CN117587099A - Amplicon library construction method based on capture probe and application thereof - Google Patents

Amplicon library construction method based on capture probe and application thereof Download PDF

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CN117587099A
CN117587099A CN202311546887.XA CN202311546887A CN117587099A CN 117587099 A CN117587099 A CN 117587099A CN 202311546887 A CN202311546887 A CN 202311546887A CN 117587099 A CN117587099 A CN 117587099A
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李凤明
陆亚红
陈志宏
刘杜鹃
陈琰
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Suzhou Shuoxing Medical Laboratory Co ltd
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Abstract

The invention discloses an amplicon library building method based on a capture probe and application thereof, and belongs to the technical field of medicine. The invention realizes the library construction technology of the same flow of probe capturing and multiplex primer amplification: the method is suitable for detection technologies such as MRD and the like requiring trace sample library establishment and mutation information amplification, can simultaneously analyze SNV/INDEL/Fusion variation on the DNA layer and Fusion variation on the RNA layer, improves detection sensitivity, reduces clinical biology background noise caused by a probe method, and improves clinical specificity.

Description

Amplicon library construction method based on capture probe and application thereof
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to an amplicon library building method based on a capture probe and application thereof.
Background
The minimal residual disease (Minimal Residue Disease, MRD) refers to a condition where a tumor patient is in clinical remission after receiving treatment, and there are no clinical symptoms but tumor cells still present in the body. Performing MRD monitoring can improve prognostic assays and provide guidance for treatment after CR, identify relapse and perform early intervention. MRD monitoring is currently divided into two main technical routes, one is Tumor-formed and one is Tumor diagnostic; the Tumor-formed technical route is monitoring of personalized gene panel, comprising the steps of carrying out tissue sequencing by utilizing wes sequencing, screening out a certain number of mutation sites to form the personalized gene panel, and carrying out MRD monitoring by utilizing blood, wherein the method has the advantages of strong individuation and high clinical sensitivity; the disadvantages are mainly that the monitoring panel is very personalized, a large number of reagent performance verifications are not passed, development is needed, the reagent performance verification is insufficient, and in addition, the TAT time is longer.
The Tumor diagnostic technical route is that the immobilized gene panel is monitored, and the immobilized panel consisting of a group of common and high-frequency mutant genes and sites of Tumor patients is used for monitoring blood of the patients. The sensitivity of the monitoring reagent performance of MRD is typically required to reach 0.02%. Therefore, neither personalized nor immobilized panel puts very high demands on the sensitivity of the banking technique. In addition, the MRD monitoring technique requires setting a clinical decision value, that is, an MRD positive determination value, and incorrect mutation detection may cause a decrease in clinical specificity, and too few mutation detection may cause a decrease in clinical sensitivity.
There are two mainstream database building methods currently applied to immobilized gene panel: one is a library construction mode based on a capture probe method, and as the probe has 15% of mismatching degree, the technical characteristics of the probe embody that the probe is favorable for finding unknown gene mutation; the other is a database-building mode based on multiplex primer amplification, and the primers are amplified and enriched within a range of a certain length of the gene, so that the technical characteristics of the method are that target fragments can be fully enriched, and the sequencing data size is reduced. The two library building application scenes are different, and the steps are also quite different.
The acquisition method is mainly used for constructing a library by 1) adding A into the end repair; 2) The joints are connected; 3) Ligation product purification, 4) library enrichment and PCR product purification; 5) Hybridization of the probe; 6) Streptavidin binding; 7) Purifying the library; 8) Library enrichment the PCR products were purified to obtain a qualified library.
Amplicon pooling requires 1) one round of PCR, 2) PCR product purification, 3) two rounds of PCR, 4) library purification.
