CN112063713A - Retinoblastoma gene mutation screening kit - Google Patents

Abstract

The invention discloses a probe set for screening retinoblastoma gene mutation, which covers the CDS region of 31 genes related to retinoblastoma; the detection of 420 gene mutation positions is covered. The invention also discloses a retinoblastoma gene mutation screening kit, which comprises a CDS region sequencing probe set of 31 genes related to retinoblastoma. The kit is based on a second-generation high-throughput sequencing platform, screens 31 genes related to the retinoblastoma, can evaluate the gene mutation condition of the retinoblastoma more accurately, can save time and labor cost, and has the characteristics of saving clinical time, short-term and high cost performance.

Description

Retinoblastoma gene mutation screening kit

Technical Field

The invention relates to a gene mutation screening kit, in particular to a retinoblastoma gene mutation screening kit.

Background

Retinoblastoma (Rb) is a rare malignancy that results from the rapid proliferation and development of photoreceptor precursor cells. Retinoblastoma is the most common intraocular malignancy in infants and young children, is common in children under 5 years of age, accounts for about three percent of all malignancies in children, and has a survival rate of only one in two ten-thousandth. The survivors eyes will lose vision and even be excised.

The onset of retinoblastoma is related to gene mutation, so that the detection and screening of retinoblastoma have very important practical significance: on one hand, screening of retinoblastoma gene mutation on a newborn can help to judge whether related gene mutation is carried, if related carrying exists, the risk can be judged, regular eyeground screening can be diagnosed as early as possible, and if no related carrying exists, follow-up diagnosis can be reduced, and medical resources can be saved; on the other hand, screening of retinoblastoma can be applied to genetic consultation and prenatal diagnosis, prenatal noninvasive screening can be performed on pregnant women carrying gene mutation and partners thereof, the birth of children suffering from retinoblastoma can be effectively avoided, and the purpose of good prenatal and postnatal care is achieved.

At present, screening for retinoblastoma gene mutation is mainly based on related hot spots, such as the c.116 site of exon 1 of RB1 gene. The screening of the hot spot mutation can only detect one concerned site at a time, and can not effectively screen all related sites, so that the mutation condition of genes related to the retinoblastoma except the concerned site can not be detected. The kit is based on a second-generation high-throughput sequencing platform, 31 gene full-length probe regions related to retinoblastoma gene mutation are designed, mutation conditions of all related genes can be effectively screened at one time, the retinoblastoma gene mutation conditions can be more accurately evaluated, and time and labor cost are saved.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a gene capture type kit for comprehensively screening retinoblastoma-related gene mutation, which is based on a second-generation high-throughput sequencing platform to screen 31 gene CDS (CDS) regions related to retinoblastoma, so that the retinoblastoma gene mutation condition can be more accurately evaluated, and the time and labor cost are saved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a probe set for screening retinoblastoma gene mutation, which covers the CDS region of 31 genes related to retinoblastoma;

the 31 retinoblastoma-related genes are as follows:

NFE2L2、TOMM20、MYC、BAP1、NF1、PTEN、HMOX1、EZH2、PIK3CB、BRAF、 CDKN2A、IDH1、NQO1、CEP290 FOXA1、NRAS、TP53、MAP2K1、FTH1、HIF1A、 CHRM1、HRAS、PPP6C、DDX3X、PTGS2、DDR1、E2F1、KRAS、ARID2、RAC1、RB1；

the probe set covers 420 gene mutation position detections, and the sequences are respectively shown in SEQ ID No. 1-SEQ ID No. 420.

The invention provides application of the probe set for screening retinoblastoma gene mutation in preparation of a screening kit for detecting retinoblastoma gene mutation.

The invention provides a retinoblastoma gene mutation screening kit, which comprises a CDS region probe set of 31 genes related to retinoblastoma;

the 31 retinoblastoma-related genes are as follows:

NFE2L2、TOMM20、MYC、BAP1、NF1、PTEN、HMOX1、EZH2、PIK3CB、BRAF、 CDKN2A、IDH1、NQO1、CEP290 FOXA1、NRAS、TP53、MAP2K1、FTH1、HIF1A、 CHRM1、HRAS、PPP6C、DDX3X、PTGS2、DDR1、E2F1、KRAS、ARID2、RAC1、RB1；

the probe set covers 420 gene mutation position detections, and the sequences are respectively shown in SEQ ID No. 1-SEQ ID No. 420.

