CN112391465A - Multi-gene mutation detection kit for lung cancer - Google Patents
Multi-gene mutation detection kit for lung cancer Download PDFInfo
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Abstract
The invention discloses a multi-gene mutation detection kit for lung cancer, which comprises a library building kit, a hybridization kit, a probe kit and a joint kit for amplifying DNA fragments of all exons and partial intron regions of ALK, BCL2L11, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, RB1, RET, ROS1, TP53 and PTEN of 15 genes. The kit is used for enriching and detecting the variation conditions of multiple genes, such as mutation, deletion, insertion, fusion and the like, by combining a multi-probe targeted capture technology with a second-generation sequencing technology.
Description
Technical Field
The invention relates to the fields of molecular biology and medicine, in particular to a method for detecting multi-gene mutation of lung cancer.
Background
Lung cancer is the most common cancer worldwide for the last decades. Lung cancer can be divided histopathologically into two main groups: non-small cell lung cancer and small cell lung cancer, non-small cell lung cancer accounts for more than 80% of all lung cancer cases. With the introduction of the "precise medical plan", China also vigorously advances the development of targeted gene detection and targeted therapeutic drugs. The lung cancer related genes mainly comprise EGFR, ALK, BRAF, PIK3CA, ROS1, NRAS and the like, and related targeting drugs comprise EGFR Tyrosine Kinase Inhibitors (TKI) aiming at the EGFR; crizotinib inhibitors against ALK, and the like. The variation condition of the gene has clinical guiding significance for patient disease monitoring, prognosis evaluation, medication guidance and the like.
Early molecular diagnostics employed a variety of technology platforms, such as quantitative PCR, FISH, immunohistochemistry, etc., and the method was limited to detecting only one or a few genetic variations at a time, with the amount of sample required increasing with the increase in the number of genes detected. Therefore, the requirement for detecting all target genes cannot be satisfied. Meanwhile, recent studies show that important target gene variations in lung cancer are not completely mutually exclusive, but coexist in a small fraction of patients. For these patients, the use of early molecular diagnostic methods can lead to missed detection of certain genetic variant events, thereby allowing the physician to make possible false positives on the patient's optimal targeted therapy. The second generation DNA-based sequencing method can parallelly detect dozens or even hundreds of genes including point mutation, insertion deletion, DNA copy number change, fusion rearrangement and other variant forms at one time, thereby detecting all gene variants related to targeted therapy at one time. Therefore, a new way is provided for breaking through the limitation of the traditional molecular diagnosis method.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a multi-gene mutation detection kit for lung cancer and application thereof.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
the kit for detecting the multi-gene mutation of the lung cancer comprises a library building kit, a hybridization kit, a probe kit and a joint kit for amplifying DNA fragments of all exons and partial intron regions of ALK, BCL2L11, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, RB1, RET, ROS1, TP53 and PTEN of 15 genes.
As a further scheme of the invention: the kit comprises components and contents of a library construction kit 1 box, a hybridization kit 1 box, a probe kit 1 box and a joint kit 1 box, wherein the detection kit is applied to 16-person detection, and the storage temperature of the kit is-25 ℃ to-15 ℃.
As a further scheme of the invention: the library construction kit can be used for DNA fragmentation end repair, ligation reaction and amplification enrichment.
As a further scheme of the invention: the library construction kit comprises 320 microliters of 1-tube end repair and A reaction solution, 80 microliters of 1-tube end repair and A enzyme, 460 microliters of 1-tube connection reaction solution, 40 microliters of 1-tube ligase, 200 microliters of 1-tube front amplification reaction solution, 20 microliters of 1-tube front amplification enzyme, 10 microliters of 1-tube dNTP mixed solution and 40 microliters of 1-tube front amplification primer.
As a further scheme of the invention: the hybridization kit can perform hybridization capture, cleaning after hybridization and enrichment after hybridization.
