CN111304331A - MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof - Google Patents

MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof Download PDF

Info

Publication number
CN111304331A
CN111304331A CN202010156441.6A CN202010156441A CN111304331A CN 111304331 A CN111304331 A CN 111304331A CN 202010156441 A CN202010156441 A CN 202010156441A CN 111304331 A CN111304331 A CN 111304331A
Authority
CN
China
Prior art keywords
pcr
amplicon
pcr reaction
amplicon product
magnetic beads
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010156441.6A
Other languages
Chinese (zh)
Inventor
唐春花
邢宽
谢珍
张鹏
袁鸣
安雪茄
高婷婷
高彧辉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Experimental Medicine Examines Co ltd
Original Assignee
Nanjing Experimental Medicine Examines Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Experimental Medicine Examines Co ltd filed Critical Nanjing Experimental Medicine Examines Co ltd
Priority to CN202010156441.6A priority Critical patent/CN111304331A/en
Publication of CN111304331A publication Critical patent/CN111304331A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)

Abstract

The invention discloses a MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and a preparation method thereof, wherein the MDS detection kit comprises all primers of an amplicon, a PCR mixed solution, a PCR reaction solution, a digestion solution, a joint P5, a joint P7, absolute ethyl alcohol, magnetic beads and a nucleic-Free Water, after 25ng and 10ng/ul of DNA is extracted by using a DNA extraction kit, the PCR mixed solution and 1ul of PCR reaction solution are subjected to PCR program operation to obtain an amplicon product, the digestion solution and the magnetic beads are added into the amplicon product for purification to obtain a pure amplicon product, finally, the joint P5, the joint P7, the PCR mixed solution and the PCR reaction solution are added into the purified amplicon product for PCR reaction, and a pure amplicon library is obtained by using the magnetic beads, so that the MDS detection kit has strong specificity, short experimental period and improves multigenic mutation screening feasibility.