Further, the test samples include blood, fresh tissue, FFPE samples and saliva.

Further, the kit comprises reagents: human Cot-1, Blocker, 2X Hybridization Buffer, Hybridization Buffer Eufastener, nucleic-Free Water, Bead Wash Buffer.

The invention provides a use method of the retinoblastoma gene mutation screening kit, which comprises the following steps:

(1) extracting a genome of a sample to be detected;

(2) and (3) DNA quality inspection: performing quality inspection on the extracted sample genome to be detected by using the Qubit;

(3) constructing a library of a sample to be tested: fragmentation + terminal repair + dA-labeling, linker, magnetic bead purification, library enrichment, purification of an enriched product, and determination of library concentration;

(4) hybridizing a sample library to be detected with the probe;

(5) separation of the captured product;

(6) enrichment of the captured product;

(7) purifying a capture product;

(8) the Qubit determines the product concentration;

(9) performing sequencing on the NGS;

(10) off-line data quality control;

(11) and (6) analyzing the data.

Further, in the step (1), the test sample comprises blood, fresh tissue, FFPE sample and saliva.

Further, in the step (4), the hybridization between the test sample library and the probe set specifically comprises the following steps:

a) taking an EP tube, adding a sample library to be detected, Human Cot-1 and Blocker, and drying;

b) the following reagents were added in sequence: 2X Hybridization Buffer, Hybridization Buffer Euhancer, Nuclear-Free Water;

c) standing at room temperature;

d) shaking and centrifuging, transferring to a new low-adsorption PCR tube, and keeping the temperature at 95 ℃ for 10 min;

e) immediately placing the mixture into an ice box, standing the mixture, and adding a retinoblastoma gene screening probe set;

f) shaking and mixing uniformly, instantly separating, and then putting into a PCR instrument for incubation for 16-20 h at 65 ℃.

Further, in the step (5), the separation of the captured product comprises the following specific steps:

a) balancing Dynabeads M-270 magnetic beads at room temperature for 30min for later use, and mixing uniformly;

b) taking magnetic beads into a centrifugal tube with low adsorption, putting the centrifugal tube into a magnetic frame, clarifying, and then discarding the supernatant;

c) adding 1X Bead Wash Buffer, shaking, mixing, centrifuging instantaneously, returning to the magnetic rack, and removing supernatant after clarification;

d) repeating the previous step once;

e) adding 1X Bead Wash Buffer, shaking, mixing, centrifuging instantaneously, and transferring into a low adsorption PCR tube;

f) placing on a magnetic frame to thoroughly enrich magnetic beads, and carefully discarding the supernatant;

g) mixing the magnetic beads with the obtained probe hybridized library, shaking and uniformly mixing, and performing instantaneous centrifugation;

h) incubating for 40-50 min at 65 ℃;

i) adding WBL, mixing, and centrifuging instantly;

j) transferring to a low adsorption tube; shaking gently and mixing uniformly, and separating instantly;

k) placing on a magnetic frame, and carefully discarding the supernatant after clarification;

l) adding SWB, and slowly and carefully blowing the mixture without bubbles; incubating at 65 ℃, putting the mixture into a magnetic frame after instantaneous dissociation, and carefully discarding the supernatant after clarification;

m) repeating the previous step once;

n) adding WBl, shaking and uniformly mixing, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and discarding supernatant after clarification;

o) adding WBll, shaking and uniformly mixing, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and discarding supernatant after clarification;

p) adding WBlll, shaking and uniformly mixing for 30s, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and removing supernatant after clarification;

q) removing the sample from the magnetic frame, and adding deionized water to resuspend the magnetic beads.

Further, in the step (7), the specific steps of purifying the captured product are as follows:

a) balancing the purified magnetic beads at room temperature for at least 30min, fully and uniformly mixing the magnetic beads before use, and instantly separating; preparing 80% ethanol at present;

b) taking magnetic beads to the enriched capture products, and incubating at room temperature;

c) after the instantaneous separation, placing the magnetic frame and discarding the supernatant;

d) adding 80% ethanol to rinse the magnetic beads, discarding the supernatant, and repeating the steps once;

e) opening a cover and drying the air;

f) taking the sample off the magnetic frame for elution;

g) adding ddH2O, shaking, mixing, instantly separating, and standing;

h) place the sample on a magnetic rack and carefully pipette the supernatant into a new PCR tube.