As a further scheme of the invention: the hybridization kit comprises 100 microliters of 1-tube sealing solution, 120 microliters of 1-tube hybridization buffer solution, 10 microliters of 1-tube nuclease blocking solution, 16ml of 1-bottle magnetic bead binding buffer solution, 14 ml of 1-bottle cleaning buffer solution, 224 ml of 1-bottle cleaning buffer solution, 200 microliters of 1-tube post-amplification reaction solution, 20 microliters of 1-tube post-amplification enzyme, 10 microliters of 1-tube dNTP mixed solution and 20 microliters of 1-tube post-amplification primer.
As a further scheme of the invention: the probe kit comprises 10 microliter of 1 tube of 15 gene probes.
As a further scheme of the invention: the joint box comprises 16P 5 end indexes, 16P 7 end indexes and 6 microliter/tube.
Compared with the prior art, the invention has the beneficial effects that: the invention has reasonable design and simple structure, and realizes the purpose of simultaneously detecting DNA fragments of all exons and partial intron regions of 15 genes by using a capture probe technology; the invention integrates the DNA fragment detection of all exons and partial intron regions of the lung cancer related gene into a reagent kit, and has more convenient use and lower cost.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The kit for detecting the multi-gene mutation of the lung cancer comprises a library building kit, a hybridization kit, a probe kit and a joint kit.
The library construction kit comprises a packaging box body, a library construction kit liner and reagent tubes, wherein the library construction kit liner is arranged inside the library construction kit packaging box body, container holes are formed in the liner, and the reagent tubes comprise 1 tube of end repair and A reaction liquid 320 microliter, 1 tube of end repair and A enzyme 80 microliter, 1 tube of connection reaction liquid 460 microliter, 1 tube of ligase 40 microliter, 1 tube of front amplification reaction liquid 200 microliter, 1 tube of front amplification enzyme 20 microliter, 1 tube of dNTP mixed liquid 10 microliter and 1 tube of front amplification primer 40 microliter.
The hybridization kit comprises a packaging box body, a hybridization kit liner, a reagent tube and a reagent bottle, wherein the hybridization kit liner is arranged inside the hybridization kit packaging box body, container holes are formed in the liner, the reagent tube comprises 100 microliters of 1-tube sealing liquid, 120 microliters of 1-tube hybridization buffer liquid, 10 microliters of 1-tube nuclease blocking liquid, 200 microliters of 1-tube post-amplification reaction liquid, 20 microliters of 1-tube post-amplification enzyme, 10 microliters of 1-tube dNTP mixed liquid and 20 microliters of 1-tube post-amplification primer, and the reagent bottle comprises 16ml of 1-bottle magnetic bead binding buffer liquid, 14 ml of 1-bottle washing buffer liquid and 224 ml of 1-bottle washing buffer liquid.
The probe kit comprises a packaging box body, a probe kit liner and a reagent tube, wherein the probe kit liner is arranged inside the packaging box body of the probe kit, container holes are formed in the liner, and the reagent tube comprises 1 tube of 15-gene probes 10 microliter.
The joint kit comprises a packaging box body, a joint kit gasket and a reagent tube, wherein the joint kit gasket is arranged inside the joint kit packaging box body, a container hole is formed in the gasket, and the reagent tube comprises 16P 5 end indexes, 16P 7 end indexes and 6 microliter/tube.
The specific linker in the kit refers to a specific sequence tag which can be added to both ends of the target fragment by reaction. In the kit, 16 adapters are provided, and different specific sequence tags are added to different samples. Specific sequence tags can be detected using second generation sequencing to distinguish between different samples.
The kit comprises the following components in percentage by weight:
building a library kit 1:
1 tube 320 microliter/tube end is repaired and added with A reaction solution,
1 tube 80 microliter/tube end repair with enzyme A,
1 tube 460 microlitre/tube of the reaction solution,
1 tube 40. mu.l/tube of ligase,
1 tube 200. mu.l/tube of pre-amplification reaction solution,
1 tube 20. mu.l/tube of preamplifying enzyme,
1 tube of 10. mu.l/tube of dNTP mixture,
1 tube 40. mu.l/tube preamplification primer.