The principle of the library construction mode of the capture probe method is a DNA complementary pairing principle, and the probes are crosslinked and combined with a DNA template, so that the probes can be purified for downstream library construction and analysis; because the probe has a mismatch degree of 15%, the capture specificity is relatively poor, and expensive and complex COTIDNA (chip on insulator) is often required to be used for sealing, but nonspecific enrichment still generates more biological noise and sequencing redundant sequences, and the specificity judgment of SNV/INDEL and the like is greatly influenced; the multiplex primer has strong enrichment capability for specific fragments due to library enrichment in a specific target section, so that the generation of biological background noise can be reduced; meanwhile, the capture probe method can cause a considerable amount of invalid and redundant sequencing data to be generated due to the mismatching degree of probes; the multiplex primer has enrichment effect on the target fragment, so that the generation of redundant data can be reduced; however, amplicon-based pooling methods have poor ability to discover unknown variations, while probe capture pooling methods have the ability to discover unknown variations.
MRD detection is a trace mutation detection technology, the sequencing depth is usually very deep and is more than or equal to 50000X, the detection is simply based on the technical means of a capturing method, a large amount of variation information can appear when the detection is based on immobilized gene panel due to the nonspecific enrichment of a probe capturing method, a large amount of redundant sequences can be generated in sequencing data, a large amount of gene variation can be generated at the same time, the variation information has no prompting effect on a patient, and the clinical significance is not realized, so that the clinical specificity of the MRD can be reduced; the enrichment of specific target fragments is carried out by using primers based on a library construction mode of amplicons, unknown mutation with guiding significance is difficult to find, missing detection of real mutation is easy to cause, and the clinical sensitivity of MRD is reduced.
MRD detection technology is very sensitive to sequencing background noise, the generation of the background noise consists of two parts, one part is chemical background noise introduced by a library building reagent, a sequencing reagent, PCR reaction and the like, the other part is biological background noise of a human body caused by factors such as environment or age, the biological background noise comprises leucocyte clonal hematopoietic variation and the like, and the biological background noise is generated spontaneously by the human body and is difficult to distinguish by technical means especially at low frequency; the existing part of technical means can establish a data base line or a sequencing data blacklist through the sequencing of the white blood cells of the healthy people for filtering, but the method can not be effectively solved. The clinical positive judgment value of MRD, the current mainstream technical mode such as sigratea is equal to or more than 2SNVs, and no fusion is included; and due to the current technical limitations, the detectability of fusion at the DNA level is inferior to that at the RNA level. However, many fusion variants are also driving mutations, and have considerable guiding value for clinical detection of MRD, especially in hematological tumor patients where there are a large number of fusion variants. Therefore, accurate and comprehensive detection of true variation including fusion of tumor patients by using limited blood samples is a technical difficulty to be solved in the current MRD monitoring technology.
Disclosure of Invention
One of the purposes of the invention is to provide a method for constructing a library of amplicons based on capture probes, wherein the method comprises reverse transcription and fragmentation after DNA/RNA co-extraction, capturing the fragmented DNA/RNA by using a set of capture probes, incubating an extended artificial sequence (the artificial sequence is a non-human sequence) in a captured library, performing non-specific and specific amplification on a target gene region by using a set of multiplex primer sets, library purification, index universal primer amplification and library purification.
Preferably, the co-extract reverse transcription is accomplished by M-MLV reverse transcriptase and random primers.
Preferably, the capture probes are a set of probes of 100-145nt length, 20nt of which is an artificial sequence, the remainder being a region of interest probe, one end of the artificial sequence of the capture probes being labeled with biotin, the capture probes being divided into sense strand capture probes and antisense strand capture probes; the multiple primer group is a primer group with the length of 35-50 nt; the primer comprises a primer amplification region and a connector connection region, wherein the connector connection region comprises a 20nt connector nucleic acid sequence, and the primer amplification region comprises a specific sequence and a non-specific sequence; the primer comprises an upstream primer and a downstream primer; the upstream primer contains a 20nt adaptor nucleic acid sequence and a specific sequence complementary with a target gene, and the length of the upstream primer is 15-30 nt; the downstream primer includes two types: one containing a 20nt linker nucleic acid sequence and a specific sequence of 15-30 nt complementary to the gene of interest, the other containing a 20nt linker nucleic acid sequence and a non-specific sequence complementary to the artificial sequence.
More preferably, the preparation method of the fragmented DNA/cDNA is a mechanical method or an enzymatic method.
More preferably, the DNA/cDNA fragment length main peak is 80-100 nt.