Compared with the prior art, the invention has the following beneficial effects:

the kit is based on a second-generation high-throughput sequencing platform, screens 31 genes related to the retinoblastoma, is a gene capture type kit for comprehensively screening the retinoblastoma-related gene mutation, designs and captures Panel for CDS regions of the 31 genes, can effectively screen all mutation conditions of the CDS regions of the 31 genes at one time, can more accurately evaluate the retinoblastoma gene mutation conditions, and saves time and labor cost. The method also has the characteristics of saving clinical time, rapid cycle and high cost performance.

Drawings

FIGS. 1 to 13 are graphs obtained by the Sanger sequencing method in the effect test example:

sample number RBM190608003 of fig. 1, sample number RBM190608007 of fig. 2,

sample number RBM190608009 of fig. 3, sample number RBM190608009 of fig. 4,

sample number RBM190608016 of fig. 5, sample number RBM190608022 of fig. 6,

sample No. RBM190608028 of fig. 7, sample No. RBM190608043 of fig. 8,

sample number RBM190608049 of fig. 9, sample number RBM190608052 of fig. 10,

sample number RBM190608059 of fig. 11, sample number RBM190608059 of fig. 12,

sample number RBM190608062 of fig. 13.

Detailed Description

The invention provides a gene capture type kit for comprehensively screening retinoblastoma related gene mutation. The kit is based on a second-generation high-throughput sequencing platform, 31 genes related to the retinoblastoma are screened, and the CDS regions of the 31 genes are designed to capture Panel, so that all mutation conditions of the CDS regions of the 31 genes can be effectively screened at one time, the retinoblastoma gene mutation conditions can be more accurately evaluated, and the time and labor cost is saved. Meanwhile, the method has the characteristics of saving clinical time, short period and high cost performance.

The specific scheme of the invention is as follows:

1. obtaining a whole genome library;

2. panel capture for retinoblastoma: CDS regions of 31 genes associated with disease;

3. high-throughput sequencing and bioinformatic data analysis.

1) The types of samples detected by the invention are various, including blood, fresh tissue, FFPE samples, saliva and the like;

2) the kit can screen the full length of CDS regions of 31 genes related to the retinoblastoma:

the mutation positions of the retinoblastoma detection genes detected by the probe set are shown in the following table 1, and the sequences are respectively shown in SEQ ID No. 1-SEQ ID No. 420.

TABLE 1 Gene List and Probe information

The invention has the following effects:

1. the CDS region coverage of all 31 genes is up to 100 percent;

2. sensitivity of gene mutation: 2 percent;

3. see the examples for details.

The invention will now be further described with reference to the accompanying drawings and specific embodiments.

Examples

The experimental steps are as follows:

1. extracting the genome of a sample to be tested

1) Adding 20ul of protease K into the bottom of a 1.5EP tube;

2) adding 200ul of blood or other body fluid;

3) adding 200ul Buffer AL, and fully mixing for 15s by vortex;

4) incubating for 10min at 56 ℃, and centrifuging the EP tube instantaneously to enable liquid on the tube wall to be concentrated to the tube bottom;

5) adding 200ul of absolute ethyl alcohol, uniformly mixing for 15s by vortex, and performing instantaneous centrifugation;

6) moving the liquid into a collecting pipe; centrifuging at 8000rpm for 1min, and discarding the collected liquid;

7) adding 500ul Buffer AW1, and centrifuging at 8000rpm for 1 min;

8) adding 500ul Buffer AW2, and centrifuging at the maximum rotation speed for 3 min;

9) the collection tube was placed in a new 1.5ml EP tube (kit not provided), 100ul DNase-free Water was added, and incubation was performed at room temperature for 5 min; centrifuge at 12000rpm for 1 min.

DNA quality inspection

And (4) performing quality inspection on the genome extracted in the last step by using the Qubit.