Description

MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof
Technical Field
The invention relates to the technical field of leukemia related gene mutation detection, in particular to a MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and a preparation method thereof.
Background
Myelodysplastic syndrome (MDS) is a group of clonal myeloid tumors characterized by ineffective hematopoiesis and a high risk of transformation into acute leukemias, the most common molecular biological abnormalities in leukemias consisting essentially of: the gene mutation, the fusion gene and the gene expression are mainly point mutation, gene fragment insertion or deletion, so necessary gene detection can provide reference for clinical diagnosis and typing, prognosis judgment, treatment guidance, Micro Residual Disease (MRD) monitoring and clone evolution. Compared with a first generation (Sanger) sequencing technology, the multiple PCR targeted sequencing technology has higher sensitivity and can quantitatively detect the gene mutation load rate; compared with the target sequencing of a capture probe, the construction process of the multiple PCR target sequencing technology library is simpler, and a more economical and rapid sequencing scheme can be provided, but most of real-time fluorescence PCR (RQ-PCR) and digital PCR are used for detecting known mutation sites in genes, the experimental period is longer, and the feasibility of screening multiple gene mutations is poorer.
Disclosure of Invention
The invention aims to provide a MDS detection kit based on multiple PCR targeted high-throughput sequencing and a preparation method thereof, which have the advantages of strong specificity and short experimental period and improve the multi-gene mutation screening feasibility.
In order to achieve the above objects, in a first aspect, the present invention provides a method for preparing a MDS detection kit based on multiple PCR targeted high throughput sequencing, comprising:
extracting 25ng and 10ng/ul DNA, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain an amplicon product;
adding digestive juice and magnetic beads into the amplicon product for purification to obtain a pure amplicon product;
and adding a joint primer, a PCR mixed solution and a PCR reaction solution into the purified amplicon product to perform PCR reaction, and obtaining a pure amplicon library by using magnetic beads.
Wherein, the step of extracting 25ng and 10ng/ul DNA, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain an amplicon product comprises the following steps:
extracting a sample to be detected by using a DNA extraction kit to obtain 25ng and 10ng/ul DNA, adding 5ul of PCR mixed solution, 1ul of PCR reaction solution and 1ul of upstream primer downstream primers into the DNA, simultaneously complementing the mixed solution to 10ul by using a nucleic-FreeWater, and then operating a PCR program to obtain an amplicon product.
Wherein, the step of extracting 25ng and 10ng/ul DNA, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain an amplicon product comprises the following steps:
the mixed solution is subjected to pre-denaturation at 95 ℃ for 15min, then is subjected to 24 cycles of 95 ℃ for 30s, 60 ℃ for 90s and 72 ℃ for 90s in sequence, is operated at 72 ℃ for 10min, and is temporarily stored at 4 ℃.
Wherein, adding digestive juice and magnetic beads into the amplicon product for purification to obtain pure amplicon product, comprising:
and adding digestive juice into the amplicon product, sequentially carrying out program operation on a PCR instrument at 37 ℃ for 30min and at 80 ℃ for 10min, recovering the amplicon product by combining magnetic beads, and washing to obtain a pure amplicon product.
Adding a joint primer, a PCR mixed solution and a PCR reaction solution into the purified amplicon product to perform PCR reaction, and obtaining a pure amplicon library by using magnetic beads, wherein the method comprises the following steps:
and adding a joint P5 and a joint P7 into the purified amplicon product, then adding a PCR mixed solution and a PCR reaction solution for PCR reaction, and recovering and washing by using magnetic beads to obtain a pure amplicon library.
In a second aspect, the invention provides a MDS detection kit based on multiple PCR targeted high-throughput sequencing, which comprises all primers of an amplicon, a PCR mixed solution, a PCR reaction solution, a digestion solution, a linker P5, a linker P7, absolute ethanol, magnetic beads and nucleic-Free Water.