Hybridization kit 1 kit:
1 tube of 100 microliter/tube of confining liquid,
1 tube of 120. mu.l/tube of hybridization buffer,
1 tube 10. mu.l/tube of RNase blocker,
1 vial of 16 ml/vial of magnetic bead binding buffer,
1 vial of 4 ml/vial of wash buffer 1,
1 vial of 24 ml/vial of wash buffer 2,
1 tube 200. mu.l/tube of post-amplification reaction solution,
1 tube 20. mu.l/tube of post-amplification enzyme,
1 tube of 10. mu.l/tube of dNTP mixture,
1 tube 20. mu.l/tube of post amplification primer.
Probe kit 1 kit:
1 tube 10. mu.l/tube 15 gene probe.
Splice closure 1 closure:
16P 5 end indexes, 1 tube, 6 microlitres of each,
16P 7 end indexes, 1 tube each, 6 microliters.
The detection kit is applied to 16-person detection, and the storage temperature of the kit is-25 ℃ to-15 ℃.
The specific embodiment of the invention is as follows:
1 extraction and fragmentation of genomic DNA
1.1 extraction of genomic DNA. Tissue extraction is performed on the subject.
1.2 fragmentation of genomic DNA. The Covaris instrument is used and the operation is carried out according to the recommended operation flow, and the fragment size is 200-300 bp.
2 preparation of Ligation master mix
2.1 thawing the Ligation Buffer, and after thawing, shaking vigorously for 15 s.
2.2, preparing according to the following table, blowing, beating and uniformly mixing for 15-20 times after the preparation is finished, centrifuging for a short time, and standing for 30-45 minutes at room temperature.
| Reagent | Volume required for 1 reaction (microlitre) |
| Ligation Buffer | 23 |
| T4 DNA Ligase | 2 |
| Total volume | 25 |
3 terminal repair reaction
3.1 unfreezing the End Repair-A labeling Buffer, and after unfreezing, violently shaking for 15 s.
3.2 preparing End Repair/dA-labeling master mix according to the following table, blowing and beating uniformly for 15-20 times after the preparation is finished, and centrifuging briefly.
| Reagent | Volume required for 1 reaction (microlitre) |
| End Repair-A Tailing Buffer | 16 |
| End Repair-A Tailing Enzyme Mix | 4 |
| Total volume | 20 |
3.3 Add 20. mu.l of End Repair/dA-labeling master mix into 50. mu.l of fragmented DNA, blow-beating and mixing for 15-20 times, and centrifuging for a short time.
3.4 the PCR machine is operated, the reaction conditions are as follows:
| temperature (. degree.C.) | Time (minutes) |
| 20 | 15 |
| 72 | 15 |
| 4 | — |
4P 5-index ligation
Note: in picking P5 (green plate) and P7 (yellow plate) for each sample, P5 and P7 of the same sample should be at the same position on the respective plates.
4.1 taking 25 microliter of Ligation master mix, adding the mixture into the reaction product in the previous step, uniformly blowing and stirring for 15-20 times, and centrifuging for a short time.
4.2 adding 5 microliter of index adapter at P5 end corresponding to SureSelect XT Low Input Dual index adapter (green plate), blowing and uniformly mixing for 15-20 times, and centrifuging for a short time.
4.3 the PCR machine is operated, the reaction conditions are as follows:
| temperature (. degree.C.) | Time (minutes) |
| 20 | 30 |
| 4 | — |
5 purification and sorting
5.1 Add 60. mu.l of magnetic beads to the ligation product from the previous step, gently blow to ensure the uniformity of the whole system, and let stand at room temperature for 5 minutes.
And 5.2, placing the centrifugal tube on a magnetic frame to separate the magnetic beads from the liquid.