3. Construction of a library of samples to be tested

1) Fragmentation + end repair + dA-tailing

The reaction system is as follows:

the reaction procedure was as follows (hot lid 70 ℃):

2) connecting joint

The following reagents were added to the product of the previous step:

reaction procedure:

22℃ 15min

4℃ Hold

3) magnetic bead purification

a) Balancing the purified magnetic beads at room temperature for at least 30min, fully and uniformly mixing the magnetic beads before use, and instantly separating; preparing 80% ethanol at present;

b) taking 80ul of magnetic beads to the product of the last step, and incubating for 5min at room temperature;

c) placing on a magnetic frame for 3min after instantaneous separation, and discarding the supernatant;

d) adding 200ul 80% ethanol to rinse the magnetic beads, discarding the supernatant after 30s, and repeating once;

e) opening the cover and drying in air for 3 min;

f) taking the sample off the magnetic frame for elution;

g) adding 22ul ddH2O, shaking, mixing, instantly separating, and standing for 5 min;

h) the sample was placed on a magnetic stand and allowed to stand for 5min, 20ul of supernatant was carefully pipetted into a new PCR tube, and no beads were pipetted.

4) Library enrichment

The reaction system is detailed in the following table:

reaction procedure, detailed in the following table:

5) purification of the enriched product

a) Balancing the purified magnetic beads at room temperature for at least 30min, fully and uniformly mixing the magnetic beads before use, and instantly separating; preparing 80% ethanol at present;

b) taking 40ul of magnetic beads, and incubating at room temperature for 5 min;

c) placing on a magnetic frame for 3min after instantaneous separation, and discarding the supernatant;

d) adding 200ul 80% ethanol to rinse the magnetic beads, discarding the supernatant after 30s, and repeating once;

e) opening the cover and drying in air for 3 min;

f) taking the sample off the magnetic frame for elution;

g) adding 35ul ddH2O, shaking, mixing, instantly separating, and standing for 5 min;

h) the sample was placed on a magnetic stand and allowed to stand for 5min, 32ul of supernatant was carefully pipetted into a new PCR tube, and no beads were pipetted.

6) Qubitdetermination library concentration

4. Hybridization of test sample library with Probe set

a) Taking a new 1.5mL EP tube, adding 500ng of the library, and adding 5ul of Human Cot-1 and 2ul of packer; drying at 60 deg.C for 1h in a vacuum drying instrument;

b) the following reagents were added sequentially to the EP tube in the previous step:

2X Hybridization Buffer 8.5ul

Hybridization Buffer Euhancer 2.7ul

Nuclease-Free Water 1.8ul

c) standing at room temperature for 5 min;

d) shaking and centrifuging, transferring to a new low-adsorption 0.2ml PCR tube, and carrying out centrifugation at 95 ℃ for 10min (hot lid 105 ℃);

e) immediately placing the mixture into an ice box, standing for 2min, and adding 4ul of retinoblastoma gene screening probe set;

f) shaking and mixing uniformly, putting into a PCR instrument after instantaneous dissociation, and incubating and hybridizing for 16h (no more than 20h) at 65 ℃;

5. separation of captured products

a) Balancing Dynabeads M-270 magnetic beads at room temperature for 30min for later use, and uniformly mixing for about 15 s;

b) taking 100ul of magnetic beads into a low-adsorption 1.5ml centrifuge tube, putting the centrifuge tube into a magnetic frame, clarifying, and then discarding the supernatant;

c) adding 200ul of 1X Bead Wash Buffer, shaking and uniformly mixing for 10s, performing instantaneous centrifugation, putting back to a magnetic rack, and discarding supernatant after clarification;

d) repeating the previous step once;

e) adding 100ul of 1X Bead Wash Buffer, shaking, mixing, performing instantaneous centrifugation, and transferring to a 0.2ml low adsorption PCR tube;

f) placing on a magnetic frame to thoroughly enrich magnetic beads, and carefully discarding the supernatant;

g) mixing the magnetic beads with the library hybridized with the probe obtained in the last step, shaking and uniformly mixing, and performing instantaneous centrifugation;

h) incubation at 65 ℃ for 45min (hot lid temperature 75 ℃);

i) adding 100ul WBL (incubating at 65 deg.C for more than 2 h), mixing, and centrifuging instantly;

j) transfer to low adsorption 1.5ml tube; shaking gently and mixing uniformly, and separating instantly;

k) placing on a magnetic frame, and carefully discarding the supernatant after clarification;

