E2 downstream external gene is RET exon and E12
RET3 fusion gene combination 3 upstream external gene is NCOA4 exon and E8 downstream external gene is RET exon and E12
The RET4 fusion gene combination 1 has the upstream external gene of CCDC6 as E8 and the downstream external gene of RET as E11;
the ALK fusion gene combination 5 has the upstream exon of NPM1 as E5 and the downstream exon of ALK as E20;
the ALK fusion gene combination 5 has the upstream exon of NPM4 as E6 and the downstream exon of ALK as E20;
the upstream exon of ALK fusion gene combination 6 is NPM4 exon and the downstream exon of E13 is ALK exon and E20;
the upstream exon of ALK fusion gene combination 6 is EML4 exon and the downstream exon of E20 exon is ALK exon and E20.
3. The kit for detecting lung cancer ROS1, RET and ALK fusion genes by using one-step RT-qPCR according to claim 2, wherein the kit is characterized in that:
1) GAPDH reference gene: CY5 fluorescence
An upstream primer: 5'-CAGGTCAACTGGTATCGTGAC-3'
A downstream primer: 5'-CCACTAGAGGGGGATTGTGA-3'
Fluorescent probe: 5'-CY5-CTACGCAGTAGCACTGCCATCG- -BHQ1-3'
2) ROS1 fusion gene combination 1: FAM fluorescence
An upstream primer: 5'-GTTTCAACTGTGCAGGCACTC-3'
An upstream primer: 5'-TCTAGTTTTTCTTGTGCTGCC-3'
An upstream primer: 5'-GGCTCCTGATACCTCTGTTAAC-3'
A downstream primer: 5'-TCAGCTTTCACCACTGTATTG-3'
Fluorescent probe: 5'-FAM-AGTCCCAAAACCAGGCATTCCCA-BHQ1-3'
3) ROS1 fusion gene combination 2: FAM fluorescence
An upstream primer: 5'-GTTTGCATGCGCCCGCACTC-3'
An upstream primer: 5'-AAGGAGACTTCATCCTGC-3'
A downstream primer: 5'-TCAGCTTTCACCACTGTATTG-3'
Fluorescent probe: 5'-FAM-AGTCCCAAAACCAGGCATTCCCA-BHQ1-3'
4) RET fusion gene combination 3: FAM fluorescence
An upstream primer: 5'-AAGAAACCCTTGCTGTAAATTATGAG-3'
An upstream primer: 5'-TGAGCTCTCCAGAAAATTGATGC-3'
An upstream primer: 5'-CTTACATACCGAGCACGGAC-3'
A downstream primer: 5'-GACCACCATTCGAAATCGCC-3'
Fluorescent probe: 5'-FAM-AATTCCTCGGAAGAACTTGTTC-BHQ1-3'
5) RET fusion gene combination 4: FAM fluorescence
An upstream primer: 5'-CCCATAGGTTTCACAACCCACTG-3'
A downstream primer: 5'-TGTGGCCAAACTTCTGGAAG-3'
Fluorescent probe: 5'-FAM-TCTCCTTCTTCGTCACCGTGCTG-BHQ1-3'
6) ALK fusion gene combination 5: FAM fluorescence
An upstream primer: 5'-TTCAGGCCCAGTCCATAATAG-3'
An upstream primer: 5'-GTTACCAAAACTGCAGACAAGC-3'
A downstream primer: 5'-GGAGCCTGCTCAGCTTGTACT-3'
Fluorescent probe: 5'-FAM-CCGCGGAAGCAAGCAGGAG-BHQ1-3'
7) ALK fusion gene combination 6: FAM fluorescence
An upstream primer: 5'-GGTGGAGTCATGCTTATATGGAG-3'
An upstream primer: 5'-TCTAACTCGGGAGACTATGAAATATTG-3'
A downstream primer: 5'-GGAGCCTGCTCAGCTTGTACT-3'
Fluorescent probe: 5'-FAM-CCGCGGAAGCAAGCAGGAG-BHQ1-3'.
4. The one-step RT-qPCR method for detecting lung cancer according to claim 1
The kit for the ROS1, RET and ALK fusion genes is characterized in that: the concentration of the primer probe of the mixed liquid primer probe in the 8-linkage PCR tube reaction strip is 0.5-3 mu M.
5. A method for detecting lung cancer ROS1, RET and ALK fusion genes by one-step RT-qPCR is characterized by comprising the following steps: comprises an amplification reaction system and a reaction amplification program shown in tables 1-2;
TABLE 1
After preparation, uniformly mixing on a vortex device, centrifuging for 15 seconds, lightly uncovering the strip cover of the 8-link PCR tube reaction strip, dividing an amplification reaction system into the 8-link PCR tube reaction strip, uniformly mixing, centrifuging, and putting into a real-time PCR instrument for detection, wherein the detection is shown in the following table;
table 2.
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