5.3 keep the centrifuge tube on the magnetic rack and after the solution is clear (about 5-10 minutes), carefully discard the supernatant taking care not to disturb the magnetic beads.
5.4 keep the centrifuge tube on a magnetic stand, add slowly 200. mu.l of freshly prepared 70% ethanol along the tube wall, take care not to disturb the magnetic beads while adding ethanol, and carefully remove the supernatant after 30 seconds of incubation.
5.5 repeat step 5.4, rinsing twice in total.
5.6 suck all residual ethanol with a 2-10 microliter pipette, decap and dry in air for 5-10 minutes.
Note: ensure that the beads are just dry, the beads now appear lusterless. If the beads are not completely air-dried, alcohol remains in the sample, which reduces the efficiency of DNA elution and may interfere with downstream reactions.
5.7 after the beads were air-dried, the centrifuge tube was taken off from the magnetic frame, 105. mu.l of sterile water was added to cover the beads, the beads were blown up by a pipette and allowed to stand at room temperature for 5 minutes.
5.8 placing the centrifuge tube on a magnetic frame after the centrifuge tube is centrifuged for a short time, and sucking 100 microliters of supernatant and placing the supernatant in a new centrifuge tube after the solution is clarified.
5.9 Add 60. mu.l of magnetic beads into the tube, gently blow and mix them, and let stand at room temperature for 5 minutes.
5.10 placing the centrifuge tube on a magnetic frame to separate the magnetic beads and the liquid, and taking 155 microlitres of supernatant to a new centrifuge tube after the solution is clarified.
5.11 Add 20. mu.l of magnetic beads into the tube, gently blow and mix, and let stand at room temperature for 5 minutes.
5.12 placing the centrifuge tube on a magnetic frame to separate the magnetic beads from the liquid.
5.13 keep the centrifuge tube on the magnetic rack and after the solution is clear (about 5-10 minutes), carefully discard the supernatant taking care not to disturb the magnetic beads.
5.14 keep the centrifuge tube on a magnetic rack, add slowly 200. mu.l of freshly prepared 70% ethanol along the tube wall, take care not to disturb the magnetic beads while adding ethanol, and carefully remove the supernatant after 30 seconds of incubation.
5.15 repeat step 5.14, rinsing twice in total.
5.16 suck all residual ethanol with a 2-10 microliter pipette, decap and dry in air for 5-10 minutes.
5.17 after the magnetic beads were air-dried, the centrifuge tube was taken off from the magnetic frame, 34.5. mu.l of sterilized water was added, the magnetic beads were blown and mixed by a pipette, and the mixture was allowed to stand at room temperature for 5 minutes.
5.18 centrifuge the tube briefly and place on magnetic rack, after the solution is clarified, suck 34.5 microliter of supernatant and place in new centrifuge tube.
6 amplification enrichment with P7-terminal index
6.1 preparing pre-capture PCR reaction mix according to the following table, blowing and uniformly mixing for 15-20 times after the preparation is finished, and centrifuging for a short time.
| Reagent | Volume required for 1 reaction (microlitre) |
| 5×Herculase II Reaction Buffer | 10 |
| 100mM dNTP Mix | 0.5 |
| Forward Primer | 2 |
| Herculase II Fusion DNA Polymerase | 1 |
| Total volume | 13.5 |
6.2 Pre-capture PCR reaction mix, SureSelect XT Low Input Index Primer (P7 end yellow plate, same position as P5 green plate used in the previous ligation step), purified product, mixed according to the following table, blown and beaten well 15-20 times, briefly centrifuged.
| Reagent | Volume (microliter) |
| pre-capture PCR reaction mix | 13.5 |
| SureSelect XT Low Input Index Primer | 2 |
| Purification of the product | 34.5 |
| Total volume | 50 |
6.3 the PCR machine is operated, the reaction conditions are as follows:
note: wherein the number of amplification cycles x of step 2 is selected according to the table below,
| amount of DNA added | Number of cycles |
| 100-200ng | 8 |
| 50ng | 9 |
| 10ng | 11 |
7 purifying the amplification product
7.1 adding 45 microliter of magnetic beads into the amplification product, gently blowing and uniformly mixing to ensure the uniformity of the whole system, and standing for 5 minutes at room temperature.