More preferably, the probe capture process comprises capturing a DNA/cDNA mixture with an antisense strand capture probe, incubating the binding with streptavidin magnetic beads, magnetically separating the supernatant; capturing the supernatant by using a sense strand capture probe, incubating and combining streptavidin magnetic beads, and magnetically separating the supernatant; the library mixture of the streptavidin magnetic beads captured by the positive/antisense strand probes realizes the artificial sequence extension of the captured fragments on the capture probes and supplements the nicks under the condition of adding commercial Klenow enzyme mixed solution.
More preferably, the capture temperature of the sense strand is 65℃and the enzyme used for the incubation extension is Klenow enzyme and the incubation extension temperature is 37 ℃.
The second object of the present invention is to provide a library constructed by the above-mentioned amplicon library construction method.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method realizes the simultaneous library construction technical flow of probe capture and multiplex primer amplification, avoids the poor specificity of the probe capture method, and retains the capability of the probe capture method for finding unknown sequences; the method not only avoids poor uniformity of the panel amplicon, but also has the capability of high sensitivity of the multiplex amplification library construction target region.
(2) The DNA and RNA co-construction is realized, and the MRD panel can detect SNV/INDEL/fusion at the DNA level and fusion at the RNA level simultaneously; through double detection of fusion variation of DNA and RNA layers, the fusion detection capability is improved, and the clinical detection sensitivity of MRD is improved.
(3) Sequencing data redundancy and biological background noise caused by nonspecific enrichment of a capture method are reduced, a bioinformatics analysis flow is simplified, the specificity of clinical detection is improved, and false positive occurrence is reduced; while avoiding the expensive fabrication of complex COTs IDNA closed redundant sequences necessary for conventional capture methods.
Drawings
FIG. 1 is a flowchart of the present invention.
FIG. 2 is a schematic diagram of the sense strand/antisense strand capture probe structure in example 1.
FIG. 3 is a schematic diagram of the structure of the primer in example 1.
Fig. 4 is a schematic diagram of the present invention.
Detailed Description
Example 1
The present example is for illustrating the method for designing a probe for amplification, the library construction process, the library quality control criteria, and the sequencing analysis provided by the present invention.
(1) Probe panel design and primer panel design:
the probes comprise a sense strand capture probe and an antisense strand capture probe, are ssDNA (single strand deoxyribonucleic acid) and have the length of 100-145nt and contain 20nt fixed artificial sequences, and the specific structure is shown in figure 2, and are synthesized in Naonda technology limited company; the primers comprise a specific primer and a non-specific primer, wherein the specific primer and the non-specific primer both contain 20nt P5 or P7 linker sequences, and the specific structure is shown in figure 3 and are synthesized in the biological engineering Co.
(2) Co-extraction of DNA and RNA
DNA and RNA are extracted by a co-extraction kit and then subjected to nanodrop measurement, and OD260/230 measurement is carried out; the sum of the concentration is more than or equal to 500ng;
(3) Co-extract reverse transcription
Reverse transcription of RNA in the DNA and RNA co-extract into cDNA by random primer under the action of M-MLV reverse transcriptase;
(4) DNA and cDNA fragmentation:
the fragmentation can be carried out by selecting a breaker for breaking, or by adopting an enzyme breaking kit for breaking; the main peak range of the fragment size is 80-100 bp, so that the probe is ensured to completely cover the segmented fragments, and the binding efficiency is improved.
(5) Probe capture phase:
the method comprises three main processes: 1) Antisense strand capture probe hybridization capture process: hybridizing the antisense strand capture probes to fragment DNA and cDNA, combining streptavidin magnetic beads with biotin probes, magnetically separating and removing supernatant, and collecting streptavidin magnetic beads carrying antisense strand capture products; 2) Supernatant sense strand capture probe hybridization capture process: hybridization of sense strand capture probe the supernatant produced in step 1) and the fragmented DNA therein, binding streptavidin magnetic beads to biotin probe, magnetically separating the supernatant, and collecting streptavidin magnetic beads carrying the capture product of sense strand capture probe; 3) Streptavidin magnetic beads carrying the positive/negative strand probe capture products are collected by TE buffer redissolution and combined into one tube, and under the condition that commercial Klenow enzyme mixed solution is added, incubation is carried out for 30min at 37 ℃, so that artificial sequence extension on the capture probes is realized, and the artificial sequence is added to the captured DNA sequence by extension.
