CN112266963A - Detection kit for combined detection of chronic granulocytic leukemia - Google Patents

Detection kit for combined detection of chronic granulocytic leukemia Download PDF

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CN112266963A
CN112266963A CN202011273236.4A CN202011273236A CN112266963A CN 112266963 A CN112266963 A CN 112266963A CN 202011273236 A CN202011273236 A CN 202011273236A CN 112266963 A CN112266963 A CN 112266963A
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孙石磊
姬云
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SUZHOU KEBEI BIOTECHNOLOGY CO Ltd
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Abstract

The invention provides a kit for jointly detecting chronic myelocytic leukemia, which comprises: special joints for constructing DNA libraries, specific composite primers for detecting gene mutation, fusion and deletion and composite primers for library amplification. The special joint is 8 dimers formed by a joint primer 1 and 8 different i5 end primers; the gene mutation, fusion and deletion are BCR/ABL fusion, FGFR1 rearrangement, JAK2 gene V617F mutation, JAK2 rearrangement, NUP98 rearrangement, PDGFRA rearrangement and PDGFRB rearrangement; the library amplification composite primers have different i7 ends, and the sequences of the primers are SEQ ID NO. 39-46. The kit provided by the invention can detect common molecular genetic variation of multiple chronic granulocytic leukemia at one time, and has the advantages of simple operation, short time and high detection efficiency.

Description

Detection kit for combined detection of chronic granulocytic leukemia
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a detection kit for jointly detecting chronic granulocytic leukemia.
Background
Chronic Myelogenous Leukemia (CML) is based on a malignant disease at the pluripotent stem cell level, and is characterized by acquired abnormal chromosomes, which are mainly characterized by the immortalization of granulocytes in bone marrow and peripheral blood. The effects of chronic myelocytic leukemia on blood and bone marrow are mainly manifested by the generation of a large number of immature leukocytes which accumulate in the bone marrow and inhibit the normal hematopoiesis of the bone marrow; and can diffuse throughout the body through blood, resulting in anemia, bleeding, infection, organ infiltration, etc.
Specific cytogenetic changes occur in about 95% of patients with chronic myelogenous leukemia, which are expressed by translocation of chromosomes 9 and 22, resulting in Philadelphia chromosome (Ph chromosome), and BCR/ABL fusion genes are generated, and corresponding fusion proteins have the characteristic of abnormally increasing tyrosine kinase activity, can catalyze and phosphorylate tyrosine residues of various substrate proteins, activate multiple signal transduction pathways, promote cell proliferation and reduce apoptosis.
Chronic myeloid leukemia is mainly diagnosed by blood routine and bone marrow aspiration biopsy, the blood routine detection is difficult to judge the early stage of the disease, and the bone marrow aspiration biopsy causes great damage to patients and is inconvenient to operate.
Studying the genetic profile of cancer enables the identification of genes that cause a malignant phenotype. These gene profiles (including gene expression and mutations) provide valuable information about the biological processes of normal and disease cells. However, there are wide variations in the genetic "characteristics" of cancers, which lead to difficulties in diagnosis and treatment, as well as in developing effective therapies.
The traditional fluorescent PCR method can only detect one site every time, and the required sample amount is large; the detection cost is high; the defects of more experiment operation quantity, long time consumption of detection procedures and the like.
Chinese patent 201710493398.0 discloses a method and kit for detecting high sensitivity of cancer-related gene mutation. The detection of cancer related gene mutation is completed by designing a group of tagged primer groups aiming at a target gene, respectively carrying out two-step amplification and then carrying out a second-generation sequencing process. The method can effectively eliminate false positive low-frequency mutation, and the detection result sensitivity and specificity are high. But the data analysis process is complicated, and the detection time is increased.
It is necessary to provide a simple and efficient chronic myelocytic leukemia detection kit.
Disclosure of Invention
The invention provides a detection kit, which is characterized in that DNA and RNA of a sample to be detected are extracted together, the RNA is subjected to reverse transcription to form cDNA, then the cDNA and the DNA are subjected to high-throughput sequencing library construction, and high-throughput sequencing is performed, so that multiple mutation types are detected at one time aiming at common molecular genetic variation of chronic myelocytic leukemia.
In one aspect, the invention provides a group of specific composite primers for detecting mutation types such as multiple gene mutations, fusions, deletions, and the like.
The sequence of the specific composite primer is shown in the following table 1.
TABLE 1 specific Complex primers
Detection site Primer name Primer sequences SEQ IDNO
BCR/ABL fusion ABL1-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCCTTCTTGGATTTGCAGCCCACCAGCT SEQ ID NO:10
BCR/ABL fusion ABL1-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGGTTTTCCTTGGAGTTCCAACGAGCGGC SEQ ID NO:11
BCR/ABL fusion ABL1-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTCAAAGTCAGATGCTACTGGCCGCTG SEQ ID NO:12
BCR/ABL fusion ABL1-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGGTTTGGGCTTCACACCATTCCCCATTGT SEQ ID NO:13
BCR/ABL fusion ABL1-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCGTCGGCCACCGTTGAATGATGATGAACC SEQ ID NO:14
BCR/ABL fusion ABL1-6 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGCTGCACCAGGTTAGGGTGTTTGATCTCT SEQ ID NO:15
FGFR1 rearrangement FGFR1-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCCGAGGCATGGAGTATCTGGCCTCCAAG SEQ ID NO:16
FGFR1 rearrangement FGFR1-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCATGGACAAGCCCAGTAACTGCACCAAC SEQ ID NO:17
FGFR1 rearrangement FGFR1-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAGATGAGGAAGGCCCCTGTGCAATAGA TGA SEQ ID NO:18
FGFR1 rearrangement FGFR1-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCCAGAGTTCATGGATGCACTGGAGTCAG CAG SEQ ID NO:19
FGFR1 rearrangement FGFR1-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCAGCCCTCTCCCAGGGGTTTGCCTAAG SEQ ID NO:20
TABLE 1 continuation
JAK2 rearrangement JAK2-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGGACTGCATCGAAGTACATACAGTCCAG TCTG SEQ ID NO:21
JAK2 rearrangement JAK2-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGTCCCACTGAGGTTGTACTCTTCATTCTC ATTT SEQ ID NO:22
JAK2 rearrangement JAK2-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAAACACCATTCGTTCTGAAGACTAGAA GGTTTG SEQ ID NO:23
JAK2 rearrangement JAK2-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGTTGACCGTAGTCTCCTACTTCTCTTCGTA CGCC SEQ ID NO:24
JAK2 rearrangement JAK2-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAAAGCTTGCTCATCATACTTGCTGCTTC AAAG SEQ ID NO:25
PDGFRA rearrangement PDGFRA-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCATCACAATCACCAACAGCACCAGGACTG SEQ ID NO:26
PDGFRA rearrangement PDGFRA-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCAGGGCTGATTGATTCAATGACCCTCCAGC SEQ ID NO:27
PDGFRA rearrangement PDGFRA-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTTCAACCACCTTCCCAAACGCTCCAG SEQ ID NO:28
PDGFRB rearrangement PDGFRB-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCGTTAGTCTCCAGCTGGCTCTCCTCTT SEQ ID NO:29
PDGFRB rearrangement PDGFRB-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCAGGATGGCTGAGATCACCACCACCTT SEQ ID NO:30
PDGFRB rearrangement PDGFRB-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCACCTTCCATCGGATCTCGTAACGTGG SEQ ID NO:31
PDGFRB rearrangement PDGFRB-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCGACATAAGGGCTTGCTTCTCACTGCT SEQ ID NO:32
PDGFRB rearrangement PDGFRB-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCTCTCACTTAGCTCCAGCACTCGGACA SEQ ID NO:33
PDGFRB rearrangement PDGFRB-6 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCATCGTGGCCTGAGAATGGCTCAGG SEQ ID NO:34
NUP98 rearrangement NUP98-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTGTTCCCAAACAAAGATGCCTGTCCAGCA SEQ ID NO:35
NUP98 rearrangement NUP98-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTGCTGGAGAACAGCCTGCTGGGCAGCAG SEQ ID NO:36
JAK2 gene V617F mutation JAK2-A-V617 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAAGCAGCAAGTATGATGAGCAAGCTTT C SEQ ID NO:37
The specific primer and the universal primer 1 are used simultaneously, and the sequence of the universal primer 1 is SEQ ID NO. 38.