7.2 placing the centrifuge tube on a magnetic frame to separate the magnetic beads from the liquid.
7.3 keep the centrifuge tube on the magnetic rack and after the solution is clear (about 5-10 minutes), carefully discard the supernatant taking care not to disturb the magnetic beads.
7.4 keep the centrifuge tube on a magnetic stand, add slowly 200. mu.l of freshly prepared 70% ethanol along the tube wall, take care not to disturb the magnetic beads while adding ethanol, and carefully remove the supernatant after 30 seconds of incubation.
7.5 repeat step 7.4, rinsing twice in total.
7.6 suck all residual ethanol with a 2-10 microliter pipette, decap and dry in air for 5-10 minutes.
Note: ensure that the beads are just dry, the beads now appear lusterless. If the beads are not completely air-dried, alcohol remains in the sample, which reduces the efficiency of DNA elution and may interfere with downstream reactions.
7.7 after the magnetic beads are dried in the air, the centrifugal tube is taken down from the magnetic frame, 18 microliters of sterilized water is added, the magnetic beads are blown and uniformly mixed by using a pipette, and the mixture is kept standing for 5 minutes at room temperature.
7.8 placing the centrifuge tube on a magnetic frame to separate the magnetic beads and the liquid, and taking 18 microliters of supernatant to a new centrifuge tube after the solution is clarified.
8 hybrid Capture
8.1 take 12. mu.l of library (1000 ng) and add 5. mu.l of SureSelect XT HS and XT Low Input Block Mix, shake at high speed for 5 seconds, centrifuge briefly.
8.2 the PCR machine is operated, the reaction conditions are as follows:
8.3 Place the 8.1 product on the 8.2 set-up PCR machine and click PLAY to start the program.
8.4 Capture Library Hybridization Mix was prepared as shown in the following table.
| Reagent | Volume (microliter) |
| Nuclease-free water | 6 |
| SureSelect Fast Hybridization Buffer | 6 |
| Capture Library≤3Mb | 0.5 |
| SureSelect RNase Block | 0.5 |
| Total volume | 13 |
8.5 when the PCR program is run to the 3 rd step, click PAUSE, PAUSE the program, add 13 microliters of Capture Library Hybridization Mix to the reaction tube on the PCR instrument (containing 17 microliters of the bottle-Library mixture), blow and beat 8-10 times of mixing, cover, click on Resume, make the program continue to run.
8.6T 1 magnetic beads were prepared (the beads were washed 3 times using a SureSelect Binding Buffer and resuspended in 200. mu.l of the SureSelect Binding Buffer).
8.7 when the PCR program was run to the 5 th step of 8.2, the hybridization product was quickly transferred to magnetic beads, pipetted 5-8 times to mix well, and shaken in a horizontal shaker at 1500rpm for 30 minutes at room temperature.
8.8 during the 8.7 step, 6 wells of SureSelect Wash Buffer 2 were prepared for each sample, 200 microliters per well, and pre-heated (with a hot lid) on a PCR instrument at 70 ℃.
8.9 after the oscillation, centrifuging, discharging the supernatant on a magnetic frame, adding 200 microliters of SureSelect Wash Buffer 1, blowing and uniformly mixing for 15-20 times, and discharging the supernatant on the magnetic frame.
8.10 carefully clean excess SureSelect Wash Buffer 2, add 25. mu.L of water to resuspend, and place temporarily in a 4 ℃ freezer.