(6) Multiplex primer amplification stage:
the method comprises 2 main processes: 1) Multiplex primer amplification: the multiplex primer panel and the hybridization capture product containing cDNA and the positive and negative sense strand are amplified under a certain pcr amplification program condition under the condition of adding commercial multiplex amplification mix; 2) Enrichment library elution: magbio magnetic beads bind, ethanol wash, library elute, and the resulting library contains partial P5 and P7 linkers.
(7) Adaptor amplification stage:
the method comprises 2 main processes: 1) Amplification of index universal primer: the index universal primer and the positive and negative strand hybridization capture library are amplified under a certain pcr amplification program condition under the condition of adding commercial high-fidelity enzyme; 2) Library elution: magbio magnetic beads are combined, ethanol is used for washing, and the library is eluted, so that the formed library is a complete library. The Index universal primers include double ended Index universal sequence primers of the Illumina sequencing platform.
(8) Library quality control and sequencing:
the quality control of the library adopts qsep or agarose gel electrophoresis, and the main peak is obvious; the sequencer can select an illuminea platform or a Huada platform, and the sequencing strategy can select PE150 or SE150.
Application example 1
The embodiment discloses a method for constructing a library and controlling the quality of the library based on a library construction technology of amplification after probe capture of a marrow blood tumor MRD immobilized panel, and analyzing sequencing data by an on-machine sequencing:
(1) Myeloid blood tumor MRD probe panel design and myeloid blood tumor MRD primer panel design:
25 hot spot areas and 3 fusion gene areas of 17 genes are screened according to NCCN guideline line and public databases COSIC, CLINVAR and the like and combined with a company self-built database, and the panel size is about 6.6kb; 64 sense strand capture probes Panel 7.7kb,67 antisense strand capture probes Panel about 8.1kb and a set of multiplex primers Panel were designed, respectively.
TABLE 1.20 25 Hot spot region of genes and 3 fusion Gene region List
TABLE 2 Probe sequence Listing
TABLE 3 multiplex primer sequence listing
The probe panel was synthesized by Nanjing Aldard Biotechnology Co., ltd, and the primer panel was synthesized by Biotechnology Co., ltd.
(2) Co-extraction of DNA and RNA
Performing DNA and RNA co-extraction on a peripheral blood whole blood sample of 10 patients confirmed to be AML, wherein the extraction kit is a Zymo DNA/RNA co-extraction kit (Cat: D7005), the extraction volume is 1.5ml of peripheral blood, and the elution volume is 20 mu L; after extraction, 1. Mu.L of DNA and RNA concentrations were determined by nanodrop.
Table 4 sample information table
(3) Reverse transcription of DNA and RNA co-extracts
Selection of 500ng (sum of DNA and RNA concentration) of DNA and RNA co-extract1st Strand Synthesis Kit (cat# 12249ES 24) reverse transcription was carried out using a reverse transcription kit, and the reverse transcription product was a mixture of DNA and cDNA.
The reverse transcription system is as follows:
TABLE 5 Table of reverse transcription System
Component (A) Dosage (mu L)
Reverse transcriptase 2
Random primers 2
Reaction buffer 2
Template 500ng
Nuclease-free water Supplement to 20 mu L
The reverse transcription procedure was:
TABLE 6 reverse transcription procedure
Sequence number Temperature (. Degree. C.) Time
1 25 5min
2 37 45min
3 85 5s
4 4 Hold
(3) DNA and cDNA fragmentation
Taking 10ul DNA and cDNA mixture of the co-extract after reverse transcription, fragmenting by QIAGEN QIAseq FX in QIAseq FX DNA Library Kit (Cat. No. 180773) for unbiased disruption,
the fragmentation system is as follows:
TABLE 7 fragmentation system
Component (A) Dosage (mu L)
Fragmenting enzyme 2
Fragmenting buffer 2
DNA and cDNA mixture 10
Nuclease-free water Supplement to 20 mu L
The fragmentation procedure was:
TABLE 8 fragmentation procedure
Sequence number Temperature (. Degree. C.) Time
1 95 10min
2 40 60min
(4) Probe capture and library incubation
The fragmented DNA/cDNA was first concentrated using a Eppendorf Concentrator plus vacuum centrifugal concentrator prior to capturing the fragmented DNA/cDNA.