The gene mutation, fusion and deletion variation types comprise: BCR/ABL fusion, FGFR1 rearrangement, JAK2 gene V617F mutation, JAK2 rearrangement, NUP98 rearrangement, PDGFRA rearrangement, PDGFRB rearrangement.
In another aspect, the invention provides a kit for combined detection of chronic myelogenous leukemia.
The kit for jointly detecting chronic granulocytic leukemia comprises the specific composite primer for detecting various mutation types, fusion types, deletion types and the like.
The kit also comprises a special joint constructed by the DNA library, wherein the special joint is 8 dimers formed by a joint primer 1 and 8 different i5 end primers. The sequences of the adaptor primer 1 and the 8 different i5 terminal primers are shown in Table 2 below.
TABLE 2 primer list for special joint
Primer name Primer sequences 5_ index sequence SEQ ID NO
Adaptor primer 1 GATCGGAAGAGCCACATACTGA - SEQ ID NO:1
Primer No. 1 at end i5 AATGATACGGCGACCACCGAGATCTACACCTCTCT ATACACTCTTTCCCTACACGACGCTCTTCCGATCT CTCT CTAT SEQ ID NO:2
Primer No. 2 at end i5 AATGATACGGCGACCACCGAGATCTACACTATCCT CTACACTCTTTCCCTACACGACGCTCTTCCGATCT TATC CTCT SEQ ID NO:3
Primer No. 3 at end i5 AATGATACGGCGACCACCGAGATCTACACGTAAGG AGACACTCTTTCCCTACACGACGCTCTTCCGATCT GTAA GGAG SEQ ID NO:4
Primer No. 4 at end i5 AATGATACGGCGACCACCGAGATCTACACACTGCA TAACACTCTTTCCCTACACGACGCTCTTCCGATCT ACTG CATA SEQ ID NO:5
Primer No. 5 at end i5 AATGATACGGCGACCACCGAGATCTACACAAGGAG TAACACTCTTTCCCTACACGACGCTCTTCCGATCT AAGG AGTA SEQ ID NO:6
Primer No. 6 at end i5 AATGATACGGCGACCACCGAGATCTACACCTAAGC CTACACTCTTTCCCTACACGACGCTCTTCCGATCT CTAA GCCT SEQ ID NO:7
Primer No. 7 at end i5 AATGATACGGCGACCACCGAGATCTACACCGTCTA ATACACTCTTTCCCTACACGACGCTCTTCCGATCT CGTC TAAT SEQ ID NO:8
Primer No. 8 at end i5 AATGATACGGCGACCACCGAGATCTACACTCTCTC CGACACTCTTTCCCTACACGACGCTCTTCCGATCT TCTC TCCG SEQ ID NO:9
The special joint has a structure shown in figure 1. Wherein, the sequence of the 5_ index is as follows: CTCTCTAT, TATCCTCT, GTAAGGAG, ACTGCATA, AAGGAGTA, CTAAGCCT, CGTCTAAT or TCTCTCTCCG.
The special joint comprises 8 joint structures shown in figure 2.
The kit also comprises a group of library amplification composite primers, and the sequences of the library amplification composite primers are shown in the following table 3.
TABLE 3 library amplification Complex primers Table
Primer name Primer sequences 7_ index sequence SEQ ID NO
Primer No. 1 at end i7 CAAGCAGAAGACGGCATACGAGATTCA CAAGCGTGACTGGAGTTCAGACGTGT TCACAAGC SEQ ID NO:39
Primer No. 2 at end i7 CAAGCAGAAGACGGCATACGAGATACTA CACGGTGACTGGAGTTCAGACGTGT ACTACACG SEQ ID NO:40
Primer No. 3 at end i7 CAAGCAGAAGACGGCATACGAGATATCG TACGGTGACTGGAGTTCAGACGTGT ATCGTACG SEQ ID NO:41
Primer No. 4 at end i7 CAAGCAGAAGACGGCATACGAGATAGA CACAGGTGACTGGAGTTCAGACGTGT AGACACAG SEQ ID NO:42
Primer No. 5 at end i7 CAAGCAGAAGACGGCATACGAGATTTGT CCTGGTGACTGGAGTTCAGACGTGT TTGTCCTG SEQ ID NO:43
Primer No. 6 at end i7 CAAGCAGAAGACGGCATACGAGATTGT GAGAGGTGACTGGAGTTCAGACGTGT TGTGAGAG SEQ ID NO:44
Primer No. 7 at end i7 CAAGCAGAAGACGGCATACGAGATAAG GTTGGGTGACTGGAGTTCAGACGTGT AAGGTTGG SEQ ID NO:45
Primer No. 8 at end i7 CAAGCAGAAGACGGCATACGAGATATTA GCCAGTGACTGGAGTTCAGACGTGT ATTAGCCA SEQ ID NO:46
The library amplification composite primer and the universal primer 2 are used simultaneously, and the sequence of the universal primer 2 is SEQ ID NO: 47.
In the kit provided by the invention, the (1) specific composite primers for detecting various mutation types such as mutation, fusion, deletion and the like, (2) special joints constructed by a DNA library, and (3) library amplification composite primer reagents are independently packaged.
The kit also comprises one or more of the following reagent groups: a reverse transcription reaction reagent group; DNA fragmentation/end repair/dA addition reagent group; a DNA ligation reagent group; a PCR amplification reagent set; high fidelity hot start PCR amplification reagent set.
The reverse transcription reaction reagent group comprises a random primer, a first chain synthesis buffer solution, a first chain synthesis enzyme mixed solution, a second chain synthesis buffer solution, a second chain synthesis enzyme mixed solution and ultrapure water.
The DNA fragmentation/end repair/dA addition reagent group comprises 10 XDNA fragmentation buffer solution, 5 Xfragmentation enzyme mixed solution and ultrapure water.
The DNA ligation reagent group comprises 5 Xligase buffer solution, TIANSeq DNA ligase, a special joint and ultrapure water.
The PCR amplification reagent group comprises 5 multiplied PCR reaction enzyme mixed liquor and PCR primers.
The high-fidelity hot-start PCR amplification reagent group comprises high-fidelity hot-start enzyme reaction liquid, PCR primers and ultrapure water.
In yet another aspect, the invention provides methods of using the aforementioned kits.
The detection method comprises the following steps:
(1) connecting and detecting sample DNA by using a special joint constructed by a DNA library;
(2) performing composite PCR amplification on the ligation product in the step (1) by using a specific composite primer for detecting various mutation types, fusion types, deletion types and the like;
(3) performing PCR amplification on the amplification product obtained in the step (2) by using a library amplification composite primer to obtain a sequencing library;
(4) sequencing the sequencing library obtained in the step (3).
As some preferred embodiments, the detection method comprises the following steps:
s1, extracting DNA and RNA in a sample;
s2, carrying out reverse transcription on the RNA obtained in the step S1 to prepare cDNA;
s3, mixing the cDNA obtained in the step S2 with the DNA obtained in the step S1, and then carrying out nucleic acid fragmentation, end repair and A addition to obtain a mixed solution of the cDNA and the DNA;
s4, performing joint connection and purification on the cDNA obtained in the step S3 and the DNA mixed solution to obtain a purified joint connection product;
s5, performing first-step specific PCR and purification on the purified adaptor connection product obtained in the step S4 to obtain a first-step PCR purified product;
s6, carrying out second-step universal PCR on the first-step PCR purified product obtained in the step S5 and purifying to obtain a sequencing library;
s7, machine sequencing;
and S8, performing data result analysis by using bioinformatics analysis software.
In some embodiments, the detection flow chart of the kit is shown in fig. 7.
In another aspect, the invention also provides the special joint for constructing the DNA library, the specific composite primer for detecting mutation types such as gene mutation, fusion, deletion and the like, and the application of the composite primer for library amplification in preparing a kit for jointly detecting chronic myelogenous leukemia.
The invention has the beneficial effects that:
1. in terms of the overall concept of the invention, the invention realizes the simultaneous detection of the specific composite primers of the variation types such as gene rearrangement, gene fusion, gene mutation and the like, and improves the detection sensitivity.
2. For the design concept of the present invention for library construction:
in the traditional amplicon library, double-end positions of the generated library are fixed due to the double-primer amplification, so that a sequencing sequence at a certain position cannot effectively remove sequence repetition caused by amplification, random mutation introduced in the amplification process cannot be identified and reduced, and the sensitivity is reduced. As shown in fig. 3.
After random fragmentation, the invention connects universal primers at two ends of DNA, one end adopts specific primer amplification, and the other end adopts universal primer, and retains the randomness of one section, thereby effectively identifying repeated sequencing sequence. As shown in fig. 4.
3. Compared with the traditional amplicon library construction detection fusion gene
The fusion gene is a chimeric gene formed by connecting the coding regions of two or more genes end to end and placing the two or more genes under the control of the same set of regulatory sequences (including promoters, enhancers, ribosome binding sequences, terminators and the like). For example, BCR/ABL fusion, where ABL is the core gene, BCR is the chaperone gene, fusion genes typically have multiple chaperone genes, and fusion breakpoints tend to be different.
Therefore, in the traditional amplicon library construction, the two genes of the chaperone gene and the core gene need to be known at the same time, and the primer design detection is carried out under the condition that the breakpoint position is also known. Easily miss the type of fusion for which the breakpoint is unknown, or for which the chaperone gene is unknown.
The invention sets a single primer on a core gene, does not need to know a breakpoint or a chaperone gene in advance, can contain a sequence in a library in the primer amplification process, and can identify the chaperone gene and the breakpoint position through sequencing. As shown in fig. 5.
4. Comparison with detection of the above mutation by arms _ PCR
The mutation operation is complex by using an arms _ PCR method, only one to three sites can be detected in each tube of reaction, and all the sites can be simultaneously detected by adding detection primers of a plurality of sites and performing high-throughput sequencing.
The arm _ PCR method cannot utilize mRNA detection, and the kit can detect.
5. Compared with the method using RT-PCR
The RT-PCR method has low detection efficiency, only one type of mutation can be detected at one time, and the kit can realize the simultaneous detection of various mutations by simultaneously adding a plurality of groups of primers. Moreover, the RT-PCR can only detect known types and limit the type of fusion genes of specific breakpoints, and the invention overcomes the defect.
6. Compared with detection by Sanger sequencing method
The Sanger sequencing method is low in sensitivity and sensitivity of detecting mutation sites, and can only reach 10%, the kit provided by the invention utilizes a high-throughput sequencing method, each mutation is sequenced in parallel more than 10000 times per sequencing, and the sensitivity can reach 0.1%. In addition, the Sanger sequencing method is complex to operate, only one site can be detected in each tube of reaction, and the kit can detect all the sites simultaneously by adding detection primers of a plurality of sites and performing high-throughput sequencing.
7. Comparison with solution-phase hybridization Capture library construction
The traditional liquid phase hybridization capture library has long process, about 24 steps are involved, the time consumption is 20-24h, the operation process is complex, the kit simplifies the operation process to 11 steps and shortens the time consumption to 6-8 h. As shown in fig. 6.
8. The whole technical scheme of the invention utilizes a high-throughput sequencing method, sequences each mutation at each time and performs parallel sequencing more than 10000 times, combines the whole concept, the design concept of library construction and (1) a special joint constructed by a DNA library; (2) detecting the fusion gene and the specific composite primer of point mutation; (3) the design concept of the library amplification composite primer realizes the simultaneous detection of all the sites (fusion gene and a plurality of mutation sites) on one hand; on the other hand, the detection sensitivity is improved from multiple aspects and angles, and the sensitivity can reach 0.1%. And the conventional detection method (such as Sanger sequencing) has low sensitivity which can only reach 10%.
Drawings
Fig. 1 shows a special joint structure.
Fig. 2 shows 8 joint structures of the dedicated joint.
FIG. 3 shows the construction of a library of conventional amplicons to detect mutations in gene loci.
FIG. 4 shows the construction of a single primer library of the present invention for detecting gene locus mutations.
FIG. 5 is a comparison of the construction of a conventional amplicon library and the detection of fusion genes with the single primer library of the present invention.
FIG. 6 is a comparison of the construction of a solution phase hybrid capture library with a single primer library of the present invention.
FIG. 7 is an experimental flow chart of the method of using the kit of the present invention.
FIG. 8 is a flow chart of the construction of the single primer amplification library of the present invention.
FIG. 9 is a positive detection map of BCR-ABL 1.
FIG. 10 is a negative detection map of BCR-ABL 1.
FIG. 11 is a positive detection map of ZYM 2-FGFR 1.
FIG. 12 is a negative detection map of ZYM 2-FGFR 1.
Figure 13 is a JAK c.1849g > T p.v617f positive detection map.
FIG. 14 is a JAK c.1849G > T p.V617F negative detection map.
FIG. 15 is a positive test image of PCM1-JAK 2.
FIG. 16 is a negative test chart for PCM1-JAK 2.
FIG. 17 is a positive test image of NUP98-HOXA 9.
FIG. 18 is a negative detection map of NUP98-HOXA 9.
FIG. 19 is a FIP1L1-PDGFRA positive detection map.
FIG. 20 is a FIP1L1-PDGFRA negative detection scheme.
FIG. 21 is a positive detection map of ETV 6-PDGFRB.
FIG. 22 is an ETV6-PDGFRB negative detection map.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
EXAMPLE 1 kit for the Combined detection of Chronic myeloid leukemia
The kit comprises:
(1) dedicated linker for DNA library construction: the special joint is 8 dimers formed by joint primer 1 and 8 different i5 terminal primers.
The sequences of the adaptor primer 1 and the 8 different i5 terminal primers are shown in Table 4 below.
Table 4 special joint article-guiding table
Primer name Primer sequences 5_ index sequence SEQ ID NO
Adaptor primer 1 GATCGGAAGAGCCACATACTGA - SEQ ID NO:1
Primer No. 1 at end i5 AATGATACGGCGACCACCGAGATCTACACCTCTCT ATACACTCTTTCCCTACACGACGCTCTTCCGATCT CTCT CTAT SEQ ID NO:2
Primer No. 2 at end i5 AATGATACGGCGACCACCGAGATCTACACTATCCT CTACACTCTTTCCCTACACGACGCTCTTCCGATCT TATC CTCT SEQ ID NO:3
Primer No. 3 at end i5 AATGATACGGCGACCACCGAGATCTACACGTAAGG AGACACTCTTTCCCTACACGACGCTCTTCCGATCT GTAA GGAG SEQ ID NO:4
TABLE 4 continuation
Primer No. 4 at end i5 AATGATACGGCGACCACCGAGATCTACACACTGCA TAACACTCTTTCCCTACACGACGCTCTTCCGATCT ACTG CATA SEQ ID NO:5
Primer No. 5 at end i5 AATGATACGGCGACCACCGAGATCTACACAAGGAG TAACACTCTTTCCCTACACGACGCTCTTCCGATCT AAGG AGTA SEQ ID NO:6
Primer No. 6 at end i5 AATGATACGGCGACCACCGAGATCTACACCTAAGC CTACACTCTTTCCCTACACGACGCTCTTCCGATCT CTAA GCCT SEQ ID NO:7
Primer No. 7 at end i5 AATGATACGGCGACCACCGAGATCTACACCGTCTA ATACACTCTTTCCCTACACGACGCTCTTCCGATCT CGTC TAAT SEQ ID NO:8
Primer No. 8 at end i5 AATGATACGGCGACCACCGAGATCTACACTCTCTC CGACACTCTTTCCCTACACGACGCTCTTCCGATCT TCTC TCCG SEQ ID NO:9
The dedicated fitting has a structure as shown in fig. 1. Wherein, the sequence of the 5_ index is as follows: CTCTCTAT, TATCCTCT, GTAAGGAG, ACTGCATA, AAGGAGTA, CTAAGCCT, CGTCTAAT or TCTCTCTCCG.
Thus, the dedicated fitting includes 8 fitting structures as shown in fig. 2.
(2) Specific composite primers and universal primers 1 for detecting mutation types such as gene mutation, fusion, deletion and the like, wherein the sequences of the specific composite primers and the corresponding detection sites thereof are shown in the following table 5.
TABLE 5 specific composite primers Table
Detection site Primer name Primer sequences SEQ IDNO
BCR/ABL fusion ABL1-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCCTTCTTGGATTTGCAGCCCACCAGCT SEQ ID NO:10
BCR/ABL fusion ABL1-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGGTTTTCCTTGGAGTTCCAACGAGCGGC SEQ ID NO:11
BCR/ABL fusion ABL1-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTCAAAGTCAGATGCTACTGGCCGCTG SEQ ID NO:12
BCR/ABL fusion ABL1-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGGTTTGGGCTTCACACCATTCCCCATTGT SEQ ID NO:13
BCR/ABL fusion ABL1-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCGTCGGCCACCGTTGAATGATGATGAACC SEQ ID NO:14
BCR/ABL fusion ABL1-6 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGCTGCACCAGGTTAGGGTGTTTGATCTCT SEQ ID NO:15
FGFR1 rearrangement FGFR1-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCCGAGGCATGGAGTATCTGGCCTCCAAG SEQ ID NO:16
FGFR1 rearrangement FGFR1-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCATGGACAAGCCCAGTAACTGCACCAAC SEQ ID NO:17
TABLE 5 continuation
FGFR1 rearrangement FGFR1-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAGATGAGGAAGGCCCCTGTGCAATAGA TGA SEQ ID NO:18
FGFR1 rearrangement FGFR1-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCCAGAGTTCATGGATGCACTGGAGTCAG CAG SEQ ID NO:19
FGFR1 rearrangement FGFR1-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCAGCCCTCTCCCAGGGGTTTGCCTAAG SEQ ID NO:20
JAK2 rearrangement JAK2-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGGACTGCATCGAAGTACATACAGTCCAG TCTG SEQ ID NO:21
JAK2 rearrangement JAK2-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGTCCCACTGAGGTTGTACTCTTCATTCTC ATTT SEQ ID NO:22
JAK2 rearrangement JAK2-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAAACACCATTCGTTCTGAAGACTAGAA GGTTTG SEQ ID NO:23
JAK2 rearrangement JAK2-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGTTGACCGTAGTCTCCTACTTCTCTTCGTA CGCC SEQ ID NO:24
JAK2 rearrangement JAK2-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAAAGCTTGCTCATCATACTTGCTGCTTC AAAG SEQ ID NO:25
PDGFRA rearrangement PDGFRA-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCATCACAATCACCAACAGCACCAGGACTG SEQ ID NO:26
PDGFRA rearrangement PDGFRA-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCAGGGCTGATTGATTCAATGACCCTCCAGC SEQ ID NO:27
PDGFRA rearrangement PDGFRA-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTTCAACCACCTTCCCAAACGCTCCAG SEQ ID NO:28
PDGFRB rearrangement PDGFRB-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCGTTAGTCTCCAGCTGGCTCTCCTCTT SEQ ID NO:29
PDGFRB rearrangement PDGFRB-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCAGGATGGCTGAGATCACCACCACCTT SEQ ID NO:30
PDGFRB rearrangement PDGFRB-3 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCACCTTCCATCGGATCTCGTAACGTGG SEQ ID NO:31
PDGFRB rearrangement PDGFRB-4 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCGACATAAGGGCTTGCTTCTCACTGCT SEQ ID NO:32
PDGFRB rearrangement PDGFRB-5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCTCTCACTTAGCTCCAGCACTCGGACA SEQ ID NO:33
TABLE 5 continuation
PDGFRB rearrangement PDGFRB-6 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCCATCGTGGCCTGAGAATGGCTCAGG SEQ ID NO:34
NUP98 rearrangement NUP98-1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTGTTCCCAAACAAAGATGCCTGTCCAGCA SEQ ID NO:35
NUP98 rearrangement NUP98-2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTGCTGGAGAACAGCCTGCTGGGCAGCAG SEQ ID NO:36
JAK2 gene V617F mutation JAK2-A-V617 GTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCGAAGCAGCAAGTATGATGAGCAAGCTTT C SEQ ID NO:37
Pairing with i5 end primer Universal primer 1 AATGATACGGCGACCACCGAGAT SEQ ID NO:38
The specific primer was used together with the universal primer 1.
(3) The sequences of the library amplification composite primers are shown in Table 6 below.
TABLE 6 library amplification composite primers
Primer name Primer sequences 7_ index sequence SEQ ID NO
Primer No. 1 at end i7 CAAGCAGAAGACGGCATACGAGATTCA CAAGCGTGACTGGAGTTCAGACGTGT TCACAAGC SEQ ID NO:39
Primer No. 2 at end i7 CAAGCAGAAGACGGCATACGAGATACTA CACGGTGACTGGAGTTCAGACGTGT ACTACACG SEQ ID NO:40
Primer No. 3 at end i7 CAAGCAGAAGACGGCATACGAGATATCG TACGGTGACTGGAGTTCAGACGTGT ATCGTACG SEQ ID NO:41
Primer No. 4 at end i7 CAAGCAGAAGACGGCATACGAGATAGA CACAGGTGACTGGAGTTCAGACGTGT AGACACAG SEQ ID NO:42
Primer No. 5 at end i7 CAAGCAGAAGACGGCATACGAGATTTGT CCTGGTGACTGGAGTTCAGACGTGT TTGTCCTG SEQ ID NO:43
Primer No. 6 at end i7 CAAGCAGAAGACGGCATACGAGATTGT GAGAGGTGACTGGAGTTCAGACGTGT TGTGAGAG SEQ ID NO:44
Primer No. 7 at end i7 CAAGCAGAAGACGGCATACGAGATAAG GTTGGGTGACTGGAGTTCAGACGTGT AAGGTTGG SEQ ID NO:45
Primer No. 8 at end i7 CAAGCAGAAGACGGCATACGAGATATTA GCCAGTGACTGGAGTTCAGACGTGT ATTAGCCA SEQ ID NO:46
Universal primer 2 AATGATACGGCGACCACCG - SEQID NO:47
The library amplification composite primer was used simultaneously with the universal primer 2.
(4) A reverse transcription reaction reagent group;
(5) DNA fragmentation/end repair/dA addition reagent group;
(6) a DNA ligation reagent group;
(7) a PCR amplification reagent set;
(8) high fidelity hot start PCR amplification reagent set.
Wherein:
the reverse transcription reaction reagent group comprises a random primer, a first chain synthesis buffer solution, a first chain synthetase mixed solution, a second chain synthesis buffer solution, a second chain synthetase mixed solution and ultrapure water;
the DNA fragmentation/end repair/dA addition reagent group comprises 10 XDNA fragmentation buffer, 5 Xfragmentation enzyme mixed liquor and ultrapure water;
the DNA ligation reagent group comprises 5 Xligase buffer solution, TIANSeq DNA ligase, a special joint and ultrapure water;
the PCR amplification reagent group comprises 5 multiplied PCR reaction enzyme mixed liquor and PCR primers;
the high-fidelity hot-start PCR amplification reagent group comprises high-fidelity hot-start enzyme reaction liquid, PCR primers and ultrapure water.
Example 2
The kit of the embodiment is the kit provided in embodiment 1, fig. 7 is an experimental flow chart of the detection method, and fig. 8 is a flow chart of the construction of the single primer amplification library. The detection method of the kit comprises the following specific steps:
first, cDNA reverse transcription
1. First Strand cDNA Synthesis
1.1 reagent preparation: slowly thawing the extracted sample RNA on ice, transferring to a liquid container, mixing, taking out the random primer first strand synthetase mixed solution and the first synthetic buffer solution (purchased from Tiangen science and technology, Biochemical technology, Inc., product type is TIANSeq cDNA synthesis module, catalog number is NG 308-T1) from-20 ℃, thawing, flicking, and mixing.
1.2 the following reaction system is established in a PCR tube and is gently blown and evenly mixed by a pipette:
RNA XμL(1000ng);
1 mu L of random primer;
ultra pure water (10-X) μ L;
the total volume was 11. mu.L.
Incubate at 1.365 ℃ for 5 minutes, then place on ice for 2 minutes.
1.4 step 1.3 after completion, the reverse transcription components listed in the following table were added to the original tube to prepare the following reaction system:
step 1.3, ending 11 mu L of reaction solution;
7 μ L of first strand synthesis buffer;
2 mu L of first-strand synthetase mixed liquor;
the total volume was 20. mu.L.
1.5 lightly blowing and beating the mixture by a pipettor, and fully and uniformly mixing the mixture to perform a first strand cDNA synthesis reaction, wherein the reaction procedure is as follows: 10min at 25 ℃; 15min at 42 ℃; 15min at 70 ℃; hold at 4 ℃. The temperature of the hot lid of the PCR machine was set at 105 ℃.
Note that: immediately after the completion of the reaction, the second strand cDNA synthesis reaction was carried out.
2. Second Strand cDNA Synthesis
2.1 taking out the second strand synthetase buffer solution and the second strand synthetase mixed solution (purchased from Tiangen science and technology Biochemical technology Co., Ltd., product type of TIANSeq cDNA Synthesis Module, catalog number of NG 308-T1) from-20 ℃, gently and uniformly mixing, establishing the following reaction system in a PCR tube, and gently blowing and beating by using a pipettor to fully and uniformly mix:
20. mu.L of synthesized first strand cDNA;
8.5 mu L of second strand synthetase buffer solution;
3.5 mu L of second strand synthetase mixed solution;
48 microliter of ultrapure water;
the total volume was 80. mu.L.
2.2 second Strand cDNA Synthesis reactions were performed in a PCR instrument using the following protocol: 60min at 16 ℃; hold at 4 ℃. The temperature of the hot cover of the PCR instrument is set to be less than or equal to 40 ℃.
Note that: after the reaction is completed, the synthesized product of the second strand of cDNA may be stored temporarily at 4 ℃ for 1 hour, but it is recommended that the next purification step be carried out immediately after the reaction is completed.
2.31.8 Xpurification magnetic beads (purchased from Tiangen technology Biochemical technology Co., Ltd., product type TIANSeq DNA fragment sorting magnetic beads, catalog No. NG 306) purified second strand cDNA synthesis product, 37 u L pure water elution, cDNA quantification.
Secondly, mixing the cDNA with DNA extracted from the sample
50ng of cDNA was mixed with 50ng of DNA extracted from the same sample in equal amounts to obtain 100ng of mixed solution of cDNA and DNA.
Thirdly, constructing a library: nucleic acid fragmentation, end repair and addition of A
1. Preparing a reaction system (the DNA loading amount is more than 10 NG) by taking 10 XDNA fragmentation buffer solution and 5 Xfragmentation enzyme mixed solution (purchased from Tiangen science and technology Biochemical technology Co., Ltd., product type of TIANSeq rapid DNA fragmentation/terminal repair/dA addition module, catalog number of NG 301), operating on ice, and after adding each component, gently sucking and beating the components to mix uniformly, and taking no need of vortex:
10 XDNA fragmentation buffer 5 u L;
5 Xfragmentation enzyme mixture 10. mu.L;
mixture of cDNA and DNA X. mu.L (100 ng);
ultra pure water (35-X) μ L;
the total volume was 50. mu.L.
2. The following reaction procedure was performed in a PCR instrument: 1min at 4 ℃; 7min at 32 ℃; 30min at 65 ℃; hold at 4 ℃. The temperature of the hot lid of the PCR instrument was set at 70 ℃.
Wherein, the selection of the fragmentation time at 32 ℃ is referred to as follows:
when the main peak of the DNA is 250bp, the fragmentation time of 10ng DNA loading at 32 ℃ is 24min, the fragmentation time of 100ng DNA loading at 32 ℃ is 16min, and the fragmentation time of 1000ng DNA loading at 32 ℃ is 14 min.
When the main peak of the DNA is 350bp, the fragmentation time at 32 ℃ of 10ng of DNA loading is 16min, the fragmentation time at 32 ℃ of 100ng of DNA loading is 10min, and the fragmentation time at 32 ℃ of 1000ng of DNA loading is 8 min.
When the main peak of the DNA is 450bp, the fragmentation time at 32 ℃ of 10ng of DNA loading is 14min, the fragmentation time at 32 ℃ of 100ng of DNA loading is 8min, and the fragmentation time at 32 ℃ of 1000ng of DNA loading is 6 min.
When the main peak of the DNA is 550bp, the fragmentation time at 32 ℃ of 10ng of DNA loading is 10min, the fragmentation time at 32 ℃ of 100ng of DNA loading is 6min, and the fragmentation time at 32 ℃ of 1000ng of DNA loading is 4 min.
3. And immediately entering the joint connection step after the reaction is finished.
Fourthly, constructing a library: joint connection
1. Preparing a reaction system from 5 XLigase Buffer and TIANeq DNA Ligase (purchased from Tiangen science and technology Biochemical technology Co., Ltd., product type of TIANeq quick connection module, catalog number NG 303) in a three-step reaction finishing tube:
step three, 50 mu L of reaction product;
5×Ligase Buffer 20μL;
TIAN Seq DNA Ligase 10μL;
2.5 muL of special joint (15 muM);
17.5 microliter of ultrapure water;
the total volume was 100. mu.L.
Wherein, special joint includes following 8 joint structures:
the joint 1:
Figure 159812DEST_PATH_IMAGE001
and (3) joint 2:
Figure 903646DEST_PATH_IMAGE002
and (3) a joint:
Figure 436259DEST_PATH_IMAGE003
and (4) connecting the joint:
Figure 838421DEST_PATH_IMAGE004
and (5) a joint:
Figure 772879DEST_PATH_IMAGE005
and (6) a joint:
Figure 969505DEST_PATH_IMAGE006
and (3) a joint 7:
Figure 989414DEST_PATH_IMAGE007
and (3) a joint 8:
Figure 178956DEST_PATH_IMAGE008
2. pipette, beat and mix evenly, put into PCR instrument, carry out the following reaction program: 15min at 20 ℃; hold at 4 ℃. Without a heat cover.
3. 1.6 Xmagnetic beads (purchased from Tiangen science and biochemistry technology Co., Ltd., product type: TIANSeq DNA fragment sorting magnetic beads, catalog number: NG 306) purification adapter-ligated product, 16. mu.L of ultra-pure water was eluted, and 15. mu.L of supernatant was taken into a new PCR tube to perform the first PCR amplification reaction.
Fifth, first step multiplex PCR amplification
1. The following PCR reaction system was prepared from 5 Xmultiplex PCR reaction enzyme mixture (purchased from Takara, product type: Multiplex PCR Assay Kit Ver.2, cat # RR 062A):
5 multiplied by 4 microlitres of the mixed solution of the multiple PCR reaction enzyme;
specific primer mixture 0.56. mu.L;
4.7. mu.L of the universal primer 1 (currently available);
step four, 10.74 mu L of purified adaptor connection product;
the total volume was 20. mu.L.
The preparation method of the specific primer mixture comprises the following steps: the primers were diluted to 100. mu.M and the mixture system was formulated as shown in Table 7 below.
TABLE 7 specific primer mixture System
Primer name SEQ IDNO Mixing amount of each primer was 100. mu.M (. mu.L) 20 μ L final concentration of primers (μ M) in the reaction System
ABL1-1 SEQ ID NO:10 1 0.1
ABL1-2 SEQ ID NO:11 1 0.1
ABL1-3 SEQ ID NO:12 1 0.1
ABL1-4 SEQ ID NO:13 1 0.1
ABL1-5 SEQ ID NO:14 1 0.1
ABL1-6 SEQ ID NO:15 1 0.1
FGFR1-1 SEQ ID NO:16 1 0.1
FGFR1-2 SEQ ID NO:17 1 0.1
FGFR1-3 SEQ ID NO:18 1 0.1
FGFR1-4 SEQ ID NO:19 1 0.1
FGFR1-5 SEQ ID NO:20 1 0.1
TABLE 7 continuation
JAK2-1 SEQ ID NO:21 1 0.1
JAK2-2 SEQ ID NO:22 1 0.1
JAK2-3 SEQ ID NO:23 1 0.1
JAK2-4 SEQ ID NO:24 1 0.1
JAK2-5 SEQ ID NO:25 1 0.1
PDGFRA-1 SEQ ID NO:26 1 0.1
PDGFRA-2 SEQ ID NO:27 1 0.1
PDGFRA-3 SEQ ID NO:28 1 0.1
PDGFRB-1 SEQ ID NO:29 1 0.1
PDGFRB-2 SEQ ID NO:30 1 0.1
PDGFRB-3 SEQ ID NO:31 1 0.1
PDGFRB-4 SEQ ID NO:32 1 0.1
PDGFRB-5 SEQ ID NO:33 1 0.1
PDGFRB-6 SEQ ID NO:34 1 0.1
NUP98-1 SEQ ID NO:35 1 0.1
NUP98-2 SEQ ID NO:36 1 0.1
JAK2-A-V617 SEQ ID NO:37 1 0.1
Specific primer Total amount of mixed liquid - 28μL -
2. After the reaction mixture was prepared, the mixture was pipetted 10 times and mixed, and placed in a PCR instrument to run the following reaction program: 2min at 99 ℃; 15s at 99 ℃, 4min at 69 ℃ and 18 cycles; 10min at 72 ℃; hold at 4 ℃. The temperature of the hot lid of the PCR machine was set at 105 ℃.
3. After the PCR is completed, electrophoresis is performed. 1.6 Xmagnetic beads (purchased from Tiangen science and biochemistry technology, Ltd., product type TIANSeq DNA fragment sorting magnetic beads, catalog number NG 306) were purified, eluted with 15. mu.L of pure water to obtain the first PCR amplification product, and quantit was quantified.
Sixth, second PCR amplification
1. Taking high-fidelity hot-start enzyme reaction liquid (purchased from Tiangen science and technology, biochemistry and technology limited company, the product type is high-fidelity PCR reaction premixed liquid, the catalog number is NG 219), and carrying out second round PCR amplification on the purified product of the first step PCR, wherein the amplification system is as follows:
25 mu L of high-fidelity hot start enzyme reaction solution;
i7-primer(10μM) 1.5μL;
1.5. mu.L of universal primer 2 (10. mu.M);
10 mu L of the product of the first PCR purification;
12 microliter of ultrapure water;
the total volume was 50. mu.L.
2. After the mixed solution of the PCR amplification reaction is prepared, a pipettor is blown and beaten for 10 times to be uniformly mixed, and the mixture is put into a PCR instrument, and the following reaction program is operated for 3min at 95 ℃; 20s at 98 ℃, 15s at 58 ℃ and 15s at 72 ℃ for 8 cycles; 1min at 72 ℃; hold at 4 ℃. The temperature of the hot lid of the PCR machine was set at 105 ℃.
3. After the PCR reaction is finished, the quantit is quantified and electrophoresed.
4. 1.3 Xmagnetic beads (purchased from Tiangen science and biochemistry technology Co., Ltd., product type TIANSeq DNA fragment sorting magnetic beads, catalog number NG 306) were purified, eluted with 10. mu.L of ultrapure water, and quantit was quantified.
Seventhly, the library is diluted to 2-3 ng/. mu.L, and the Agilent 4150 TapeStation system carries out detection.
Eighthly, sequencing and processing.
And ninthly, feeding back sequencing data.
Test example 1
The method described in example 2 of the present invention was used to detect a sample.
Samples including patients with chronic myeloid leukemia and healthy persons were approved.
1. Sample 1: the source is a clinical sample with the tumor hospital number of 198272_ KTD072 in Yunnan province.
The detection result is shown in FIG. 9, and the sample is positive to BCR-ABL 1.
2. Sample 2: the source is a clinical sample with the tumor hospital number of 196547_ KTD031 in Yunnan province.
The detection result is shown in FIG. 10, and the sample is negative to BCR-ABL 1.
3. Sample 3: the source is a clinical sample with the tumor hospital number of 198276_ KTD069 in Yunnan province.
As a result of the detection, ZYM 2-FGFR1 was positive in this sample, as shown in FIG. 11.
4. Sample 4: the source was a clinical sample with accession number 191561_ NW685 in tumor hospital, Yunnan province.
As a result of the detection, ZYM 2-FGFR1 was negative in this sample, as shown in FIG. 12.
5. Sample 5: the source was a clinical sample with tumor hospital number 191463_ NW636 in Yunnan province.
The results are shown in FIG. 13, and the sample was JAK c.1849G > T.V617F positive.
6. Sample 6: the source is a clinical sample with the tumor hospital number of 198273_ KTD071 in Yunnan province.
As shown in FIG. 14, the sample was JAK c.1849G > T.V617F-negative.
7. Sample 7: the source was a clinical sample with tumor hospital number 191242_ NW608 in Yunnan province.
As a result of the detection, the sample was positive for PCM1-JAK2, as shown in FIG. 15.
8. Sample 8: the source is a clinical sample with the tumor hospital number of 198277_ KTD073 in Yunnan province.
As a result of the detection, the sample was negative for PCM1-JAK2, as shown in FIG. 16.
9. Sample 9: the source is a clinical sample with the tumor hospital number of 197548_ KTD094 in Yunnan province.
The detection result is shown in FIG. 17, and the sample is NUP98-HOXA9 positive.
10. Sample 10: the source is a clinical sample with the tumor hospital number of 197550_ KTD095 in Yunnan province.
The detection results are shown in FIG. 18, and the sample is NUP98-HOXA9 negative.
11. Sample 11: the source is a clinical sample with the tumor hospital number of 200888_ KTD029 in Yunnan province.
The detection result is shown in FIG. 19, and the sample is FIP1L1-PDGFRA positive.
12. Sample 12: the source is a clinical sample with the tumor hospital number of 297013_ KTD091 in Yunnan province.
The results are shown in FIG. 20, and the sample is FIP1L1-PDGFRA negative.
13. Sample 13: the source is a clinical sample with the tumor hospital number of 197527_ KTD054 in Yunnan province.
The detection result is shown in FIG. 21, and the sample is positive to ETV 6-PDGFRB.
14. Sample 14: the source is a clinical sample with the tumor hospital number of 197884_ KTD117 in Yunnan province.
The detection result is shown in FIG. 22, and the sample is negative to ETV 6-PDGFRB.
Test example 2 accuracy test of a kit for combined detection of chronic myelogenous leukemia
The positive samples provided in test example 1 were verified by Sanger sequencing, and the detection results were consistent with those of the method provided by the present invention, indicating that the kit provided by the present invention has good accuracy.
Test example 3 stability test of kit for combined detection of Chronic myelocytic leukemia
The stability test was performed on the kit provided in example 1 by the following method:
the test kits were placed at 4 ℃ and 20 ℃ for 7 days, 14 days, and 21 days, respectively, and then the samples of test example 1 were tested by the test method provided in example 2, and the results are shown in table 8 below.
TABLE 8 stability test results
Sample(s) 7 days at 4 DEG C At 4 ℃ for 14 days At 4 ℃ for 21 days 7 days at 20 DEG C 14 days at 20 DEG C At 20 ℃ for 21 days
Sample 1 Y Y Y Y Y Y
Sample 2 Y Y Y Y Y Y
Sample 3 Y Y Y Y Y N
Sample 4 Y Y Y Y Y Y
Sample 5 Y Y Y Y Y Y
Sample 6 Y Y Y Y Y Y
TABLE 8 continuation
Sample 7 Y Y Y Y Y N
Sample 8 Y Y Y Y Y Y
Sample 9 Y Y Y Y Y Y
Sample 10 Y Y Y Y Y Y
Sample 11 Y Y Y Y Y Y
Sample 12 Y Y Y Y Y N
Sample 13 Y Y Y Y Y Y
Sample 14 Y Y Y Y Y Y
Wherein, Y represents that the detection result is accurate, and N represents that the detection result is inaccurate. From the above data, the stability of the kit provided by the present application is good, and the kit can be stored at least at 4 ℃ for 21 days and at 20 ℃ for 14 days.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Suzhou Kobe Biotechnology Ltd
<120> detection kit for combined detection of chronic myelocytic leukemia
<130> 20201012
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<213> Artificial sequence (artificial sequence)
<400> 19
gtgactggag ttcagacgtg tgctcttccg atcccagagt tcatggatgc actggagtca 60
gcag 64
<210> 20
<211> 60
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 20
gtgactggag ttcagacgtg tgctcttccg atccagccct ctcccagggg tttgcctaag 60
<210> 21
<211> 65
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 21
gtgactggag ttcagacgtg tgctcttccg atcggactgc atcgaagtac atacagtcca 60
gtctg 65
<210> 22
<211> 66
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 22
gtgactggag ttcagacgtg tgctcttccg atcgtcccac tgaggttgta ctcttcattc 60
tcattt 66
<210> 23
<211> 67
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 23
gtgactggag ttcagacgtg tgctcttccg atcgaaacac cattcgttct gaagactaga 60
aggtttg 67
<210> 24
<211> 67
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 24
gtgactggag ttcagacgtg tgctcttccg atcgttgacc gtagtctcct acttctcttc 60
gtacgcc 67
<210> 25
<211> 66
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 25
gtgactggag ttcagacgtg tgctcttccg atcgaaagct tgctcatcat acttgctgct 60
tcaaag 66
<210> 26
<211> 61
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 26
gtgactggag ttcagacgtg tgctcttccg atcatcacaa tcaccaacag caccaggact 60
g 61
<210> 27
<211> 62
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 27
gtgactggag ttcagacgtg tgctcttccg atcagggctg attgattcaa tgaccctcca 60
gc 62
<210> 28
<211> 59
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 28
gtgactggag ttcagacgtg tgctcttccg atcttcaacc accttcccaa acgctccag 59
<210> 29
<211> 60
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 29
gtgactggag ttcagacgtg tgctcttccg atccgttagt ctccagctgg ctctcctctt 60
<210> 30
<211> 60
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 30
gtgactggag ttcagacgtg tgctcttccg atccaggatg gctgagatca ccaccacctt 60
<210> 31
<211> 60
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 31
gtgactggag ttcagacgtg tgctcttccg atccaccttc catcggatct cgtaacgtgg 60
<210> 32
<211> 60
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 32
gtgactggag ttcagacgtg tgctcttccg atccgacata agggcttgct tctcactgct 60
<210> 33
<211> 60
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 33
gtgactggag ttcagacgtg tgctcttccg atcctctcac ttagctccag cactcggaca 60
<210> 34
<211> 58
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 34
gtgactggag ttcagacgtg tgctcttccg atccatcgtg gcctgagaat ggctcagg 58
<210> 35
<211> 62
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 35
gtgactggag ttcagacgtg tgctcttccg atctgttccc aaacaaagat gcctgtccag 60
ca 62
<210> 36
<211> 61
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 36
gtgactggag ttcagacgtg tgctcttccg atctgctgga gaacagcctg ctgggcagca 60
g 61
<210> 37
<211> 62
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 37
gtgactggag ttcagacgtg tgctcttccg atcgaagcag caagtatgat gagcaagctt 60
tc 62
<210> 38
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 38
aatgatacgg cgaccaccga gat 23
<210> 39
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 39
caagcagaag acggcatacg agattcacaa gcgtgactgg agttcagacg tgt 53
<210> 40
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 40
caagcagaag acggcatacg agatactaca cggtgactgg agttcagacg tgt 53
<210> 41
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 41
caagcagaag acggcatacg agatatcgta cggtgactgg agttcagacg tgt 53
<210> 42
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 42
caagcagaag acggcatacg agatagacac aggtgactgg agttcagacg tgt 53
<210> 43
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 43
caagcagaag acggcatacg agatttgtcc tggtgactgg agttcagacg tgt 53
<210> 44
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 44
caagcagaag acggcatacg agattgtgag aggtgactgg agttcagacg tgt 53
<210> 45
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 45
caagcagaag acggcatacg agataaggtt gggtgactgg agttcagacg tgt 53
<210> 46
<211> 53
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 46
caagcagaag acggcatacg agatattagc cagtgactgg agttcagacg tgt 53
<210> 47
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 47
aatgatacgg cgaccaccg 19

Claims (9)

1. A group of specific composite primers for detecting gene mutation, fusion and deletion mutation types is characterized in that the gene mutation, fusion and deletion types are BCR/ABL fusion, FGFR1 rearrangement, JAK2 gene V617F mutation, JAK2 rearrangement, NUP98 rearrangement, PDGFRA rearrangement and PDGFRB rearrangement; the nucleotide sequence of the specific composite primer is shown in SEQ ID NO. 10-SEQ ID NO. 37.
2. The specific composite primer for detecting the types of gene mutation, fusion and deletion mutation as claimed in claim 1, wherein the specific composite primer further comprises a universal primer 1, and the sequence of the universal primer 1 is SEQ ID NO. 38.
3. A kit for combined detection of chronic myeloid leukemia, wherein the kit comprises the specific composite primer for detecting types of gene mutation, fusion and deletion mutation of claim 1.
4. The kit for the combined detection of chronic myeloid leukemia according to claim 3, further comprising: a special joint for constructing a DNA library, wherein the special joint is 8 dimers formed by joint primers 1 and 8 different i5 end primers;
the sequence of the joint primer 1 is SEQ ID NO. 1;
the sequences of the 8 different i5 end primers are SEQ ID NO. 2-SEQ ID NO. 9.
5. The kit for the combined detection of chronic myeloid leukemia according to claim 4, further comprising: a group of composite primers for library amplification, wherein the library amplification composite primers have different i7 ends, and the sequences of the primers are SEQ ID NO. 39-SEQ ID NO. 46.
6. The kit for combined detection of chronic myeloid leukemia according to claim 5, wherein said composite primers for library amplification further comprise universal primer 2, and the sequence of universal primer 2 is SEQ ID NO. 47.
7. The kit for combined detection of chronic myeloid leukemia according to claim 6, wherein the kit further comprises one or more of reverse transcription reagent set, DNA fragmentation/end repair/dA addition reagent set, DNA ligation reagent set, PCR amplification reagent set, and high fidelity hot start PCR amplification reagent set.
8. The kit for combined detection of chronic myeloid leukemia according to claim 7, wherein the reverse transcription reagent set comprises random primers, first strand synthesis buffer, first strand synthase mixture, second strand synthesis buffer, second strand synthase mixture and ultra pure water; the DNA fragmentation/end repair/dA addition reagent group comprises 10 multiplied by DNA fragmentation buffer solution, 5 multiplied by fragmentation enzyme mixed solution and ultrapure water; the DNA ligation reagent group comprises 5 Xligase buffer solution, TIANSeq DNA ligase, a special joint and ultrapure water; the PCR amplification reagent group comprises 5 multiplied PCR reaction enzyme mixed liquor and PCR primers; the high-fidelity hot-start PCR amplification reagent group comprises high-fidelity hot-start enzyme reaction liquid, PCR primers and ultrapure water.
9. Use of the specific composite primer for detecting gene mutation, fusion and deletion variation types as claimed in claim 1 in the preparation of a kit for combined detection of chronic myeloid leukemia.
CN202011273236.4A 2020-11-13 2020-11-13 Detection kit for combined detection of chronic granulocytic leukemia Active CN112266963B (en)

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CN113481289A (en) * 2021-06-22 2021-10-08 天津见康华美医学诊断技术有限公司 Primer composition for detecting sideroblastic red blood cell anemia and application thereof

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CN113481289A (en) * 2021-06-22 2021-10-08 天津见康华美医学诊断技术有限公司 Primer composition for detecting sideroblastic red blood cell anemia and application thereof
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