9 post-hybridization amplification
9.1 post-capture PCR Reaction Mix was prepared as shown in the following table.
| Reagent | Volume (microliter) |
| Nuclease-free water | 12.5 |
| 5x Herculase II Reaction Buffer | 10 |
| Herculase II Fusion DNA Polymerase | 1 |
| 100mM dNTP Mix | 0.5 |
| SureSelect Post-Capture Primer Mix | 1 |
| Total volume | 25 |
9.2 Add 25. mu.l of post-capture PCR Reaction Mix to 25. mu.l of the product from the previous step. Blow-beating and mixing for 15-20 times, and then centrifuging for a short time.
9.3, processing. The reaction procedure was as follows:
9.4 after off-machine the PCR product was placed on a magnetic rack, the supernatant was transferred to a new 1.5ml centrifuge tube, 45. mu.l of magnetic beads were added and purified 2 steps with 70% ethanol, eluted with 25. mu.l of water and quantitatively prepared on-machine.
While the foregoing is directed to the preferred embodiment of the present invention, and not to any one of the preceding forms or essential limitations thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (8)
1. A multi-gene mutation detection kit for lung cancer is characterized in that: comprises a library construction kit, a hybridization kit, a probe kit and a joint kit for amplifying DNA fragments of all exons and partial intron regions of ALK, BCL2L11, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, RB1, RET, ROS1, TP53 and PTEN, 15 genes.
2. The detection kit according to claim 1, characterized in that: the kit comprises components and contents of a library construction kit 1 box, a hybridization kit 1 box, a probe kit 1 box and a joint kit 1 box, wherein the detection kit is applied to 16-person detection, and the storage temperature of the kit is-25 ℃ to-15 ℃.
3. The detection kit according to claim 1, characterized in that: the library building kit comprises a terminal repair and A reaction solution and a terminal repair and A enzyme which can carry out DNA fragmentation terminal repair, a ligation reaction solution and a ligase of a ligation reaction, a pre-amplification reaction solution, a pre-amplification enzyme, a dNTP mixed solution and a pre-amplification primer of an amplification enrichment reaction.
4. The detection kit according to claim 1, characterized in that: the components and contents of the library construction kit comprise 1 tube of 320 mu l/tube end repair and A reaction liquid, 1 tube of 80 mu l/tube end repair and A enzyme, 1 tube of 460 mu l/tube connection reaction liquid, 1 tube of 40 mu l/tube ligase, 1 tube of 200 mu l/tube pre-amplification reaction liquid, 1 tube of 20 mu l/tube pre-amplification enzyme, 1 tube of 10 mu l/tube dNTP mixed liquid, 1 tube of 40 mu l/tube pre-amplification primer, 1 outer package box and 1 inner liner.
5. The detection kit according to claim 1, characterized in that: the hybridization kit comprises hybridization capture of DNA sequences, washing after hybridization and enrichment after hybridization.
6. The detection kit according to claim 1, characterized in that: the components and contents of the hybridization kit comprise 1 tube of 100 mul/tube sealing liquid, 1 tube of 120 mul/tube hybridization buffer solution, 1 tube of 10 mul/tube ribonuclease blocking liquid, 1 bottle of 16 ml/bottle magnetic bead binding buffer solution, 1 bottle of 4 ml/bottle washing buffer solution 1, 1 bottle of 24 ml/bottle washing buffer solution 2, 1 tube of 200 mul/tube post-amplification reaction liquid, 1 tube of 20 mul/tube post-amplification enzyme, 1 tube of 10 mul/tube dNTP mixed liquid, 1 tube of 20 mul/tube post-amplification primer, 1 outer package box and 1 inner liner.
7. The detection kit according to claim 1, characterized in that: the components and contents of the probe kit comprise 15 gene probes of 1 tube and 10 mu l/tube, 1 outer package box and 1 inner liner.
8. The detection kit according to claim 1, characterized in that: the components and contents of the joint kit comprise 16P 5 end indexes, 1 pipe of each type, 6 mu l of 1 pipe, 16P 7 end indexes, 1 pipe of each type, 6 mu l of 1 pipe, 1 outer packing box and 1 inner lining.
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