The concentrated product was first captured by an antisense strand capture probe, after capture, the capture fragments were captured using streptavidin magnetic beads, the capture system is shown in table 9, and the capture procedure is shown in table 10. The working concentration of the antisense strand capture probe was 10 pmol/. Mu.L.
The process of combining and capturing the product by using streptavidin magnetic beads is as follows: a. after the capture procedure is completed, 50 μl of streptavidin magnetic beads are immediately added to the antisense strand capture probe capture product; b. adding 1ml of reaction buffer, combining for 30 minutes, and shaking and uniformly mixing every 5 minutes during the period; c. and magnetically separating by using a magnetic frame, and collecting streptavidin magnetic beads. 4.8 μl of supernatant was transferred for sense strand capture probe hybridization.
The supernatant was subjected to sense strand capture probe capture, after capture, the capture fragments were captured using streptavidin magnetic beads, the capture system is as shown in Table 9, and the capture procedure is as shown in Table 10. The working concentration of the sense strand capture probe was 10 pmol/. Mu.L. Hyb#1 and Hyb#2 buffers were purchased from Nanjna Aldard Biotechnology Inc.
The process of combining and capturing the product by using streptavidin magnetic beads is as follows: a. after the capture procedure is completed, 50 μl of streptavidin magnetic beads are immediately added to the sense strand capture probe capture product; b. adding 1ml of reaction buffer, combining for 30 minutes, and shaking and uniformly mixing every 5 minutes during the period; c. magnetic separation is carried out by using a magnetic frame, supernatant is discarded, and streptavidin magnetic beads are collected.
Table 9 Capture System
The forward/antisense strand hybrid capture procedure is:
table 10 Capture procedure
Sequence number Temperature (. Degree. C.) Time
1 95 30s
2 65 60min
3 65 hold
Two tubes of streptavidin magnetic beads containing the capture product were each resuspended in 10. Mu.L TE buffer and combined into a tube of capture product with a total volume of 20. Mu.L.
And (3) incubation of capture products:
TABLE 11 amplification System
Sequence number Component (A) Dosage (mu L)
1 Capturing the product 20
2 10 XERAbufer (containing Klenow enzyme) 3
3 RNasefreeH2O 7
The incubation procedure was 30min at 37 ℃.
10 XERA buffer (containing Klenow enzyme) was purchased from Tiangen Biochemical technologies Co., ltd., product number: NG302.
(5) Multiplex primer amplification
Taking 30 mu L of incubation products for amplification, wherein the working concentration of each primer in the multiplex primer panel is 5nM, and the total amplification volume is 50 mu L; ringap enzyme and Ringap buffer were purchased from Xiamen Fangshuo Biotechnology Inc.
The amplification system is as follows:
table 12 amplification System
Sequence number Component (A) Dosage (mu L)
1 Ringap enzyme 5
2 Ringap buffer 5
3 Multiplex primer panel 2
4 Incubation of the product 30
5 Nuclease-free water 8
The amplification procedure is shown in Table 13.
TABLE 13 amplification procedure
The amplified product was subjected to purification operation using magbio beads 1.2 X60. Mu.L in 70% ethanol and washed twice, and eluted with 22. Mu.L TE buffer to obtain an enriched library. magbio magnetic beads were purchased from south genia-biosciences, inc.
(6) Adaptor amplification
Amplifying 20 mu L of the enrichment library, wherein the total amplification volume is 40 mu L;
the amplification system is as follows:
TABLE 14 amplification System
Sequence number Component (A) Dosage (mu L)
1 Ringap enzyme 5
2 Ringap buffer 5
3 Index universal primer 2
4 Enrichment library 20
5 Nuclease-free water 8
The amplification procedure is shown in Table 15.
TABLE 15 amplification procedure
The amplified product was subjected to purification procedure using magbio beads 1.2 X60. Mu.L, washed twice with 70% ethanol, and eluted with 22. Mu.LTE buffer.