The invention relates to a MDS detection kit based on multiple PCR targeted high-throughput sequencing and a preparation method thereof, the kit comprises all primers of an amplicon, PCR mixed solution, PCR reaction solution, digestive juice, a joint P5, a joint P7, absolute ethyl alcohol, magnetic beads and nucleic-Free Water, after 25ng and 10ng/ul DNA is extracted by using the DNA extraction kit, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain amplicon products, then adding digestive juice and magnetic beads into the amplicon product for purification to obtain pure amplicon product, finally adding joint P5, joint P7, PCR mixed solution and PCR reaction solution into the purified amplicon product for PCR reaction, and a pure amplicon library is obtained by using the magnetic beads, so that the specificity is strong, the experimental period is short, and the multi-gene mutation screening feasibility is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of steps of a method for preparing a MDS detection kit based on multiplex PCR targeted high throughput sequencing.
FIG. 2 is a schematic diagram of the multiplex PCR targeted sequencing detection technique provided by the present invention.
FIG. 3 is a diagram showing the library detection results of the multiplex PCR detection technique provided in the present invention.
FIG. 4 is a first generation sequencing verification peak diagram of the mutation sites provided by the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Referring to fig. 1 and 2, the present invention provides a method for preparing a MDS detection kit based on multiple PCR targeted high throughput sequencing, comprising:
s101, extracting 25ng and 10ng/ul DNA, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain an amplicon product.
Specifically, a DNA extraction kit is used for extracting a sample to be detected to obtain 25ng and 10ng/ul DNA, 5ul of PCR mixed solution, 1ul of PCR reaction solution and 1ul of upstream primer downstream primers are added into the DNA, meanwhile, a PCR program is operated after the mixed solution is complemented to 10ul by using a nucleic-Free Water to obtain an amplicon product, a commercial DNA extraction kit is used for extracting the DNA from the sample to be detected, the DNA concentration is ensured to be more than 10ng/ul through quality inspection, an electrophoresis strip has no obvious tailing to ensure the integrity of the DNA, and after the PCR program is operated, the upstream 5' ends of all amplicon primers are provided with a same universal sequence T1; the 5' ends of the downstream primers are all provided with another identical universal sequence T2. The T1 sequence is typically part of the 3' end of the P5 linker sequence; the T2 sequence is part of the 3' end of the P7 linker sequence. Therefore, by the round of multiplex PCR reaction, not only can the amplicon products of a plurality of target nucleotides be obtained, but also the universal sequences T1 and T2 are respectively and successfully introduced to two sides of the amplicon products, so that preparation is provided for introducing a complete adaptor sequence for subsequent PCR reaction.
S102, adding digestive juice and magnetic beads into the amplicon product for purification to obtain a pure amplicon product.
Specifically, the obtained amplicon product is purified, and in addition to the amplicon product generated during the PCR reaction, a small amount of various impurities such as enzyme, primer, dNTP, salt ions, byproducts and the like remain. Partial amplification impurities can be removed by adding digestive juice, and a program is operated on a PCR instrument: 30min at 37 ℃ and 10min at 80 ℃. However, the digestion solution cannot completely remove the double-stranded by-product caused by amplification, and therefore, it is necessary to collect the amplicon product by binding the target fragment with magnetic beads, and wash the amplicon product to remove other components in the reaction, thereby obtaining a pure amplicon product.
S103, adding a joint primer, a PCR mixed solution and a PCR reaction solution into the purified amplicon product to perform PCR reaction, and obtaining a pure amplicon library by using magnetic beads.
Specifically, the purified amplicon product is used as a template, a linker primer required by high-throughput sequencing, namely a linker P5 and a linker P7 are added, the linker P5 is a universal linker, the linker P7 is a section of special sequence for identifying a sample, and PCR reaction is carried out by adding a PCR mixed solution and a PCR reaction solution. Linker P5 and linker P7 linker primers were complementary paired to the T1 and T2 sequences, respectively, flanking the amplicon product. Therefore, after the PCR reaction is finished, the linker sequences P5 and P7 are respectively introduced to two sides of the amplicon product, and a new amplicon library is obtained. And then, the amplicon product is recovered by combining the magnetic beads with the target fragment, other components in the reaction are washed to remove, a pure amplicon library is obtained, and the library can be subjected to quantitative determination and quality inspection to perform on-machine sequencing, so that the specificity is strong, the experimental period is short, and the multi-gene mutation screening feasibility is improved.
