CN116287162A - Kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof and application method - Google Patents

Abstract

The invention provides a kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof and a use method, wherein the kit comprises 4crRNA probes for targeting BCR gene cleavage sites, tyrosine kinase mutation regions and promoter regions and 4 qPCR primers for verifying targeting efficiency of the corresponding crRNA probes. The crRNA probe is used as a core component of a targeted gene region of interest, RNPs are assembled by the crRNA probe and transactivation crRNA and endonuclease protein Cas9 to precisely cut target sites, so that the aim of enriching the gene fragments of interest is fulfilled, and the targeting efficiency of 4crRNA probes in each genome DNA sample is controlled by qPCR experiment quality. The invention realizes the simultaneous detection of the fusion site, tyrosine kinase region mutation and promoter methylation level of the BCR-ABL1 fusion gene by adopting a CRISPR/Cas 9targeted enrichment system-based nanopore gene sequencing technology with long sequencing read length and high sequencing depth so as to fully and comprehensively study the mutation related to the fusion gene.

Description

Kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof and application method

Technical Field

The invention relates to the technical field of detection of genetic variation and epigenetic variation, in particular to a kit for detecting BCR-ABL1 fusion gene and tyrosine kinase zone mutation and promoter methylation thereof and a use method.

Background

The BCR-ABL1 fusion gene is formed by t (9:22) (q 34:11) translocation, i.e., a gene rearrangement event caused by translocation of the ABL1 gene in chromosome 9, q34, to the BCR gene in chromosome 22, q11, whereby the translocated chromosome 22 is also referred to as the Philadelphia chromosome (ph). BCR-ABL1 fusion genes are found in more than 90% of Chronic Myelogenous Leukemia (CML), 25-30% of adult Acute Lymphoblastic Leukemia (ALL), 3% -5% of pediatric ALL, and a minority of Acute Myelogenous Leukemia (AML) patients, whose expressed fusion products have constitutive tyrosine kinase activity, leading to uncontrolled cell proliferation and the occurrence of the corresponding leukemia. The BCR-ABL1 fusion gene is used as a specific molecular marker for diagnosing clinical related diseases as a plurality of leukemia driving factors and marker molecular events, and molecular targeted drugs Tyrosine Kinase Inhibitors (TKIs) comprising imatinib, nilotinib, dasatinib and Bo Su Tini are developed aiming at the carcinogenesis mechanism. However, during the course of treatment, some patients still exhibit primary or secondary resistance to TKIs treatment (dominant effect) and lead to increased risk of disease progression or recurrence.

Mutations in the ABL1 tyrosine Kinase Domain (KD) directly associated with the BCR-ABL1 fusion gene have been shown to be the most prominent drug resistance mechanism for TKIs (about 60% of secondary drug resistance). Currently, more than 100 different KD mutations have been identified that result in more than 50 amino acid changes, and different mutations exhibit varying degrees of sensitivity to TKIs. In addition, epigenetic modification changes, such as abnormal DNA methylation, that occur on BCR-ABL1 molecules can also affect disease progression, therapeutic response, and overall prognosis in CML patients. A study of the correlation of the methylation (5 mC) modification level of the BCR-ABL1 gene promoter and the efficacy of imatinib for CML patients shows that the increase of the methylation of the BCR promoter DNA indicates that the prognosis of the patients is good and the responsiveness to imatinib is better. This shows that methylation abnormality on the promoter of the BCR-ABL1 fusion gene is expected to be potential as an epigenetic biomarker for predicting TKIs sensitivity, drug resistance and disease progression risk, and can provide a stable and reliable reference index for evaluating and guiding the application of epigenetic drugs in CML two-line therapeutic drugs.

As described above, the BCR-ABL1 fusion gene is taken as a gene structural variation, is a mechanism of occurrence of related leukemia, and KD mutation (single nucleotide variation) and promoter 5mC modification abnormality (epigenetic modification change) on the molecule are closely related to TKIs resistance and disease progression. According to NCCN and ELN latest guidelines, the definite diagnosis basis of CML and Ph+ALL is that typical clinical manifestations are combined to be Ph positive or BCR-ABL1 fusion gene positive; and in the treatment process of two diseases, the adjustment and replacement of the TKI dosage needs to refer to the drug resistance mechanism related to BCR-ABL 1. Thus, there is a need to make a correct diagnosis and to implement an individualized treatment regimen by detecting three molecular events, BCR-ABL1 fusion gene and its KD mutation and promoter 5mC abnormality, to meet the full knowledge of the clinician on the patient. However, there is no suitable clinical method for detecting three molecular events, i.e., genetic structural variation, single nucleotide variation and DNA epigenetic change, simultaneously in one experiment, and it is often necessary to use different detection techniques. For example, detection of BCR-ABL1 fusion genes relies primarily on chromosomal banding techniques in conventional cytogenetic methods and Fluorescent In Situ Hybridization (FISH) techniques in molecular cytogenetic methods, as well as real-time fluorescent quantitative polymerase chain reaction (qPCR) for monitoring disease conditions; detection of KD mutation of ABL1 gene is routinely performed using sanger sequencing or second generation sequencing (NGS); and the detection of the modification level of 5mC of the BCR gene promoter mainly comprises the step of carrying out first-generation or second-generation sequencing on the products after bisulfite conversion. Therefore, the detection of the three molecular events is time-consuming, complicated in process and high in accumulated cost.

In recent years, a nanopore sequencing technology represented by a third generation sequencing technology in the emerging sequencing market has the advantages of high sequencing throughput, long ultra-long reading time, less time consumption, low cost and the like, and can directly detect a DNA base sequence and modification thereof without any pretreatment. These advantages make it incomparable in detecting large structural variations such as fusion genes and 5mC modification of DNA. However, the higher error rate (5% -10%) of the nanopore gene sequencing technology limits the application of the nanopore gene sequencing technology in mutation detection. Therefore, researchers develop a nanopore gene sequencing technology 'nanopore Cas9Targeted-Sequencing (nCATS)' for performing deep sequencing on a gene region of interest (ROI), which corrects a sequencing error rate by increasing the sequencing depth of the ROI, thereby realizing accurate detection of mutation and effectively reducing noise interference caused by other irrelevant background genes. Numerous studies have demonstrated that this technique can simultaneously analyze three molecular events, gene structural variation, single nucleotide variation, and 5mC modification of DNA in one experiment. The method provides an excellent technical means for simultaneously detecting three molecular events related to the BCR-ABL1 fusion gene in one experiment.

Disclosure of Invention

Based on this, the present invention aims to provide a kit for detecting mutation of BCR-ABL1 fusion gene and tyrosine kinase region thereof and methylation of promoter and a use method thereof, so as to at least solve the shortcomings in the related art.

The invention provides a kit for detecting BCR-ABL1 fusion gene and tyrosine kinase zone mutation and promoter methylation thereof, which comprises the following components:

crRNA probes derived from CRISPR, comprising a BCR-23177704crRNA probe with a target site upstream of the BCR gene small break region and upstream of the BCR gene promoter, two probes BCR-23285486crRNA and BCR-23286143crRNA with a target site upstream of the BCR gene major break region, and an ABL1-1308887888crRNA probe with a target site downstream of the ABL1 kinase mutation region;

a transactivation crRNA that is universal and anneals to a crRNA probe to a chimeric single-stranded guide RNA;

and, CRISPR-associated protein Cas9, said protein Cas9 having endonuclease activity, assembling ribonucleoprotein complex RNPs with single-stranded guide RNA, and cleaving a target site in genomic double-stranded DNA under the guidance of said single-stranded guide RNA, breaking the double-stranded DNA;

the CRISPR/Cas 9targeted enrichment system is formed by the reagents so as to enrich the DNA fragments of interest.

Further, the length of complementary pairing of the 5 'end of the 4crRNA probes and the target site sequence is 20 nucleotides, and the 3' end sequence adjacent to the 4crRNA probe target sites is a PAM motif, and the identification numbers, the base sequences and the PAM motif of the corresponding target sites of the 4crRNA probe sequences are as follows:

BCR-23177704crRNA：5’-CAAGGGAGAAAGCCACTATC-3’；5’-TGG-3’；

BCR-23285486crRNA：5’-CACGGGATACTTCTTAGACC-3’；5’-TGG-3’；

BCR-23286143crRNA：5’-CCATACAAGCTACCCTGATG-3’；5’-GGG-3’；

ABL1-130888788crRNA：5’-ACTGGCCCAGTGTGACCAAT-3’；5’-TGG-3’。

furthermore, the targeting efficiency of the 4crRNA probes to their respective target sites in each genomic DNA sample was quality controlled by real-time fluorescent quantitative polymerase chain reaction experiments.

Furthermore, the primer pairs used for controlling the targeting efficiency of the 4crRNA probes in the real-time fluorescence quantitative polymerase chain reaction experiment are BCR-23177704FP/RP, BCR-23285486FP/RP, BCR-23286143FP/RP and ABL1-130888788FP/RP, and the sequences of the primer pairs are as follows:

BCR-23177704FP：5’-CCAACCCAACCCTCCAGAA-3’；

BCR-23177704RP：5’-GTCACAGGTCAGACAACTAAGCA-3’；

BCR-23285486FP：5’-CCAACCCAACCCTCCAGAA-3’；

BCR-23285486RP：5’-GTCACAGGTCAGACAACTAAGCA-3’；

BCR-23286143FP：5’-AGTGGAGGGCTTGGAGTAGT-3’；

BCR-23286143RP：5’-GGGGCTTGTCCTCACCATTT-3’；

ABL1-130888788FP：5’-TCGTGTCAGATGTGGACGGT-3’；

ABL1-130888788RP：5’-GACACCCATGAAAGATCCCCA-3’。

further, the parameters of the real-time fluorescent quantitative polymerase chain reaction experimental reaction program are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 15sec, annealing at 55℃for 15sec, elongation at 72℃for 20sec,40 cycles.

Further, the kit also comprises a sample genome DNA extraction reagent, a targeted nanopore gene sequencing library construction reagent and a nanopore gene sequencing chip excitation reagent.

The invention also provides a use method of the kit, which is applied to the kit for detecting the mutation of the BCR-ABL1 fusion gene and the tyrosine kinase region and the methylation of the promoter, and the use method of the kit comprises the following steps:

step one: extracting genomic DNA from a sample;

step two: dephosphorylation of the extracted genomic DNA sample;

step three: targeting enrichment of DNA fragments of interest from genomic DNA that has been treated in step two by the CRISPR/Cas9 targeting enrichment system;

step four: the targeting efficiency of BCR-23177704crRNA, BCR-23285486crRNA, BCR-23286143crRNA and ABL1-130888788crRNA probes is respectively and qualitatively controlled through a real-time fluorescent quantitative polymerase chain reaction experiment by using BCR-23177704FP/RP, BCR-23285486FP/RP, BCR-23285486FP/RP and ABL1-130888788FP/RP primer pairs;

step five: and (3) preparing a targeted sequencing library of the sample subjected to the quality control in the fourth step, and sequencing by using a nanopore gene sequencing technology, wherein the obtained sequencing data can be analyzed by using nanopore gene sequencing data analysis software, so that the fusion site of the BCR-ABL1 fusion gene, the mutation of the ABL1 tyrosine kinase region and the methylation level of the BCR gene promoter are detected.

Further, the sample is selected from a human peripheral blood sample or a bone marrow sample, and a human leukemia-related cell line.

The invention has the beneficial effects that:

the invention provides a kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof, wherein the target ROIs regions (shown in figure 1) and actions of 4crRNA probes are respectively as follows:

(1) BCR-23177704crRNA forward probes (the crRNA sequence is in the 5 'to 3' direction of the forward strand of the targeted gene region and is completely in the 5 'to 3' direction of the base sequence of 20bp of the targeted gene region, so that the crRNA probes are called forward crRNA probes, and conversely are called reverse crRNA probes), target the secondary fragmentation site region (m-BCR, about 54.4 kb) of the BCR gene and the upstream of the BCR gene promoter, and detect the DNA sequence downstream of the target site by forward sequencing, namely the crRNA probes, namely BCR-ABL1 fusion genes for detecting that the BCR gene fragmentation point is located in m-BCR, the fragmentation region is found in ALL patients of about 2/3Ph+, and are also used for targeting DNA fragments enriched in CpG islands (a DNA region rich in CG dinucleotides, the CpG islands being about 2 kb) containing the BCR gene promoter to detect methylation level;

(2) BCR-23285486crRNA and BCR-23286143crRNA are targeted to the upstream of a main fragmentation region (M-BCR, about 5.8 kb) of the BCR gene, the DNA sequence at the downstream of a target site is detected by forward sequencing, and the BCR-ABL1 fusion gene with the fragmentation point of the BCR gene at the M-BCR is detected, the fragmentation region is found in most Ph+ CML patients and 1/3Ph+ ALL patients, so that 2 crRNA probes are targeted to the upstream of the M-BCR to improve the targeting degree of the most common fragmentation region of the BCR gene and the depth of subsequent sequencing data, thereby reducing the negative rate of detection;

(3) The ABL1-130888788crRNA reverse probe targets the downstream of the ABL1 gene KD, and the DNA sequence upstream of the target site is detected by reverse sequencing for detecting the known or unknown drug resistance mutation in the ABL1 gene KD.

Further, because there are two crRNA probes targeting upstream of the BCR gene M-BCR, the depth of sequencing data covering this region and the corresponding downstream region is very high; and because the nanopore gene sequencing technology has the characteristic of long reading length, i.e. the full-length sequence of single-stranded DNA passing through the nanopore can be detected theoretically. Thus, the kit and method disclosed in the present invention can detect some rare Ph+ samples having a breakpoint located mainly in a third cleavage region (μ -BCR, about 1.3 kb) about 16kb away from the M-BCR region of the BCR gene.

Further, all of the 3 forward crRNA probes used to detect the BCR-ABL1 fusion site are located upstream of the 3 BCR gene fragmentation regions where the fragmentation regions are relatively conserved, and the fragmentation regions are not large in span, rather than the fragmentation regions spanning approximately 140kb of the ABL1 gene. Therefore, the 3crRNA forward probes not only ensure that most types of BCR-ABL1 fusion genes are detected, but also ensure that the subsequent nanopore gene sequencing starts from the BCR gene detection of the fusion head part with relatively conserved cleavage sites, and sequence the ABL1 with relatively non-conserved cleavage regions of the fusion tail part, so as to ensure that the accurate cleavage sites of the BCR genes and the ABL1 genes are successfully detected.

Further, the 3 forward crRNA probes cleave the corresponding target sites by cleaving the RNPs assembled with transactivation crRNA (tracrRNA) and Cas9 proteins, resulting in double-stranded DNA breaks and two blunt-ended breaks. However, since the DNA fragment at the 5 '-side of the crRNA probe still has the Cas9 protein attached to it (the DNA fragment upstream of the breakpoint), the sequencing adapter preferentially attaches to the DNA fragment at the 3' -side of the crRNA probe (the DNA fragment downstream of the breakpoint). The working principle of the RNPs ensures that the direction of nanopore gene sequencing is from the BCR gene at the fusion head to the ABL1 gene at the fusion tail. The method not only effectively avoids the difficulty of detecting the BCR/ABL1 fusion gene caused by the ABL1 gene which is positioned at the tail part of the fusion and has complex and changeable cleavage sites, but also can realize the detection of some fusion partners which are positioned at the tail part of the BCR gene and are unknown (as shown in figure 2). The reverse probe ABL1-130888788crRNA is exactly opposite to the working principle of the forward crRNA probe, so that the upstream DNA fragment of the break formed by RNPs assembled by the ABL1-130888788crRNA probe at the target site is preferentially connected with a sequencing joint, and the detected gene region is exactly the KD region of the ABL1 gene.

Further, the primer pairs BCR-23177704FP/RP, BCR-23285486FP/RP, BCR-23286143FP/RP and ABL1-130888788FP/RP are respectively used for quality control on the targeting efficiency of BCR-23177704crRNA, BCR-232854816 crRNA, BCR-23286143crRNA and ABL1-130888788crRNA probes in each sample, so that the success of the subsequent nanopore genetic sequencing experiment can be effectively ensured (as shown in FIG. 3).

Furthermore, the invention combines a CRISPR/Cas9 targeting enrichment system and a nanopore gene sequencing technology, and the obtained sequencing data has the characteristics of long reading length, high sequencing depth, single base resolution and the like, and the advantages of the characteristics in the invention are reflected in ensuring the accuracy of detection of BCR-ABL1 fusion genes, KD region mutation of ABL1 genes and 5mC modification level of BCR gene promoters. For example, the high length of reading and the high depth of sequencing can greatly reduce the negative rate of detection of the BCR-ABL1 fusion gene, and can accurately detect any mutation in exons and introns in the KD region, including the identified TKIs drug-resistant mutation and the novel drug-resistant mutation site; single base resolution then makes it possible to assess the methylation level of each CG motif in CpG islands of about 2kb in length, including the BCR promoter.

Furthermore, the invention detects the modification level of 5mC of the BCR gene promoter based on the nanopore gene sequencing technology capable of directly sequencing natural DNA without bisulphite treatment of DNA samples and subsequent PCR amplification, thereby not only reducing bias and loss of modification information caused by DNA degradation and PCR amplification in the experimental process, but also greatly simplifying the experimental method and saving a large amount of time.

Furthermore, the kit and the application method disclosed by the invention are used for detecting the BCR/ABL1 fusion gene based on the DNA level, and the experimental result is reliable and accurate.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram of the major cleavage sites of the BCR and ABL1 genes, the BCR promoter, the KD region of the ABL1 gene, and the targeting sites of 4crRNA probes;

FIG. 2 is a schematic diagram of the principle of cleavage of a target site by a CRISPR/Cas 9targeted enrichment system;

FIG. 3 is a schematic diagram of the principle of detecting the targeting efficiency of crRNA probes by a real-time fluorescent quantitative polymerase chain reaction (qPCR) experiment;

FIG. 4 is a schematic illustration of the principle of nanopore gene sequencing technology based on a CRISPR/Cas 9targeted enrichment system;

FIG. 5 is a graph showing the purity and fragment size of gDNA of the K562 cell line according to the first embodiment of the present invention;

FIG. 6 is a schematic diagram showing the structure of BCR-ABL fusion gene in gDNA of K562 cell line according to the first embodiment of the present invention;

FIG. 7 is a schematic diagram showing the single nucleotide variation in KD region of ABL1 gene in gDNA of K562 cell line detected according to the present embodiment of the present invention;

FIG. 8 is a schematic diagram showing the level of modification of BCR gene promoter 5mC in gDNA of K562 cell line according to the present embodiment.

Detailed Description

The implementation of the present invention will be described in detail below in order to provide a more thorough understanding of the features and technical aspects of the present invention. The experimental procedure described in the following examples is only used to demonstrate the feasibility of the invention, and the application of the invention is not limited thereto. The experimental procedures mentioned in the examples, unless otherwise specified, are all conventional experimental methods; the reagent consumable materials mentioned, unless otherwise specified, are all conventional reagent consumable materials.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In this embodiment, the reagent used in the CRISPR/Cas 9targeted enrichment system is purchased from IDT company, the nanopore gene sequencing reagent and sequencing instrument are purchased from ONT company, and the other reagents or instruments used are not noted to the manufacturer and are all conventional products available through commercial purchase.

The kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof and the use method thereof based on a CRISPR/Cas 9targeted enrichment system by using the nanopore gene sequencing technology are specifically and specifically described below.

Example 1

Detecting BCR-ABL1 fusion gene and KD mutation and promoter 5mC modification level in K562 cell line gDNA:

the invention designs 4crRNA probes, and 4 pairs of qPCR primers (see table 1) for quality control of the targeting efficiency of the corresponding crRNA probes, so that target gene fragments and targeting efficiency quality control are enriched through a genome DNA (gDNA) dephosphorylation, CRISPR/Cas9 targeting enrichment system, a target gene fragment sequencing library is constructed, and nanopore gene sequencing is carried out, wherein the existence of BCR-ABL1 fusion genes or unknown fusion genes positioned at the tail of the BCR, mutation occurring in the ABL1 gene KD and the modification level of 5mC of a BCR gene promoter are detected in a gDNA sample qualified in quality control, and the complete experimental flow is shown in figure 4.

TABLE 1

(1) Human chronic myelogenous leukemia cell (K562) culture and gDNA extraction

K562 cells were grown in suspension in RPMI 1640 medium containing 10% fetal bovine serum, 100U/m penicillin and 100. Mu.g/ml streptomycin, and cultured in an incubator saturated with 5% CO2 and humidity at 37 ℃. While the K562 cells were in the logarithmic growth phase, they were transferred into centrifuge tubes and cell counts were performed, followed by centrifugation at 800rpm for 5min, and the supernatant was discarded. The K562 cell line gDNA was then extracted according to the instructions of the gDNA extraction kit (QIAGEN, inc., QIAamp DNA Blood Mini Kit): cell pellet (cell number < 5×10≡6, maximum initial cell number of the gDNA extraction kit) was resuspended with 200. Mu.L PBS, and mixed with 20. Mu.L proteinase K and 4. Mu.L RNA A enzyme; 200 mu L of lysate AL is added and is fully and uniformly vortex, and cells are lysed for 10min at 56 ℃; adding 200 mu L of absolute ethyl alcohol, shaking by vortex for 15sec, transferring the reaction solution to a filter column, and centrifuging at 8000rpm for 1min; adding 500. Mu.L of buffer AW1, centrifuging at 8000rpm for 1min; then 500. Mu.L of buffer AW2 was added thereto and the mixture was centrifuged at 14000rpm for 3min; the filter column was placed on a new collection tube, 50. Mu.L to 200. Mu.L of eluent AE was added, left standing at room temperature for 3min, centrifuged at 8000rpm for 1min, and the eluted gDNA was collected and stored in a-80℃refrigerator. When gDNA was used, its concentration was determined with a fluorescent quantitator Qubit and its fragment size and purity was analyzed with a fully automated capillary analyzer Qsep 100. The concentration of gDNA of the K562 cell line used in example 1 was 216 ng/. Mu.L as measured by Qubit, the fragment size and purity as measured by Qsep100 are shown in FIG. 5, and the fragment of the gDNA of the K562 cell line is mainly distributed at about 10kb, and the number of hetero peaks within 20bp to 1000bp is small, which indicates that the length and purity of the gDNA of the proposed K562 cell line are all available.

(2) BCR-ABL1 fusion site, KD mutation and promoter analysis, and design and synthesis of crRNA probe for targeting the three molecular events and qPCR primer for quality control of corresponding targeting efficiency

On the BCR gene, the cleavage site occurs mainly in the M-BCR region of about 5.8kb (including e12-e 16), and is found in most of Ph+ (over 90% of CML patients Ph+) and 1/3 of Ph+ (over 30% of ALL patients Ph+); also occurring in the m-bcr region (approximately 54.4kb long) comprising e1-e2, the fragmentation region is found in ALL of 2/3Ph+ (more than 30% of ALL patients Ph+); and occurs in the μ -bcr region (approximately 1.3kb long) including e19-e21, which is commonly found in chronic neutrophilic leukaemia with very low incidence (see FIG. 1). Whereas the region where the ABL1 gene may be disrupted is within the 1 st and 2 nd intron regions of about 140kb in length (see FIG. 1). It can be seen that, for the BCR-ABL1 fusion gene, the ABL1 gene at the fusion tail part has large, changeable and unstable span of the cleavage site, while the cleavage site of the BCR gene at the fusion head part is relatively conserved, and is mainly concentrated in 3 cleavage areas with small span. In addition, cpG island portions including the promoter of the BCR gene overlap with the μ -BCR region (see FIG. 1), and KD is located at the entire length of the ABL1 gene downstream of about 33kb, in which TKIs resistance mutation occurs mainly on exons 4 to 8.

For the above reasons, the present invention is based on the design principle of crRNA probe, and applies online design software chopchopchop for M-BCR and M-BCR of BCR gene (5 kb gene region (chr 22:23283000-23288000 and chr22:23175500-23180500, hg38) respectively designing 4 and 2 alternative forward crRNA probes, and designing 2 alternative reverse crRNA probes in a 5kb gene region (chr9: 130888675-chr9:130893675, hg38) outside about 1kb at the 3' -end of the ABL1 gene, respectively, since the M-BCR region is the most common cleavage site of the BCR gene, 4 alternative crRNA probes are designed upstream of the region, 2 crRNA probes with the best targeting efficiency are prepared to be screened out from the region by a subsequent qPCR experiment, the sequencing depth of the region and the downstream region can be improved, and in addition, because the sequencing technology of the nanopore gene has the characteristic of long reading length, i.e. the whole-length sequence of single-stranded DNA passing through the nanopore can be detected theoretically, on the premise of ensuring the sequencing depth and the length of the corresponding downstream region, the 2 crRNA probes can be screened out from the BCR 1 gene region, the 2 crRNA probes with the optimal targeting efficiency, and the 2 crRNA probes can be screened out from the region right side of the region, and the BCR 1 gene region can be detected for the region with the fusion site being about 16kb at the downstream of the region, and the BCR 1.2 gene can be detected as the right region, and the gene region with the gene cleavage probe can be completely satisfied by the BCR 1, this region-targeted enriched DNA fragment can be used for evaluation of BCR gene promoter 5mC levels. In addition, 2 inverted crRNA probes are designed at the downstream of the tail end of the ABL1 gene and are used for targeting and enriching the upstream DNA fragment containing the KD of the ABL gene so as to detect the TKIs drug-resistant mutation.

The 8 candidate crRNA probes designed above were synthesized by IDT company.

The targeting efficiency of 8 alternative crRNA probes to target sites in each gDNA sample can be controlled by qPCR experiments, and the principle is that qPCR primers including amplification products are designed for the target sites of crRNA probes, so that a gene fragment including the target sites of crRNA probes cannot be used as a template to complete the amplification process after RNPs cleavage (as shown in fig. 3). Therefore, compared with the control group using gDNA cleaved by RNPs as a template, the CT value of the experimental group was higher than that of the control group, and the targeting efficiency of the corresponding crRNA was judged by calculating the ΔCT value (experimental group-control group) (see FIG. 3). Therefore, 2 pairs of corresponding qPCR primers are designed by Primer Premier 5 software aiming at the target sites of the 8crRNA probes to be replaced, gDNA of a K562 cell line is used as an experimental sample, the specificity and the amplification efficiency of each pair of qPCR primers are verified, and finally, the qPCR primers with good specificity and high amplification efficiency, which are respectively used for the targeting efficiency of the 8crRNA probes to be replaced, are selected.

The screened 8 pairs of qPCR primers are used for verifying the targeting efficiency of the corresponding 8 alternative crRNA probes, and the process can be completed through a simple in vitro cleavage experiment and a qPCR experiment: first, 1. Mu.L of crRNA probe (20. Mu.M), 1. Mu.L of tracrRNA (20. Mu.M) and 8. Mu.L of enzyme-free duplex buffer were annealed to 10. Mu.L of single-stranded guide RNA (gRNA) (2. Mu.M) after heating at 95℃for 5min, 1. Mu.L of the latter were incubated with 0.8. Mu.L of Cas9 protein (6.2. Mu.M), 1. Mu.L of 10 Xreaction buffer and 7.2. Mu.L of enzyme-free water for 30min at room temperature to assemble 8 kinds of 10. Mu.L RNPs at a concentration of 1. Mu.M; then, 5. Mu.g of the gDNA of the K562 cell line was subjected to simple in vitro cleavage in 8 reaction systems of 42. Mu.L containing 1 Xreaction buffer, 10. Mu.L of RNPs, at 37℃for 30min; finally, 20ng of K562 cell line gDNA after and without RNPs are used as templates of qPCR test group and control group respectively, the total is 8 groups of control experiments before and after RNPs are cut, and the primers used in each group of control experiments are the quality control qPCR primers of the screened corresponding crRNA probe targeting efficiency. Finally, 4crRNA probes with high targeting efficiency are screened out, wherein the forward probes of BCR-23177704crRNA target the upstream region of M-BCR and BCR gene promoters, the two forward probes of BCR-23285486crRNA BCR-23286143crRNA target the upstream region of M-BCR, and the reverse probes of ABL1-130888788crRNA target the downstream of the 3' -end of the ABL1 gene (see figure 1). The PAM motifs of the 4crRNA probe sequences and the corresponding target sites are shown below:

BCR-23177704crRNA：5’-CAAGGGAGAAAGCCACTATC-3’；5’-TGG-3’；

BCR-23285486crRNA：5’-CACGGGATACTTCTTAGACC-3’；5’-TGG-3’；

BCR-23286143crRNA：5’-CCATACAAGCTACCCTGATG-3’；5’-GGG-3’；

ABL1-130888788crRNA：5’-ACTGGCCCAGTGTGACCAAT-3’；5’-TGG-3’。

the qPCR primers used for controlling the targeting efficiency and quality of the 4crRNA probes are BCR-23177704FP/RP, BCR-23285486FP/RP, BCR-23286143FP/RP and ABL1-130888788FP/RP, and the primer sequences are as follows:

BCR-23177704FP：5’-CCAACCCAACCCTCCAGAA-3’；

BCR-23177704RP：5’-GTCACAGGTCAGACAACTAAGCA-3’；

BCR-23285486FP：5’-CCAACCCAACCCTCCAGAA-3’；

BCR-23285486RP：5’-GTCACAGGTCAGACAACTAAGCA-3’；

BCR-23286143FP：5’-AGTGGAGGGCTTGGAGTAGT-3’；

BCR-23286143RP：5’-GGGGCTTGTCCTCACCATTT-3’；

ABL1-130888788FP：5’-TCGTGTCAGATGTGGACGGT-3’；

ABL1-130888788RP：5’-GACACCCATGAAAGATCCCCA-3’。

the sample qualified in quality control of the crRNA probe targeting efficiency is obtained through the early pre-experiment: the delta CT of BCR-231777044crRNA is more than or equal to 2, and the delta CT of ABL1-130888788crRNA and BCR-23285486crRNA or BCR-23286143crRNA is more than or equal to 2; namely, in one sample, qPCR quality control results of 1 crRNA probe (BCR-23177704 crRNA) targeting the M-BCR region and upstream of the BCR gene promoter and 1 crRNA probe (ABL 1-130888788 crRNA) targeting the 3' -end of the ABL1 gene must satisfy ΔCT not less than 2, and one of qPCR quality control results of 2 crRNA probes (BCR-23285486 crRNA and BCR-23286143 crRNA) targeting the M-BCR region must satisfy ΔCT not less than 2.

(2) Assembly of RNPs and gDNA dephosphorylation

(1) Assembling RNPs: first, a 10. Mu.L gRNA pool (final concentration: 2. Mu.M) was synthesized by taking 1. Mu.L of BCR-23177704crRNA (20. Mu.M), 1. Mu.L of BCR-23285486crRNA (20. Mu.M), 1. Mu.L of BCR-23286143crRNA (20. Mu.M), 1. Mu.L of ABL1-130888788crRNA (20. Mu.M) and 4. Mu.L of tracrrna (20. Mu.M), and supplementing to 10. Mu.L with 2. Mu.L of enzyme-free duplex buffer, heating at 95℃for 5min and cooling to 4 ℃. Next, 20. Mu.L RNPs (final concentration 1. Mu.M) were assembled, 10. Mu.L of gRNA pool, 1.6. Mu.L of LCas9 (6.2. Mu.M), 2. Mu.L of 10 Xreaction buffer and 6.4. Mu.L of enzyme-free water, incubated at room temperature for 30min and then transferred to ice for later use, and the remaining 10. Mu.L of RNPs were allowed to stand at-80℃for 1 month; during the RNPs incubation, the next step of gDNA dephosphorylation was continued.

(2) gDNA dephosphorylation: the initial input of gDNA required is 3-10. Mu.g, 5. Mu.g is recommended; the 30. Mu.L dephosphorylation reaction system was: 10. Mu. L K562 cell line gDNA (495 ng/. Mu.L. Times.10. Mu.L=4.95. Mu.g), 3. Mu.L of 10 Xreaction buffer, 3. Mu.L of CIP rapid phosphatase and 14. Mu.L of enzyme-free water were reacted at 37℃for 15min to dephosphorylate the 5' end of the gDNA, followed by heating at 80℃for 2min to inactivate the rapid phosphatase.

The 5' phosphate group in the double stranded gDNA end has been removed, and even if the 3' hydroxyl group in the end can be added with a deoxyribose adenylate (dATP) by the subsequent Taq DNA polymerase, the genomic double stranded DNA end cannot be attached to a nanopore gene sequencing adapter containing the T residue end by means of A/T complementation pairing in view of the 5' end having been dephosphorylated. The treatment effectively removes a large number of non-targeted genomic background genes, avoids the production of invalid sequencing data, and reduces the burden of subsequent data analysis.

(3) Preparation of RNPs for cleavage of corresponding crRNA Probe target sites and novel cleavage Ends

After equilibration of the dephosphorylated 30 μ L K562 cell line gDNA to room temperature, 10 μl RNPs assembled in step 2 were added thereto; in addition, 1. Mu.L of dATP and 1. Mu.L of Taq DNA polymerase were added for adding one A to the 3' -end of the double-stranded DNA. Then, 42 μl of the reaction solution was incubated at 37 ℃ for 30min, allowing the RNPs to cleave the corresponding crRNA probe target site in the double-stranded gDNA, and adding one dATP to the 3' end of the double-stranded gDNA to form a dA tail, followed by reaction at 72 ℃ for 5min to inactivate the Cas9 protein, and finally transferring to ice for preservation.

In the former reaction, the two DNA fragments generated after cleavage of double-stranded DNA by RNPs are blunt-ended, but the fragment located on the 5 'side of crRNA probe forms an RNA-DNA hybrid with crRNA probe, so Cas9 protein in RNPs is not exposed due to adhesion, and the fragment located on the 5' side of crRNA is exposed. Therefore, in the subsequent reaction of adding dA tail, the gene fragment which is positioned at the 5' side of the crRNA probe after RNPs are cut and is attached with Cas9 at the broken end cannot add one dATP by the action of Taq DNA polymerase, and a nanopore gene sequencing joint containing the tail end of a T residue cannot be connected in the reaction of adding the joint by an A/T complementary pairing mode; the DNA fragment located at the 3' side of the crRNA probe can be preferentially connected to the nanopore gene sequencing adaptor in the adaptor-adding reaction due to the exposed broken end. Thus, this procedure ensures that the gene segments downstream of the forward crRNA probe and the gene segments downstream of the reverse crRNA probe are preferentially ligated to the sequencing adaptors.

(4) quality control of qPCR experiments on targeting efficiency of each crRNA probe in each sample

The concentration of 42. Mu.L gDNA samples prepared after cleavage by RNPs and preparation of the nascent cleaved ends was: (495 ng/. Mu.L. Times.10. Mu.L)/(42. Mu.L= 117.86 ng/. Mu.L). 1.5. Mu.L of the above samples were diluted 10-fold to 11.786 ng/. Mu.L and used as templates for the experimental group (after cleavage of RNPs), and 1. Mu.L was further diluted 11.786 ng/. Mu.L from the original gDNA sample of the K562 cell line and used as templates for the control group (before cleavage of RNPs). The qPCR reaction was 20. Mu.L, where 1.5. Mu.L (about 17.8 ng) of template, 10. Mu.L of 2 XSYBR Green fluorescent dye, 2. Mu.L of 2. Mu.M upstream and downstream primers, and 4.5. Mu.L of enzyme-free water were used for each reaction, 1 multiplex well was performed, and then the targeting efficiency of each crRNA probe was judged based on the DeltaCT value (experimental CT-control CT value). The results are shown in Table 2, and in this example 1, the targeting efficiency of 4crRNA probes in gDNA samples of the K562 cell line was acceptable (ΔCT > 2), and subsequent sequencing experiments were performed.

TABLE 2

K562 gDNA sample	CT value before cutting	CT value after cutting	Delta CT value
				BCR-23177704crRNA	25.79	29.57	3.78
BCR-23285485crRNA	26.23	31.61	5.38
				BCR-23286143crRNA	23.08	26.62	3.54
ABL1-130888788crRNA	22.08	26.13	4.05

(5) Construction of a sequencing library targeting enriched fragments of interest

The method comprises the following specific operations of constructing a sequencing library of target enriched target fragments by using a target nanopore gene sequencing library-building kit (SQK-CS 9109) of ONT company:

(1) ligation sequencing adaptors: firstly, preparing 38 mu L of sequencing joint mixture, wherein the mixture comprises 10 mu L of joint connection buffer solution, 10 mu L of T4 ligase, 5 mu L of nanopore gene sequencing joint and 3 mu L of enzyme-free water; then, the method comprises the steps of. Adding the mixed solution into gDNA samples which are cut by RNPs and prepared by newly generated broken ends twice, wherein 20 mu L of the mixed solution is added for the first time, and the rest 18 mu L of the mixed solution is added for the second time; finally, 80. Mu.L of the sample as a whole (1.5. Mu.L of sample for crRNA probe targeting efficiency index was negligible) was incubated at room temperature for 10min.

(2) Magnetic bead purification gDNA: the procedure is to purify the gDNA sample in the ligation reaction mixture by adsorption of magnetic beads, which aims to remove excess sequencing adaptors, some short DNA fragments and substances introduced during the above-described experiments (e.g.crRNA probes, tracrRNA, various enzymes and ions, etc.), and to concentrate the gDNA sample in preparation for sequencing. The operation process is as follows: firstly, adding an equal volume (80 mu L) of SPRI dilution buffer into a gDNA sample, flicking and uniformly mixing, then adding AMPure XP magnetic beads (0.3×160 mu L=48 mu L) which are 0.3 times of the total volume of the diluted sample, flicking and uniformly mixing the same, and performing short instantaneous separation, and incubating at room temperature for 10min; secondly, transferring the incubated sample to a magnetic rack, agglomerating magnetic beads adsorbed with the sample, clarifying the solution, and discarding the supernatant; then adding 250 mu L of long fragment cleaning buffer solution, re-placing the magnetic beads in a magnetic rack after the magnetic beads are resuspended by a light-elastic test tube, and repeating the cleaning process once after the magnetic beads adsorbed with the sample are clustered and the solution is clarified; finally, taking out the clustered magnetic beads adsorbed with the sample from the magnetic rack, adding 14 mu L of elution buffer, placing the magnetic beads in a room temperature for incubation for 10min after the light-elastic test tube is completely resuspended, placing the sample on the magnetic rack after incubation is finished, sucking 13 mu L (losing 1 mu L) of supernatant containing the gDNA sample into a new 1.5mL centrifuge tube after the clustered magnetic beads of the gDNA sample are removed, taking 1 mu L of supernatant and carrying out concentration measurement by using a fluorescent quantitative instrument Qubit to determine the total amount of gDNA finally sequenced on the machine (generally about half of the initial gDNA amount, because the recovery rate of gDNA in the purification process is about 50% -60%), and placing the rest 12 mu L of liquid which is a sequencing library containing target enriched fragments on ice for standby. The total amount of sequencing library targeted to enrich for the fragment of interest in this example 1 is: 246 ng/. Mu.L.times.12. Mu.L= 2.952. Mu.g.

(6) Sequencing on a machine:

(1) activating a sequencing chip: after quality inspection, the sequencing chip can be activated by using FLO-MINI106D chips with nano holes larger than 800 and using nano hole gene sequencing chip activation buffer (FLT and FB), and the method comprises the following steps: firstly, preparing chip activation mixed solution, namely adding 30 mu L of FLT into a tube FB, and uniformly mixing; then, the sequencing chip's priming hole is opened, and after the air bubble is discharged by the gun head, 800. Mu.L of chip activation mixed solution is taken and rapidly added into the flow cell in the sequencing chip from the priming hole, and the mixture is incubated for 5min at room temperature, so that the preparation of the on-machine library can be performed.

(2) Preparing a library sequenced on-press: preparing 75 mu L of sequencing library, adding 37.5 mu L of nano-pore gene sequencing buffer and 25.5 mu L of nano-pore gene sequencing chip loading magnetic beads into 12 mu L of sequencing library of target enrichment target fragments, and uniformly mixing a flick test tube.

(3) Completing activation of the chip and loading the on-machine sequencing library: after the cover of the SpotON sample injection hole of the sequencing chip is opened, 200 mu L of chip activation mixed solution is quickly added into a flow cell in the sequencing chip from a priority hole to complete activation of the sequencing chip, then 75 mu L of prepared on-machine library is added into the flow cell of the sequencing chip from the SpotON sample injection hole in a dropwise manner, and finally the SpotON hole and the priority hole are closed.

(4) Sequencing: and mounting the chip carrying the sequencing library to a nanopore genetic tester MinION or GriION for sequencing, and acquiring sequencing data in real time through a driving operation software MinKNOW of a nanopore genetic sequencing platform.

(7) Ending the experiment: the sequencing can be ended when the data output of the to-be-tested sequence is basically saturated or no. For the used sequencing chip, the sequencing chip cleaning kit Flow Cell Wash Kit of ONT company can be used for cleaning, then the cleaned chip is stored at 4 ℃, and the available nano holes need to be checked before the next use, if the number is more than 800, the chips can be reused.

(8) Analysis of targeted sequencing data: data analysis is largely divided into two major categories, genetic variation analysis (structural and mononucleotide variations such as fusion genes) and DNA epigenetic variation (5 mC modification level changes):

analysis of genetic variation: during sequencing, a nanopore gene sequencing instrument is controlled through MinKNOW software, the running state of a sequencing chip is monitored, and electric signal data output by sequencing are collected. After the machine is started, converting electric signal data (stored in a fast5 file) collected during sequencing into base sequence data (stored in a fastq file) by using a Guppy high-precision base recognition mode, and performing quality control on the base sequence data in the fastq file by using a NanoPlot to filter reads of which the quality value of a score (Qscore) is not in accordance with the requirement; then, comparing the filtered data meeting the requirements with a human reference genome sequence GRCh38 (Hg 38) through minimum 2 software, and storing the compared result in a sam file format; finally, analyzing structural variations such as fusion genes from the sam file by using NanoSV, and converting the sam file into a file format (bam file) which can be checked by using a visualization software IGV by using Samtools; single nucleotide variations were then analyzed from the bam file using bcftools.

Analysis of 5mC modification level of DNA: the fast5 file was analyzed mainly by megalon software and calls Guppy, minimap and samtools simultaneously, and the final 5mC modification analysis results were visualized by IGV.

(9) Data analysis results

(1) Sequencing depth:

table 3 shows that the targeting efficiency of 4crRNA probes in the gDNA sample of the K562 cell line in the embodiment 1 is extremely high, and the number of the reads enriched for the targeted target gene fragment is far higher than the minimum requirement of 30 reads, which indicates that the targeted target gene fragment is successfully subjected to high-quality deep sequencing in the embodiment 1.

TABLE 3 Table 3

(2) BCR-ABL1 fusion gene:

the BCR-ABL1 fusion gene present in the K562 cell line was successfully detected by analysis of NanoSV software, and cleavage sites of BCR gene and ABL1 gene were (Hg) respectively: chr22-23290555 and chr9-130731760. The BCR-ABL1 fusion gene of the k562 cell line gDNA of this example 1 was examined with visualization software IVG as shown in fig. 6: the cleavage site for the BCR gene occurs in the M-BCR region downstream of the BCR-23285486crRNA and BCR-23286143crRNA probes, and the cleavage site for the ABL1 gene occurs in intron No. 1 of the ABL1 gene.

(3) Single nucleotide variation occurring in ABL1 gene KD:

as shown in FIG. 7, a total of 5 single nucleotide variations were occurred in the ABL1 gene KD of K562 cells, and all were homozygous mutations, but all of these 5 variations were occurred in the intron region of the ABL1 gene, and any TKIs drug resistant mutation site occurred in the exon was not detected.

(4) BCR gene promoter 5mC modification level:

table 4 shows the sequencing depth of C and the percentage of 5mC occurring (percentage of 5mC in C sequencing depth) for each CG motif in CpG islands (chr 22: 23180364-23182278) containing BCR gene promoter, and FIG. 8 shows the IGV visualization of 5mC sequencing data. As shown in the figure: the BCR-23177704crRNA probe targets most reads completely cover CpG islands including BCR gene promoter, and the methylation state of C in any CG motif in this region can be detected, wherein the methylation state of CpG islands is: the first half assumes a hypomethylated state and the second half assumes a hypermethylated state, whereas the BCR gene promoter is upstream of the CpG island, so that the entire promoter assumes a hypomethylated state.

TABLE 4 Table 4

The result of the 5mC modification level of the BCR gene promoter in the gDNA of the K562 cell line is verified by bisulfite amplicon sequencing (BSAS), and the result is reliable.

By the description of the embodiment 1, the kit and the method provided by the invention realize high-quality deep sequencing of the BCR-ABL1 fusion gene and KD region mutation thereof and promoter 5mC modification level, and the real result is accurate.

The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims (8)

1. A kit for detecting BCR-ABL1 fusion gene and tyrosine kinase domain mutation and promoter methylation thereof, comprising:

crRNA probes derived from CRISPR, comprising a BCR-23177704crRNA probe with a target site upstream of the BCR gene small break region and upstream of the BCR gene promoter, two probes BCR-23285486crRNA and BCR-23286143crRNA with a target site upstream of the BCR gene major break region, and an ABL1-1308887888crRNA probe with a target site downstream of the ABL1 kinase mutation region;

a transactivation crRNA that is universal and anneals to a crRNA probe to a chimeric single-stranded guide RNA;

and, CRISPR-associated protein Cas9, said protein Cas9 having endonuclease activity, assembling ribonucleoprotein complex RNPs with single-stranded guide RNA, and cleaving a target site in genomic double-stranded DNA under the guidance of said single-stranded guide RNA, breaking the double-stranded DNA;

the CRISPR/Cas 9targeted enrichment system is formed by the reagents so as to enrich the DNA fragments of interest.

2. The kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof according to claim 1, wherein the 5 'end of the 4crRNA probes are complementarily paired with the target site sequence and the 3' end sequence adjacent to the 4crRNA probe target site is PAM motif, and the 4crRNA probe sequence identification number, base sequence and PAM motif of the corresponding target site are as follows:

BCR-23177704crRNA：5’-CAAGGGAGAAAGCCACTATC-3’；5’-TGG-3’；

BCR-23285486crRNA：5’-CACGGGATACTTCTTAGACC-3’；5’-TGG-3’；

BCR-23286143crRNA：5’-CCATACAAGCTACCCTGATG-3’；5’-GGG-3’；

ABL1-130888788crRNA：5’-ACTGGCCCAGTGTGACCAAT-3’；5’-TGG-3’。

3. the kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof according to claim 2, wherein the targeting efficiency of the 4crRNA probes to their respective target sites in each genomic DNA sample is quality controlled by a real-time fluorescent quantitative polymerase chain reaction experiment.

4. The kit for detecting BCR-ABL1 fusion gene and tyrosine kinase domain mutation and promoter methylation thereof according to claim 3, wherein the primer pairs used for the targeting efficiency of 4crRNA probes for the real-time fluorescence quantitative polymerase chain reaction experiment are BCR-23177704FP/RP, BCR-23285486FP/RP, BCR-23286143FP/RP and ABL1-130888788FP/RP, respectively, and the sequences of the primer pairs are as follows:

BCR-23177704FP：5’-CCAACCCAACCCTCCAGAA-3’；

BCR-23177704RP：5’-GTCACAGGTCAGACAACTAAGCA-3’；

BCR-23285486FP：5’-CCAACCCAACCCTCCAGAA-3’；

BCR-23285486RP：5’-GTCACAGGTCAGACAACTAAGCA-3’；

BCR-23286143FP：5’-AGTGGAGGGCTTGGAGTAGT-3’；

BCR-23286143RP：5’-GGGGCTTGTCCTCACCATTT-3’；

ABL1-130888788FP：5’-TCGTGTCAGATGTGGACGGT-3’；

ABL1-130888788RP：5’-GACACCCATGAAAGATCCCCA-3’。

5. the kit for detecting BCR-ABL1 fusion gene and tyrosine kinase domain mutation and promoter methylation thereof according to claim 4, wherein the parameters of the real-time fluorescent quantitative polymerase chain reaction experimental reaction procedure are as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 15sec, annealing at 55℃for 15sec, elongation at 72℃for 20sec,40 cycles.

6. The kit for detecting BCR-ABL1 fusion gene and tyrosine kinase region mutation and promoter methylation thereof according to claim 5, wherein the kit further comprises a sample genomic DNA extraction reagent, a targeted nanopore gene sequencing library construction reagent and a nanopore gene sequencing chip excitation reagent.

7. A method of using a kit for detecting BCR-ABL1 fusion gene and tyrosine kinase domain mutation and promoter methylation thereof according to any one of claims 1-6, the method of using the kit comprising:

step one: extracting genomic DNA from a sample;

step two: dephosphorylation of the extracted genomic DNA sample;

step three: targeting enrichment of DNA fragments of interest from genomic DNA that has been treated in step two by the CRISPR/Cas9 targeting enrichment system;

step four: the targeting efficiency of BCR-23177704crRNA, BCR-23285486crRNA, BCR-23286143crRNA and ABL1-130888788crRNA probes is respectively and qualitatively controlled through a real-time fluorescent quantitative polymerase chain reaction experiment by using BCR-23177704FP/RP, BCR-23285486FP/RP, BCR-23285486FP/RP and ABL1-130888788FP/RP primer pairs;

step five: and (3) preparing a targeted sequencing library of the sample subjected to the quality control in the fourth step, and sequencing by using a nanopore gene sequencing technology, wherein the obtained sequencing data can be analyzed by using nanopore gene sequencing data analysis software, so that the fusion site of the BCR-ABL1 fusion gene, the mutation of the ABL1 tyrosine kinase region and the methylation level of the BCR gene promoter are detected.

8. The method of claim 7, wherein the sample is selected from the group consisting of a human peripheral blood sample or a bone marrow sample, and a human leukemia-associated cell line.

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