(7) Library quality control and sequencing
1 mu L of library is taken for qubit detection, the concentration is larger than 1 ng/. Mu.L, 1 mu L of library is taken for agarose gel electrophoresis detection by adopting a qsep instrument, and the main peak is located near 200bp and is qualified for library quality inspection.
Library quality control results are shown in table 16.
TABLE 16 library quality control results
The sequencing platform selects the illumine sequencer Nexstseq550 and the sequencing strategy is PE 150. The results of this MRD monitoring of 10 patients with myeloid hematological neoplasms are shown in Table 17.
TABLE 17 MRD monitoring results for patients with medullary hematological tumors
The fusion variant detection results expected in Table 17 were verified by Fluorescence In Situ Hybridization (FISH).
As can be seen from the data analysis of Table 17, the multiplex amplicon library building process based on the capture probe can enable all ultra-low frequency mutation and ultra-low copy fusion to be stably detected, and the fusion detection of the RNA layer increases the detection capability of the fusion.
In conclusion, the capture probe and the multiplex amplicon library construction method designed by the application are suitable for next-generation high-throughput sequencing, can be used for simultaneously and accurately detecting DNA and RNA, have a fusion detection effect superior to that of single DNA fusion detection, effectively utilize a blood sample, avoid the need of single extraction and/or library establishment of the DNA and RNA of the blood sample, have strong operability and wide application range, improve the positive rate of MRD detection, and have better applicability.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (9)

1. The amplicon library construction method based on the capture probes is characterized by comprising reverse transcription and fragmentation after DNA/RNA co-extraction, capturing fragmented DNA/cDNA by using a group of capture probes, combining capture libraries, carrying out nonspecific and specific amplification on a target gene region by using a group of multiplex primer groups, library purification, index universal primer amplification and library purification.
2. The amplicon pooling method of claim 1, wherein said co-extract reverse transcription is accomplished by an M-MLV reverse transcriptase and random primers.
3. The amplicon pooling method of claim 1, wherein said capture probes are a set of probes of length 100-145nt, 20nt of which are artificial sequences, the remainder being region of interest probes, said capture probes having a biotin label at one end, said capture probes being divided into sense and antisense strand probes; the multiple primer group is a primer group with the length of 35-50 nt; the primer comprises a primer amplification region and a connector connection region, wherein the connector connection region comprises a 20nt connector nucleic acid sequence, and the primer amplification region comprises a specific sequence and a non-specific sequence; the primer comprises an upstream primer and a downstream primer; the upstream primer contains a 20nt adaptor nucleic acid sequence and a specific sequence complementary with a target gene, and the length of the upstream primer is 15-30 nt; the downstream primer includes two types: one containing a 20nt linker nucleic acid sequence and a specific sequence of 15-30 nt complementary to the gene of interest, the other containing a 20nt linker nucleic acid sequence and a non-specific sequence complementary to the artificial sequence.
4. The method for constructing a pool of amplicons according to claim 3, wherein the preparation method of the fragmented DNA/cDNA is a mechanical method or an enzymatic method.
5. The method for constructing a pool of amplicons according to claim 4, wherein the main peak of DNA/cDNA fragmentation length is 80-100 nt.
6. The amplicon pooling method of claim 5, wherein the probe capture process comprises antisense strand capture probe capture, streptavidin magnetic bead incubation binding, magnetic separation to remove supernatant; capturing a sense strand capture probe of the supernatant, incubating and combining streptavidin magnetic beads, and magnetically separating the supernatant; streptavidin magnetic beads carrying the positive/negative strand probe capture products are reconstituted and combined into a tube, and the positive/negative strand probe capture library is mixed and incubated for extension.
7. The method of claim 6, wherein the capture temperature of the sense strand is 65℃and the enzyme used for the incubation and extension is Klenow enzyme and the incubation and extension temperature is 37 ℃.
8. The amplicon pooling method of claim 7, wherein the capture procedure of the plus/minus sense strand is as set forth in the following table:
9. library constructed by the amplicon banking method of claims 1-8.
CN202311546887.XA 2023-11-20 2023-11-20 Amplicon library construction method based on capture probe and application thereof Pending CN117587099A (en)

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