l) adding 200ul of SWB, and slowly and carefully beating for 10 times without bubbles during beating; incubating at 65 deg.C for 5min, instantly separating, placing in a magnetic frame, clarifying, and carefully discarding the supernatant;

m) repeating the previous step once;

n) adding 200ul WBl, shaking and mixing uniformly for 2min, centrifuging instantaneously, placing on a magnetic frame, and carefully absorbing and removing supernatant after clarification;

o) adding 200ul WBll, shaking and uniformly mixing for 1min, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and removing supernatant after clarification;

p) adding 200ul of WBlll, shaking and uniformly mixing for 30s, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and removing supernatant after clarification;

q) taking the sample off the magnetic frame, and adding 20ul of deionized water to resuspend the magnetic beads;

6. enrichment of captured products

The concrete system is as follows:

the specific procedure is as follows:

7. purification of the captured product

a) Balancing the purified magnetic beads at room temperature for at least 30min, fully and uniformly mixing the magnetic beads before use, and instantly separating; preparing 80% ethanol at present;

b) taking 75ul of magnetic beads, and incubating for 5min at room temperature;

c) placing on a magnetic frame for 3min after instantaneous separation, and discarding the supernatant;

d) adding 200ul 80% ethanol to rinse the magnetic beads, discarding the supernatant after 30s, and repeating once;

e) opening the cover and drying in air for 3 min;

f) taking the sample off the magnetic frame for elution;

g) adding 22ul ddH2O, shaking, mixing, instantly separating, and standing for 5 min;

h) the sample was placed on a magnetic stand and allowed to stand for 5min, 32ul of supernatant was carefully pipetted into a new PCR tube, and no beads were pipetted.

Determination of product concentration by Qubit

NGS on-machine sequencing

10. Offline data quality control

11. Data analysis

Effect test example 1

In the same batch, 13 samples are selected for detection, the data of the 13 samples are subjected to QC, and the average QC of 31 genes is shown in Table 2 below.

Average QC of 231 genes in Table

Effect test example 2

11 samples were subjected to sequencing screening according to the example protocol, and the results are shown in table 3 below.

TABLE 3 summary of the results of the embodiments

Claims (10)

1. A probe set for screening retinoblastoma gene mutation, comprising: the probe set covers the CDS region of 31 retinoblastoma-related genes;

the 31 retinoblastoma-related genes are as follows:

NFE2L2、TOMM20、MYC、BAP1、NF1、PTEN、HMOX1、EZH2、PIK3CB、BRAF、CDKN2A、IDH1、NQO1、CEP290 FOXA1、NRAS、TP53、MAP2K1、FTH1、HIF1A、CHRM1、HRAS、PPP6C、DDX3X、PTGS2、DDR1、E2F1、KRAS、ARID2、RAC1、RB1；

the probe set covers 420 gene mutation position detections, and the sequences are respectively shown in SEQ ID No. 1-SEQ ID No. 420.

2. Use of the probe set for screening retinoblastoma gene mutation as set forth in claim 1 in the preparation of a screening kit for detecting retinoblastoma gene mutation.

3. A retinoblastoma gene mutation screening kit is characterized in that: the kit comprises a set of CDS region probes for 31 retinoblastoma-related genes;

the 31 retinoblastoma-related genes are as follows:

NFE2L2、TOMM20、MYC、BAP1、NF1、PTEN、HMOX1、EZH2、PIK3CB、BRAF、CDKN2A、IDH1、NQO1、CEP290 FOXA1、NRAS、TP53、MAP2K1、FTH1、HIF1A、CHRM1、HRAS、PPP6C、DDX3X、PTGS2、DDR1、E2F1、KRAS、ARID2、RAC1、RB1；

the probe set covers 420 gene mutation position detections, and the sequences are respectively shown in SEQ ID No. 1-SEQ ID No. 420.

4. The kit of claim 3, wherein: the test samples include blood, fresh tissue, FFPE samples and saliva.

5. The kit of claim 3, comprising reagents: human Cot-1, Blocker, 2X Hybridization Buffer, Hybridization Buffer Eufastener, nucleic-Free Water, BeadWash Buffer.