In a second aspect, the invention provides a MDS detection kit based on multiple PCR targeted high-throughput sequencing, which comprises all primers of an amplicon, a PCR mixed solution, a PCR reaction solution, a digestion solution, a linker P5, a linker P7, absolute ethanol, magnetic beads and nucleic-Free Water.
In this embodiment, the kit for MDS kit based on multiplex PCR targeted sequencing comprises all primers of amplicon, PCR mixture, PCR reaction solution, digestion solution, linker P5, linker P7, absolute ethanol, magnetic beads and nucleic-Free Water, wherein the PCR mixture, PCR reaction solution, digestion solution, linker P5 and linker P7 are commercial PCR kits, which are convenient to use and reduce the cost, and the kit mainly aims at the following classical transcripts of 32 genes related to myelodysplastic syndrome (MDS): ASXL (NM _), BCOR (NM _), BCORL (NM _), CALR (NM _), CBL (NM _), DDX (NM _), DNMT 3(NM _), ETV (NM _), EZH (NM _), FLT (NM), GATA (NM _), IDH (NM _), JAK (NM), KRAS (NM), MPL (NM _), NF (NM _), NPM (NM _), NRAS (NM _), PHF (NM), NM 1 (NM), RUNX (NM _), SETBP (NM _, SF3B (NM _), SRSF (NM _), STATG (NM _), TETP (WT 2 AF), STAT (WT), and TM). The designed primer Tm value is 60 +/-2 ℃, the designed primer is reasonably distributed into 4 tubes after being subjected to optimization test, the specificity of the primer and the uniformity of amplified fragments are ensured, and the proportion of non-target fragments is reduced. Based on the principle of primer design, the invention designs 32 gene exon primers related to myelodysplastic syndrome (MDS) by using related primer design software, and the corresponding primer sequences are as follows:
Figure BDA0002404209560000051
Figure BDA0002404209560000061
Figure BDA0002404209560000071
Figure BDA0002404209560000081
Figure BDA0002404209560000091
Figure BDA0002404209560000101
Figure BDA0002404209560000111
the kit provided by the invention is used for specifically detecting 32 gene mutations in MDS through 20 clinical samples.
20 cases (numbers M1-M20) of myeloid leukemia patient samples with initial diagnosis, difficult treatment and relapse are screened and detected, the flow of the detection method is strictly executed, a self-established birth communication flow is used for analysis and interpretation by professional genetic counseling unscrambler, and meanwhile, the 20 cases of leukemia patient samples are verified by generation sequencing (Sanger), wherein the library detection result by the multiplex PCR detection technology is shown in FIG. 3, the result by generation sequencing verification is shown in FIG. 4, and the specific gene loci are as follows: ASXL1(NM _015338) c.1900_1922del 23; p.e635fs 15, currently the first generation sequencing (Sanger) is also used as the gold standard for gene mutation detection, and the results of detection by the two methods are compared as follows:
summary of the results of the multiplex PCR assay for patients with accession numbers M1-M10
Figure BDA0002404209560000112
Figure BDA0002404209560000121
Summary of the results of the multiplex PCR assay for patients with accession Nos. M10 to M20 (II)
Figure BDA0002404209560000131
Figure BDA0002404209560000141
Summary of results of the patient test methods of generation No. M1-M10
Figure BDA0002404209560000142
Figure BDA0002404209560000151
Summary of the results of the patient's first generation sequencing assay methods with numbers M11-M20 (II)
Figure BDA0002404209560000152
Figure BDA0002404209560000161
The positive rates of 32 genes in 20 cases (No. M1-M20) of patients with primary diagnosis, refractory treatment and recurrent myeloid leukemia detected by multiplex PCR targeted sequencing are shown in the following table:
multiplex PCR targeted sequencing detection 32 gene positive rate table
Mutant genes Number of abrupt change Mutation ratio Mutant genes Number of abrupt change Mutation ratio
ASXL1 7 35.00％ NF1 1 5.00％
BCOR 0 0.00％ NPM1 0 0.00％
BCORL1 0 0.00％ NRAS 1 5.00％
CALR 0 0.00％ PHF6 2 10.00％
CBL 0 0.00％ PPM1D 0 0.00％
DDX41 0 0.00％ RUNX1 6 30.00％
DNMT3A 5 25.00％ SETBP1 6 30.00％
ETV6 1 5.00％ SF3B1 3 15.00％
EZH2 6 30.00％ SRSF2 5 25.00％
FLT3 0 0.00％ STAG2 0 0.00％
GATA2 0 0.00％ STAT3 0 0.00％
IDH1 4 20.00％ TET2 7 35.00％
IDH2 3 15.00％ TP53 3 15.00％
JAK2 1 5.00％ U2AF1 5 25.00％
KRAS 0 0.00％ WT1 1 5.00％
MPL 0 0.00％ ZRSR2 6 30.00％
The positive rates of 32 genes in 20 cases (Nos. M1-M20) of patients with primary diagnosis, refractory treatment and recurrent myeloid leukemia were determined by first-generation sequencing as follows:
primary sequencing detection 32 gene positive rate table
Figure BDA0002404209560000162
Figure BDA0002404209560000171