6. The method of using the retinoblastoma gene mutation screening kit of claim 3, comprising the steps of:

(1) extracting a genome of a sample to be detected;

(2) and (3) DNA quality inspection: performing quality inspection on the extracted sample genome to be detected by using the Qubit;

(3) constructing a library of a sample to be tested: fragmentation + terminal repair + dA-labeling, linker, magnetic bead purification, library enrichment, purification of an enriched product, and determination of library concentration;

(4) hybridizing a sample library to be detected with the probe;

(5) separation of the captured product;

(6) enrichment of the captured product;

(7) purifying a capture product;

(8) the Qubit determines the product concentration;

(9) performing sequencing on the NGS;

(10) off-line data quality control;

(11) and (6) analyzing the data.

7. Use according to claim 6, characterized in that: in the step (1), the detection sample comprises blood, fresh tissue, an FFPE sample and saliva.

8. The use method according to claim 6, wherein in the step (4), the hybridization between the library of the test sample and the probe set comprises the following steps:

a) taking an EP tube, adding a sample library to be detected, Human Cot-1 and Blocker, and drying;

b) the following reagents were added in sequence: 2X Hybridization Buffer, Hybridization Buffer Euhancer, Nuclear-Free Water;

c) standing at room temperature;

d) shaking and centrifuging, transferring to a new low-adsorption PCR tube, and keeping the temperature at 95 ℃ for 10 min;

e) immediately placing the mixture into an ice box, standing the mixture, and adding a retinoblastoma gene screening probe set;

f) shaking and mixing uniformly, instantly separating, and then putting into a PCR instrument for incubation for 16-20 h at 65 ℃.

9. The use method according to claim 6, wherein in the step (5), the separation of the captured product comprises the following specific steps:

a) balancing Dynabeads M-270 magnetic beads at room temperature for 30min for later use, and mixing uniformly;

b) taking magnetic beads into a centrifugal tube with low adsorption, putting the centrifugal tube into a magnetic frame, clarifying, and then discarding the supernatant;

c) adding 1X BeadWash Buffer, shaking, mixing, centrifuging instantaneously, returning to the magnetic rack, and discarding the supernatant after clarification;

d) repeating the previous step once;

e) adding 1X BeadWash Buffer, shaking, mixing, centrifuging instantaneously, and transferring into a low adsorption PCR tube;

f) placing on a magnetic frame to thoroughly enrich magnetic beads, and carefully discarding the supernatant;

g) mixing the magnetic beads with the obtained probe hybridized library, shaking and uniformly mixing, and performing instantaneous centrifugation;

h) incubating for 40-50 min at 65 ℃;

i) adding WBl preheated at 65 ℃, mixing gently, and centrifuging instantly;

j) transferring to a low adsorption tube; shaking gently and mixing uniformly, and separating instantly;

k) placing on a magnetic frame, and carefully discarding the supernatant after clarification;

l) adding SWB, and slowly and carefully blowing the mixture without bubbles; incubating at 65 deg.C for 5min, instantly separating, placing in a magnetic frame, clarifying, and carefully discarding the supernatant;

m) repeating the previous step once;

n) adding room-temperature WBl, shaking and uniformly mixing for 2min, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and removing supernatant after clarification;

o) adding room-temperature WBll, shaking and uniformly mixing for 1min, performing instantaneous centrifugation, placing on a magnetic rack, and carefully sucking and removing supernatant after clarification;

p) adding room-temperature WBlll, shaking and uniformly mixing for 30s, performing instantaneous centrifugation, placing on a magnetic frame, and carefully absorbing and removing supernatant after clarification;

q) removing the sample from the magnetic frame, and adding deionized water to resuspend the magnetic beads.

10. The use method according to claim 6, wherein in the step (7), the captured product is purified by the following steps:

a) balancing the purified magnetic beads at room temperature for at least 30min, fully and uniformly mixing the magnetic beads before use, and instantly separating; preparing 80% ethanol at present;

b) taking magnetic beads to the enriched capture products, and incubating at room temperature;

c) after the instantaneous separation, placing the magnetic frame and discarding the supernatant;

d) adding 80% ethanol to rinse the magnetic beads, discarding the supernatant, and repeating the steps once;

e) opening a cover and drying the air;

f) taking the sample off the magnetic frame for elution;

g) adding ddH2O, shaking, mixing, instantly separating, and standing;

h) place the sample on a magnetic rack and carefully pipette the supernatant into a new PCR tube.

Images