By summarizing and comparing the results of the two detection methods, the multiple PCR targeted detection technology has higher detection rate, the first generation sequencing is limited by the sensitivity (10-15%) and methodology, the gene variation condition which is lower than 10-15% is directly judged to be negative, the result of the detected sample cannot quantify the gene mutation load rate, the multiple PCR sensitivity can reach more than 1%, and the quantitative result can be given to all detected gene mutations.
The invention relates to a MDS detection kit based on multiple PCR targeted high-throughput sequencing and a preparation method thereof, the kit comprises all primers of an amplicon, PCR mixed solution, PCR reaction solution, digestive juice, a joint P5, a joint P7, absolute ethyl alcohol, magnetic beads and nucleic-Free Water, after 25ng and 10ng/ul DNA is extracted by using the DNA extraction kit, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain amplicon products, then adding digestive juice and magnetic beads into the amplicon product for purification to obtain pure amplicon product, finally adding joint P5, joint P7, PCR mixed solution and PCR reaction solution into the purified amplicon product for PCR reaction, and a pure amplicon library is obtained by using the magnetic beads, so that the specificity is strong, the experimental period is short, and the multi-gene mutation screening feasibility is improved.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A preparation method of a MDS detection kit based on multiple PCR targeted high-throughput sequencing is characterized by comprising the following steps:
extracting 25ng and 10ng/ul DNA, and carrying out PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain an amplicon product;
adding digestive juice and magnetic beads into the amplicon product for purification to obtain a pure amplicon product;
and adding a joint primer, a PCR mixed solution and a PCR reaction solution into the purified amplicon product to perform PCR reaction, and obtaining a pure amplicon library by using magnetic beads.
2. The method for preparing the MDS detection kit based on the multiple PCR targeted high throughput sequencing of claim 1, wherein 25ng and 10ng/ul DNA is extracted and subjected to PCR program operation with 5ul of PCR mixed solution and 1ul of PCR reaction solution to obtain an amplicon product, and the method comprises the following steps:
extracting a sample to be detected by using a DNA extraction kit to obtain 25ng and 10ng/ul DNA, adding 5ul of PCR mixed solution, 1ul of PCR reaction solution, 1ul of each upstream primer and downstream primer into the DNA, simultaneously complementing the mixed solution to 10ul by using a nucleic-FreeWater, and then operating a PCR program to obtain an amplicon product.
3. The method for preparing the MDS detection kit based on the multiple PCR targeted high throughput sequencing of claim 2, wherein 25ng and 10ng/ul of DNA are extracted and subjected to PCR program operation with 5ul of PCR mixture and 1ul of PCR reaction solution to obtain amplicon products, further comprising:
the mixed solution is subjected to pre-denaturation at 95 ℃ for 15min, then is subjected to 24 cycles of 95 ℃ for 30s, 60 ℃ for 90s and 72 ℃ for 90s in sequence, is operated at 72 ℃ for 10min, and is temporarily stored at 4 ℃.
4. The method for preparing the MDS detection kit based on the multiplex PCR targeted high throughput sequencing of claim 3, wherein the amplicon product is purified by adding a digestive fluid and magnetic beads, and the method comprises:
and adding digestive juice into the amplicon product, sequentially carrying out program operation on a PCR instrument at 37 ℃ for 30min and at 80 ℃ for 10min, recovering the amplicon product by combining magnetic beads, and washing to obtain a pure amplicon product.
5. The method for preparing the MDS detection kit based on the multiple PCR targeted high throughput sequencing of claim 4, wherein the step of adding a linker primer, a PCR mixture and a PCR reaction solution to the purified amplicon product to perform PCR reaction and obtaining a pure amplicon library by using magnetic beads comprises:
and adding a joint P5 and a joint P7 into the purified amplicon product, then adding a PCR mixed solution and a PCR reaction solution for PCR reaction, and recovering and washing by using magnetic beads to obtain a pure amplicon library.
6. The MDS detection kit based on the multiple PCR targeted high-throughput sequencing is characterized by comprising all primers of amplicons, PCR mixed liquor, PCR reaction liquor, digestion liquor, a joint P5, a joint P7, absolute ethyl alcohol, magnetic beads and nucleic-Free Water.
CN202010156441.6A 2020-03-09 2020-03-09 MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof Pending CN111304331A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010156441.6A CN111304331A (en) 2020-03-09 2020-03-09 MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010156441.6A CN111304331A (en) 2020-03-09 2020-03-09 MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof

Publications (1)

Publication Number Publication Date
CN111304331A true CN111304331A (en) 2020-06-19

Family

ID=71149594

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010156441.6A Pending CN111304331A (en) 2020-03-09 2020-03-09 MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof

Country Status (1)

Country Link
CN (1) CN111304331A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106521012A (en) * 2016-12-29 2017-03-22 天津协和华美医学诊断技术有限公司 Detection reagent kit for detecting MDS (myelodysplastic syndrome)-related gene group
CN106566875A (en) * 2016-09-20 2017-04-19 上海荻硕贝肯医学检验所有限公司 Primers, kit and method for detecting myelodysplastic syndromes (MDS) gene mutation
CN107868823A (en) * 2017-10-31 2018-04-03 天津协和华美医学诊断技术有限公司 A kind of detection kit of detection MDS/MPN related gene groups

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106566875A (en) * 2016-09-20 2017-04-19 上海荻硕贝肯医学检验所有限公司 Primers, kit and method for detecting myelodysplastic syndromes (MDS) gene mutation
CN106521012A (en) * 2016-12-29 2017-03-22 天津协和华美医学诊断技术有限公司 Detection reagent kit for detecting MDS (myelodysplastic syndrome)-related gene group
CN107868823A (en) * 2017-10-31 2018-04-03 天津协和华美医学诊断技术有限公司 A kind of detection kit of detection MDS/MPN related gene groups

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
宋陆茜等: "二代测序技术在骨髓增生异常综合征诊治中的临床应用" *

Similar Documents

Publication Publication Date Title
US20230220458A1 (en) Methods and systems for detecting methylation changes in dna samples
CN110628880B (en) Method for detecting gene variation by synchronously using messenger RNA and genome DNA template
CN107142320B (en) Gene marker for detecting liver cancer and application thereof
CN111154881A (en) Detection kit for gene mutation in acute myeloid leukemia and application
CN108642153B (en) High-sensitivity mutation site detection system, method and application
CN109486912A (en) A kind of probe primer combination and design method for digital pcr amplification
WO2019142193A1 (en) Kits and methods for diagnosing lung cancer
WO2014125421A1 (en) Methods and kits for identifying and adjusting for bias in sequencing of polynucleotide samples
WO2023159817A1 (en) Genetic diagnosis probes and use thereof
CN113846168B (en) Detection reagent for molecular marker of primary liver cancer and application thereof
CN111304331A (en) MDS detection kit based on multiple PCR (polymerase chain reaction) targeted high-throughput sequencing and preparation method thereof
CN113430272B (en) Reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application of reagent and kit
CN105400877B (en) Genome SNP locus detection method based on immune enzyme-linked reaction
CN117512085B (en) Primer group and kit for detecting HLA-DPB1 genotyping
CN110628899A (en) Primer pair and kit for detecting methylation of multiple genes of non-small cell lung cancer
WO2024119481A1 (en) Method for rapidly preparing multiplex pcr sequencing library and use thereof
JP7447155B2 (en) Method for detecting methylation of SDC2 gene
CN116287227A (en) Reagent and kit for diagnosing prostate cancer
CN116926167A (en) Target nucleic acid capture probe, kit, method and application in targeted capture urine sample
CN116497107A (en) Single-chain preparation primer pair based on symmetrical PCR technology, application thereof and single-chain preparation method
CN117625782A (en) Marker for early screening of lung cancer, probe composition and application thereof
CN117512085A (en) Primer group and kit for detecting HLA-DPB1 genotyping
CN117004713A (en) Biomarkers and kits for diagnosing or aiding in diagnosing gastric cancer
CN117867106A (en) Marker for cervical cancer screening, probe composition and application thereof
WO2024117970A1 (en) Method for efficient multiplex detection and quantification of genetic alterations

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination