CN111471755A - Biomarker combination for microsatellite instability detection, kit and application thereof - Google Patents
Biomarker combination for microsatellite instability detection, kit and application thereof Download PDFInfo
- Publication number
- CN111471755A CN111471755A CN202010420704.XA CN202010420704A CN111471755A CN 111471755 A CN111471755 A CN 111471755A CN 202010420704 A CN202010420704 A CN 202010420704A CN 111471755 A CN111471755 A CN 111471755A
- Authority
- CN
- China
- Prior art keywords
- oci
- primer
- artificial sequence
- microsatellite
- seq
- 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.)
- Granted
Links
- 208000032818 Microsatellite Instability Diseases 0.000 title claims abstract description 29
- 239000000090 biomarker Substances 0.000 title claims abstract description 10
- 238000001514 detection method Methods 0.000 title abstract description 27
- 239000000203 mixture Substances 0.000 claims abstract description 18
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 18
- 238000007481 next generation sequencing Methods 0.000 claims abstract description 9
- 108091092878 Microsatellite Proteins 0.000 claims description 53
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 35
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000005516 engineering process Methods 0.000 abstract description 9
- 230000004907 flux Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 77
- 230000003321 amplification Effects 0.000 description 15
- 238000003199 nucleic acid amplification method Methods 0.000 description 15
- 239000011324 bead Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 230000001186 cumulative effect Effects 0.000 description 5
- 230000007547 defect Effects 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 230000033607 mismatch repair Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000012151 immunohistochemical method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002969 morbid Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000012935 Averaging Methods 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- 101100107522 Mus musculus Slc1a5 gene Proteins 0.000 description 1
- 229910003298 Ni-Ni Inorganic materials 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007274 generation of a signal involved in cell-cell signaling Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012956 testing procedure Methods 0.000 description 1
- 238000001926 trapping method Methods 0.000 description 1
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
- C12Q1/6869—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
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- 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
- C12Q2600/156—Polymorphic or mutational markers
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 relates to a biomarker combination and discloses a primer composition, a method and a kit for microsatellite instability detection based on a next generation sequencing technology. The primer composition of the invention includes an upstream primer set and a downstream primer set, corresponding to 30 microsatellite instability sites. The method of the invention simplifies the detection operation process to a great extent and reduces the detection cost, and has the characteristics of high flux, high sensitivity and high specificity.
Description
Technical Field
The invention relates to a biomarker combination, in particular to a primer composition, a method and a kit for microsatellite instability detection based on a next generation sequencing technology.
Background
Microsatellites are single or multiple tandem repeat nucleotide sequences widely distributed in the human genome, consisting of 1-6 nucleotides, highly polymorphic, most of which are usually 15-60 repeats, usually located in the intergenic region, promoter, UTR and coding region, during normal human cell replication, with the help of the mismatch repair system, the molecular structure of the Microsatellite remains stable, avoiding genetic changes, ensuring high fidelity of DNA replication, but under the influence of certain factors, such as HNPCC (lean syndrome) or the DNA of a Microsatellite in cancer, mismatches, insertions or deletions of bases of a double-stranded molecule, causing structural changes of the Microsatellite, which is called Microsatellite Instability (MSI), the Microsatellite Instability status is classified into three, high uncertainty Microsatellite Instability (MSI-H) and low degree Microsatellite Instability (MSI-L) and Microsatellite stability (msiite), the clinical diagnosis of colorectal cancer has been recommended by clinical guidelines for detecting morbid cancer, and the clinical diagnosis of colorectal cancer has been carried out with the clinical guidelines for detecting morbid cancer using microstation L.
Currently, the commonly used MSI detection techniques mainly fall into the following two main categories: DNA mismatch repair defect detection: the related genes causing MSI, mainly DNA mismatch repair system (MMR) genes, are subjected to gene mutation detection, or the expressed protein level is detected by an immunohistochemical method. And 2, PCR detection (MSI-PCR), namely selecting specific primers, taking self normal tissues as a control, carrying out PCR or multiple fluorescence PCR amplification on the microsatellite locus in vitro, and analyzing the size of the amplified product through gel electrophoresis development or Sanger fragment size to determine the state of the MSI, wherein the product fragment has the change of mobility. In the two types of technologies commonly used at present, for the detection of the DNA mismatch repair gene defect, the traditional gene sequencing method, such as Sanger sequencing and the like, has the problems of high cost, low flux, low precision, high requirements on experimental conditions and the like, is not popularized yet in clinical application, and for an immunohistochemical method, the defects of high requirements on sample quality, complex operation, low flux, excessive result definition depending on the subjective opinion of a reader and the like exist.
The PCR method has become a common detection method at present and has been proved to be the most effective primary screening means. However, the method for detecting five micro-satellite genes by common PCR tube separation and performing gel electrophoresis on the product has the defects of complex operation, high cost and the like. However, the common PCR method has the problems of complicated operation procedure, long time consumption, low sensitivity, high uncertainty of detection results and the like; in the multiplex PCR detection, the interference relationship among different primers is very complex, the amplification product has high mixing degree, the requirements on the selection and concentration of the primers are high, the number of detectable microsatellite loci in single detection is limited, and the discrimination between MSI-H and MSS is not high.
In order to solve the problems of limited quantity of the microsatellite loci, low sensitivity, high uncertainty of detection results and the like, the invention enriches 30 microsatellite loci with stronger discrimination by using a multiplex PCR method, performs sequencing by using a next generation sequencing technology, and simultaneously evaluates the mutation states of a plurality of microsatellite loci by combining with a bioinformatics analysis means. The method has the advantages of simple operation, short time consumption, high flux, improved detection sensitivity and accuracy, and solves the problems of the conventional detection method.
Disclosure of Invention
The invention relates to a biomarker combination, and a primer composition, a method and a kit for microsatellite instability detection based on a next generation sequencing technology.
A biomarker combination comprising a combination of 30 microsatellite loci selected from the group consisting of L oci _19, L oci _483, L0 oci _162, L oci _62, L oci _127, L oci _420, L4 oci _328, L oci _231, L6 oci _262, L oci _46, L oci _481, L oci _376, L oci _9, L oci _316, L oci _387, L2 oci _245, L oci _213, L oca _402, L oci _270, L oci _140, L oci _339, L oci _260, L oci _203, L oci _486, L _94, L _ NR _ L, L _ 72, L _ OCi _ 82, L _486 _ NR _ 72.
A primer composition, which comprises an upstream primer set and a downstream primer set, wherein each primer in the upstream primer set specifically binds to the upstream of each microsatellite locus, and each primer in the downstream primer set specifically binds to the downstream of each microsatellite locus.
Preferably, each primer of the upstream primer set and/or each primer of the downstream primer set is a rhPCR primer (RNase H-Dependent PCR, see U.S. Pat. No. US12433896, publication No. US20090325169A 1). Preferably, the primer composition, the upstream primer set thereof, is selected from the group consisting of SEQ ID nos: 1-30; the downstream primer group is selected from a primer group consisting of SEQ ID No: 31-60 primers.
A kit for detecting the state of a microsatellite based on a next-generation sequencing platform comprises the primer composition.
Further, the detection of microsatellite status uses Next Generation Sequencing (NGS) technology.
Further, the microsatellite status includes an altitude microsatellite instability (MSI-H) and a microsatellite stability (MSS) type.
The application of a primer composition in preparing a kit for detecting the state of a microsatellite comprises an upstream primer group and a downstream primer group, wherein each primer of the upstream primer group and/or each primer of the downstream primer group is a rhPCR primer.
Further, the upstream primer group consists of a primer with a sequence of SEQ ID No: 1-30 of primer composition; the downstream primer group consists of a primer with a sequence of SEQ ID No: 31-60.
Use of a biomarker comprising the 30 microsatellite loci as defined above in the preparation of a kit for detecting the status of a microsatellite. Further, the microsatellite status includes an altitude microsatellite instability (MSI-H) and a microsatellite stability (MSS) type.
The invention uses the rhPCR primer and the amplification method, avoids the interference between different primers, can simultaneously detect more microsatellite loci in single detection, improves the discrimination of MSI-H and MSS, and has simple operation, short time consumption and high flux.
The invention uses the amplification method to enrich the microsatellite locus, compared with the capture method, the amplification method has no obvious preference to the length of the microsatellite, when the length of the microsatellite locus is increased, effective signal enrichment is still kept, the MSI-H and MSS can be distinguished more conveniently, and the sensitivity is high.
The combination of 30 microsatellite loci is used for detecting the state of the microsatellite, and has high accuracy and sensitivity, high discrimination of MSI-H and MSS and good detection stability.
Drawings
FIG. 1 is a graph of correlation of length-depth of microsatellite loci from capture to the present invention
FIG. 2. verification of the reliability results of the present invention by PCR gold Standard
FIG. 3 detection sensitivity of the present invention
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
The invention uses an amplicon multiplex PCR method to enrich 30 MSI loci with stronger discrimination, uses the next generation sequencing technology to carry out sequencing, and combines the bioinformatics analysis means to carry out the judgment of the microsatellite state.
Example 1 preference of Capture and amplification methods for microsatellite site Length
Optionally, the same microsatellite loci are respectively subjected to capture method and amplicon method for library construction, and second-generation sequencing analysis is carried out.
The results are shown in FIG. 1: in the capture method, the capture depth is gradually reduced along with the increase of the length of the microsatellite locus, and the capture depth is more seriously reduced after the length reaches 24 bp; the amplification method can effectively avoid the defect of the capture method relatively, and in the aspect of signal enrichment of the microsatellite loci, the longer the microsatellite loci are better in the aspect of signal enrichment, so that the MSI-H and the MSS can be distinguished more conveniently. This is consistent with the length of 5 microsatellite loci (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) in the gold standard PCR method being >20bp, so that the amplification method has stronger discrimination ability in the aspect of microsatellite instability detection.
Example 2 selection of microsatellite loci
Screening microsatellite loci with better performance in an intron (intron) area from WES sequencing data, screening loci with better performance in the whole exon range from MOSAIC paper, wherein the number of candidate loci is 108, and finally screening optimal 30 loci for calculating the msi-value by performing performance evaluation (the evaluation method is shown in the following model establishment) on each locus.
Establishing a model:
(1) extracting a repeat length distribution of each site (i);
(2) converting the repeat length distribution condition into proportional distribution;
(3) calculating the cumulative distribution of the repeat length of the position (i) according to the proportional distribution in the step (2);
(4) and (3) respectively averaging the cumulative distribution of MSI-H and MSS samples in the training set aiming at each site (i), and then finding repeat length (the length is defined as cutpoint) when the difference of the two types of sample cumulative distribution average values is maximum. Classifying according to the size relationship between repeat length of reads and cutpoint, wherein MSI reads are defined as being smaller than or equal to cutpoint, MSS reads are defined as being larger than cutpoint, and the average value of the cumulative probability of MSS samples corresponding to cutpoint is pi(defined as baseline probability);
(5) based on the training model, we assume that site (i) has NiThe stripe probability of the site is pi, and the cutpoint is Ci. Finding out n MSI reads number of the site according to the cutpointiAnd the MSS reads number is Ni-Ni. We find the probability of this site to be P by the following formula.
If the cumulative probability P (X ≧ n) of the sitei) Is significantly less than 0.001, we believe that this point supports MSI-H. Based on the discriminative power of the loci in the training sample, the locus sensitivities are ranked, and the optimal 30 loci (as shown in table 1) are selected.
(6) Finally, 30 sites are integrated to calculate MSI-value, namely the ratio of the number of the sites supporting MSI-H to the total number of the sites; and obtaining cutoff according to MSI-value distribution of samples in the training set, wherein the cutoff is set to be 0.4, MSI-H is higher than 0.4, and MSS is lower than 0.4.
The microsatellite loci selected based on the above model are shown in Table 1 below, wherein the mutation type refers to the reference sequence of the locus.
TABLE 1 microsatellite locus information
Example 3 reliability verification
For the 30 microsatellite loci shown in Table 1, the upstream primer sequences and the downstream primer sequences used were designed as shown in tables 2 and 3, respectively. All primers used were blocking-cleavable rhPCR primers (from Integrated DNA Technologies, IDT) with rA, rG, rC, rU in the sequence each representing one RNA base, 3SpC3 attached to the 3 'end of each sequence as a means for 3' end nucleic acid blocking (Blocker).
TABLE 2 sequence information Table of upstream primers
| Position of | Type of mutation | Numbering | Upstream primer sequence 5'-3' |
| Loci_19 | (T)11 | SEQ ID No.1 | CTATACCTTTCGAATTTCCTCCTACAArGTGTTG |
| Loci_483 | (A)11 | SEQ ID No.2 | GTGTGTGATCATTCGTTCCTAGrGGAAAT |
| Loci_162 | (A)11 | SEQ ID No.3 | CCTCTGGGTGAACTTCATCTTTTrATTTTA |
| Loci_62 | (A)12 | SEQ ID No.4 | TCCACACTTACCCTGATACATrCACAGC |
| Loci_127 | (A)12 | SEQ ID No.5 | CAAGGAAATTTTCACTTCACGGGrAAAAAT |
| Loci_420 | (T)12 | SEQ ID No.6 | TGCTCTACAGGGTCAATATTGAAAAArGAACTA |
| Loci_328 | (A)12 | SEQ ID No.7 | GCCCAATTCACTTTGACTTCCTArAAAAAT |
| Loci_231 | (A)12 | SEQ ID No.8 | AAAGAGGCATTTACATTCCAGArCTTGGT |
| Loci_262 | (A)12 | SEQ ID No.9 | CAGACCCCTGATATCTGTGGrAAAAAT |
| Loci_46 | (A)12 | SEQ ID No.10 | AGATTGGCACTGATAAACCTTGArGGTGTC |
| Loci_481 | (T)13 | SEQ ID No.11 | ACATATTTTGGAATGAAGCAATTTCAGrCACAGC |
| Loci_376 | (A)13 | SEQ ID No.12 | CTCTGCACTCTTCTACTGAGGrCTTTNT |
| Loci_9 | (A)14 | SEQ ID No.13 | GTCTCAGGATCTGGCArGAGCCG |
| Loci_316 | (T)14 | SEQ ID No.14 | CATAACAAATCCCTCAAGCCTAGrCATACT |
| Loci_387 | (T)14 | SEQ ID No.15 | AGCTCTCTGTTCCCACArCAGAGT |
| Loci_245 | (A)14 | SEQ ID No.16 | ATGGATTTCATGTTGAAGAATAGTTrUCTGCT |
| Loci_213 | (A)15 | SEQ ID No.17 | CTTAATCCTCGACCACATTTACCrGACAAC |
| Loci_402 | (T)15 | SEQ ID No.18 | GCCAGGAGCTATTAATATTTGAGTCrUAAGGT |
| Loci_270 | (A)15 | SEQ ID No.19 | TAGATAAATACTGAGTACCTGTCTGrGGCTGT |
| Loci_140 | (A)15 | SEQ ID No.20 | TCATTGACTCATTCCCTTATTTTCATArCCAAGG |
| Loci_339 | (A)15 | SEQ ID No.21 | TTTCTCTCTTTCAGGGTGATCTTrGGAAAT |
| Loci_348 | (T)15 | SEQ ID No.22 | CTGCAGCTTGAATGAGAATATCArCTGTCG |
| Loci_260 | (A)15 | SEQ ID No.23 | GACATCAAATCCAAGCTTTTGAAGrGTAGGG |
| Loci_203 | (T)16 | SEQ ID No.24 | AAGGACTTCAGGTAAGCTCArGGAGCA |
| Loci_83 | (A)16 | SEQ ID No.25 | AAGTATATGTGTGTTAGAGATTCAAGTAArAAAAAT |
| Loci_94 | (T)17 | SEQ ID No.26 | AGACTCCAGATAAATCAAAAGTAGTTTCTrCAAGGA |
| Loci_486 | (T)20 | SEQ ID No.27 | GTGTTTTCTCTATTTTATGTGGAGAGTrUTTTCT |
| Loci_430 | (A)22 | SEQ ID No.28 | GTAACTCTTTCTACTGGCACTAAGrCGATTC |
| NR24 | (T)23 | SEQ ID No.29 | CCTGGGCCCAGTCCTATrUTTTTA |
| NR27 | (A)26 | SEQ ID No.30 | AAGTCTGCAGTTGAAAAGCCrCAACGA |
TABLE 3 sequence information of downstream primers
| Position of | Type of mutation | Numbering | Downstream primer sequence 5'-3' |
| Loci_19 | (T)11 | SEQ ID No.31 | CTTTTAAACTTTTGTGAATGAGAGGGArAAAAAT |
| Loci_483 | (A)11 | SEQ ID No.32 | AGTACTCGTGGAAGATGTTTGrCTTGGT |
| Loci_162 | (A)11 | SEQ ID No.33 | GTCTCTGCCTCTTTTATTGGGArCAGTTA |
| Loci_62 | (A)12 | SEQ ID No.34 | GTTTCTGATTGCACATTTTGGGrGCTTTA |
| Loci_127 | (A)12 | SEQ ID No.35 | AATTTGGATCCCGTACCACTTAGArCATCTT |
| Loci_420 | (T)12 | SEQ ID No.36 | AGCTTCTGTATTTTCCCCTGAGTAArGACACC |
| Loci_328 | (A)12 | SEQ ID No.37 | GGTATCTCCTTCAGTGTTTCATCTrUAAACT |
| Loci_231 | (A)12 | SEQ ID No.38 | GTCAGACTGACTTGGAAGTATCArCTCATA |
| Loci_262 | (A)12 | SEQ ID No.39 | GGAGCTGGTGTTTCTTCCArCATTAC |
| Loci_46 | (A)12 | SEQ ID No.40 | GTGAAAATGATCTTTGTTGGGTTTTrUCCTTA |
| Loci_481 | (T)13 | SEQ ID No.41 | CGTAGAATTTGTTCTGAAATTCTACCCTrAAAAAT |
| Loci_376 | (A)13 | SEQ ID No.42 | AGATCACTATGTTGTGCACCArGGATCA |
| Loci_9 | (A)14 | SEQ ID No.43 | TTCCCCTATTACTTGACAGTrGTTGTA |
| Loci_316 | (T)14 | SEQ ID No.44 | GAAGCATTTGGTGTTTTGGTAGAAATrCTTTAT |
| Loci_387 | (T)14 | SEQ ID No.45 | GAGGCTTAGCTGGAATAACArCCANAT |
| Loci_245 | (A)14 | SEQ ID No.46 | TTTTTGGGATCTACAAGGTAACTTrUTTTTA |
| Loci_213 | (A)15 | SEQ ID No.47 | GCCATTCTGAACATGCTTAATAAAAAGrATGCTA |
| Loci_402 | (T)15 | SEQ ID No.48 | TAGAACATTAGCCCCAGATCGAArCCTGAT |
| Loci_270 | (A)15 | SEQ ID No.49 | CCATGGTCTACAAATCATGTATTTTArACTTTA |
| Loci_140 | (A)15 | SEQ ID No.50 | TTTTTCTGTATAGCTCATTTGAAAGATTTrUTTTTA |
| Loci_339 | (A)15 | SEQ ID No.51 | GAGATTGCCATAACTTGCTTTAAArCAATTA |
| Loci_348 | (T)15 | SEQ ID No.52 | TCGCAAATGAGATTCTCCAAAAGArAAGGAT |
| Loci_260 | (A)15 | SEQ ID No.53 | CATGCTTGTGACTTATTCATGGTTTrCCTTTA |
| Loci_203 | (T)16 | SEQ ID No.54 | CAACAGCTATTTCCTTTAAGTGGGrGNAAAT |
| Loci_83 | (A)16 | SEQ ID No.55 | CTGATTTCTGTGGCTTGTTTAGAGrCTATGC |
| Loci_94 | (T)17 | SEQ ID No.56 | CCGTTCCCAGCTAAATCAGAAAAArAAAAAT |
| Loci_486 | (T)20 | SEQ ID No.57 | GCAATACCAATCTTTCAGAGCTGrUGTTNT |
| Loci_430 | (A)22 | SEQ ID No.58 | CTTCTTTACTGGGTTTCTATTTCAAAArUCTTTA |
| NR24 | (T)23 | SEQ ID No.59 | ATTGTGCCATTGCATTCCAArCCTGGC |
| NR27 | (A)26 | SEQ ID No.60 | TGACCAATAAGCAAGTCACTGrUGGCTA |
That is, in the primer composition of the present embodiment, the upstream primer set is selected from the group consisting of the primers having the sequences of SEQ ID Nos: 1-30; the downstream primer group is selected from a primer group consisting of SEQ ID No: 31-60 primers.
30 MSI-H and 60 MSS clinical FFPE DNA were selected as experimental samples.
The detection steps are as follows:
1) preparation of experimental samples: a ReliaPrep FFPE gDNA Miniprep System (promega, Cat. NO: A2352) kit was used to extract DNA from paraffin-embedded specimens (formalin-embedded, FFPE).
2) Target region amplification 20u L reaction systems were prepared as follows (MSI _ FWD and MSI _ REV refer to forward and reverse primer mixtures, respectively, i.e., primer sets of tables 2 and 3, both from IDT corporation):
TABLE 4 target region amplification reaction System
Volume of DNA X ul: the amount of DNA put into the library is 50ng, the volume of the DNA put into the library is determined as the ratio of the total amount of the DNA to the concentration of the DNA according to the concentration of the extracted DNA, and X is used for expressing the input volume of different samples.
A first round of amplification against the target region was performed using a ProFlex PCR System PCR instrument (Applied biosystems by life technologies, model: ProFlex) following the reaction procedure;
TABLE 5 target region amplification procedure
3) Purification of the target amplification product: the Agencour AMPure XP beads are selected and used for purifying a target product by using 80% ethanol (which is prepared for use).
The specific purification steps are as follows:
a. adding 30 μ l of magnetic beads (1.5x magnetic beads) into the PCR tube containing the reaction solution, shaking and mixing uniformly, centrifuging for a short time, and incubating at room temperature for 10 min;
b. standing on a magnetic frame until the liquid is clear, and removing the supernatant by using a pipette (taking care not to touch the magnetic beads and keeping the PCR tube on the magnetic frame all the time);
c. adding 200 mul of 80% ethanol into the PCR tube, standing for 30s, and removing the supernatant;
d. repeating c;
e. sucking liquid in the PCR tube as clean as possible by using a 10-microliter gun head, and standing at room temperature until the magnetic beads are dried;
f. adding 22 mul of nuclease-free water, taking down the PCR tube from the magnetic frame, shaking and mixing uniformly, and centrifuging for a short time;
g. incubating at room temperature for 5min, placing the PCR tube on a magnetic frame, standing until the liquid is clear, sucking 20 μ l of supernatant, and adding into a new PCR tube;
h. adding 30 mul of magnetic beads into the PCR tube filled with the supernatant, shaking and uniformly mixing, centrifuging for a short time, and incubating for 10min at room temperature;
i. standing on a magnetic frame until the liquid is clear, and removing the supernatant by using a pipette (taking care not to touch the magnetic beads and keeping the PCR tube on the magnetic frame all the time);
g. adding 200 mul of 80% ethanol into the PCR tube, standing for 30s, and removing the supernatant;
k. repeating g;
l, sucking liquid in the PCR tube as clean as possible by using a 10-microliter gun head, and standing at room temperature until the magnetic beads are dried;
m. adding 20ul of reaction liquid prepared according to the table 5 into the dried magnetic beads, shaking and uniformly mixing, incubating at room temperature for 3min, and then placing on a PCR instrument for PCR reaction.
4) Amplification of target product (Index PCR) reaction system was prepared according to table 6:
TABLE 6 target product amplification reaction System
Amplification was performed according to the following reaction procedure;
5) purification of the target library: the method comprises the steps of selecting Agencour AMPure XP beads, purifying a target product by using 80% ethanol (which is used for preparation now), and carrying out quantitative quality inspection on a purified library.
The specific purification steps are as follows:
a. adding 20 mul of magnetic beads (1.0x magnetic beads) into the PCR tube containing the reaction solution, shaking and uniformly mixing, centrifuging for a short time, and incubating at room temperature for 10 min;
b. standing on a magnetic frame until the liquid is clear, and removing the supernatant by using a pipette (taking care not to touch the magnetic beads and keeping the PCR tube on the magnetic frame all the time);
c. adding 200 mul of 80% ethanol into the PCR tube, standing for 30s, and removing the supernatant;
d. repeating c;
e. sucking liquid in the PCR tube as clean as possible by using a 10-microliter gun head, and standing at room temperature until the magnetic beads are dried;
f. adding 22 mul of nuclease-free water, taking down the PCR tube from the magnetic frame, shaking and mixing uniformly, and centrifuging for a short time;
g. and (3) incubating at room temperature for 5min, placing the PCR tube on a magnetic frame, standing until the liquid is clear, sucking 20 mu l of supernatant, and adding the supernatant into a new 1.5ml EP tube to obtain the library.
h. The constructed library was subjected to Qubit quantification and lab chip fragment analysis.
6) The library is subjected to on-machine sequencing and subjected to signal generation process analysis.
Msi-values were calculated according to the model of example 2 to determine microsatellite status of clinical samples.
The experimental results are as follows:
the results of the tests were performed on 30 MSI-H and 60 MSS clinical FFPE DNA samples and compared to the results using PCR gold standard assays.
The results are shown in FIG. 2, with a cutoff value of 0.4, MSS below 0.4, and MSI-H above 0.4; the detection results of 90 samples tested are all consistent with the gold standard, and the consistency rate is 100%.
Example 4 sensitivity comparison
The MSI samples were tested using the method of the present invention using extracted DNAs from the MSI-H cell line (SW48, Co115, HCT-8, L S180, AN3CA) and MSS cell line (HT55) mixed, assuming that the MSI-H cell line has a tumor content of 100% and the MSS cell line has a tumor content of 0% against the MSS cell line DNA, and the MSI-H cell line DNA was diluted to simulate a tumor content of 0.1%, 1%, 5%, 10%, respectively, and the testing procedure was as described in example 2 and example 3.
The comparative example used a conventional trapping method.
As a result, as shown in FIG. 3, the method L OD was detectable at 5% or less, whereas the conventional capture method L OD, which is a prior art method, was only detectable at 10%, and therefore, the sensitivity of the present invention was significantly advantageous.
Sequence listing
<110> Shanghai thinking Dibiomedical science and technology Co., Ltd
<120> biomarker combinations for microsatellite instability detection, kits and uses thereof
<130>LT2003002F
<160>60
<170>SIPOSequenceListing 1.0
<210>1
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
ctataccttt cgaatttcct cctacaagtg ttg 33
<210>2
<211>28
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gtgtgtgatc attcgttcct agggaaat 28
<210>3
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
cctctgggtg aacttcatct tttatttta 29
<210>4
<211>27
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tccacactta ccctgataca tcacagc 27
<210>5
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
caaggaaatt ttcacttcac gggaaaaat 29
<210>6
<211>32
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
tgctctacag ggtcaatatt gaaaaagaac ta 32
<210>7
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gcccaattca ctttgacttc ctaaaaaat 29
<210>8
<211>28
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
aaagaggcat ttacattcca gacttggt 28
<210>9
<211>26
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
cagacccctg atatctgtgg aaaaat 26
<210>10
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
agattggcac tgataaacct tgaggtgtc 29
<210>11
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
acatattttg gaatgaagca atttcagcac agc 33
<210>12
<211>27
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
ctctgcactc ttctactgag gctttnt 27
<210>13
<211>22
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
gtctcaggat ctggcagagc cg 22
<210>14
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
cataacaaat ccctcaagcc tagcatact 29
<210>15
<211>23
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
agctctctgt tcccacacag agt 23
<210>16
<211>32
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
tgctctacag ggtcaatatt gaaaaagaac ta 32
<210>17
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
cttaatcctc gaccacattt accgacaac 29
<210>18
<211>31
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gccaggagct attaatattt gagtcuaagg t 31
<210>19
<211>31
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
tagataaata ctgagtacct gtctgggctg t 31
<210>20
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
tcattgactc attcccttat tttcatacca agg 33
<210>21
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
tttctctctt tcagggtgat cttggaaat 29
<210>22
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
ctgcagcttg aatgagaata tcactgtcg 29
<210>23
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gacatcaaat ccaagctttt gaaggtaggg 30
<210>24
<211>26
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
aaggacttca ggtaagctca ggagca 26
<210>25
<211>35
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
aagtatatgt gtgttagaga ttcaagtaaa aaaat 35
<210>26
<211>35
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
agactccaga taaatcaaaa gtagtttctc aagga 35
<210>27
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
gtgttttctc tattttatgt ggagagtutt tct 33
<210>28
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
gtaactcttt ctactggcac taagcgattc 30
<210>29
<211>23
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
cctgggccca gtcctatutt tta 23
<210>30
<211>26
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
aagtctgcag ttgaaaagcc caacga 26
<210>31
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
cttttaaact tttgtgaatg agagggaaaa aat 33
<210>32
<211>27
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
agtactcgtg gaagatgttt gcttggt 27
<210>33
<211>28
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gtctctgcct cttttattgg gacagtta 28
<210>34
<211>28
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
gtttctgatt gcacattttg gggcttta 28
<210>35
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
aatttggatc ccgtaccact tagacatctt 30
<210>36
<211>31
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
agcttctgta ttttcccctg agtaagacac c 31
<210>37
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
ggtatctcct tcagtgtttc atctuaaact 30
<210>38
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
gtcagactga cttggaagta tcactcata 29
<210>39
<211>25
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
ggagctggtg tttcttccac attac 25
<210>40
<211>31
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
gtgaaaatga tctttgttgg gttttucctt a 31
<210>41
<211>34
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
cgtagaattt gttctgaaat tctaccctaa aaat 34
<210>42
<211>27
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
agatcactat gttgtgcacc aggatca 27
<210>43
<211>26
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
ttcccctatt acttgacagt gttgta 26
<210>44
<211>32
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
gaagcatttg gtgttttggt agaaatcttt at 32
<210>45
<211>26
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gaggcttagc tggaataaca ccanat 26
<210>46
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
tttttgggat ctacaaggta acttutttta 30
<210>47
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
gccattctga acatgcttaa taaaaagatg cta 33
<210>48
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
tagaacatta gccccagatc gaacctgat 29
<210>49
<211>32
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
ccatggtcta caaatcatgt attttaactt ta 32
<210>50
<211>35
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
tttttctgta tagctcattt gaaagatttu tttta 35
<210>51
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gagattgcca taacttgctt taaacaatta 30
<210>52
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
tcgcaaatga gattctccaa aagaaaggat 30
<210>53
<211>31
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
catgcttgtg acttattcat ggtttccttt a 31
<210>54
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
caacagctat ttcctttaag tggggnaaat 30
<210>55
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
ctgatttctg tggcttgttt agagctatgc 30
<210>56
<211>30
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
ccgttcccag ctaaatcaga aaaaaaaaat 30
<210>57
<211>29
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
gcaataccaa tctttcagag ctgugttnt 29
<210>58
<211>33
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
cttctttact gggtttctat ttcaaaauct tta 33
<210>59
<211>26
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
attgtgccat tgcattccaa cctggc 26
<210>60
<211>27
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
tgaccaataa gcaagtcact guggcta 27
Claims (10)
1. A biomarker combination comprising a combination of 30 microsatellite loci selected from the group consisting of L oci _19, L oci _483, L oci _162, L oci _62, L2 oci _127, L oci _420, L4 oci _328, L oci _231, L6 oci _262, L oci _46, L oci _481, L oci _376, L oci _9, L oci _316, L1 oci _387, L oci _245, L3 oci _213, L oci _402, L oci _270, L oci _140, L oci _339, L oci _260, L oci _203, L oci _486, L oci _83, L oci _ L, L _ NR _ L, L _ NR _ 72.
2. A primer composition comprising an upstream primer set and a downstream primer set, wherein each primer in the upstream primer set specifically binds upstream of the microsatellite site of claim 1 and each primer in the downstream primer set specifically binds downstream of the microsatellite site of claim 1.
3. The primer composition of claim 2, wherein each primer of the set of forward primers and/or each primer of the set of reverse primers is a rhPCR primer.
4. The primer composition of claim 3, wherein the set of forward primers consists of a primer having the sequence of SEQ ID No: 1-30 of primer composition; the downstream primer group consists of a primer with a sequence of SEQ ID No: 31-60.
5. A kit for detecting the state of a microsatellite based on a next generation sequencing platform, which comprises the primer composition according to any one of claims 2 to 4.
6. The kit of claim 5, wherein the microsatellite status includes a high microsatellite instability (MSI-H) and microsatellite stability (MSS) type.
7. The application of the primer composition in the preparation of the kit for detecting the microsatellite status comprises an upstream primer group and a downstream primer group, wherein each primer of the upstream primer group and/or each primer of the downstream primer group is a rhPCR primer.
8. The use of claim 7, wherein the set of forward primers consists of a primer having the sequence of SEQ ID No: 1-30 of primer composition; the downstream primer group consists of a primer with a sequence of SEQ ID No: 31-60.
9. Use of a biomarker combination according to claim 1 in the manufacture of a kit for detecting microsatellite status.
10. The application of claim 9, wherein the microsatellite status includes an altitude microsatellite instability (MSI-H) and a microsatellite stability (MSS) type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010420704.XA CN111471755B (en) | 2020-05-18 | 2020-05-18 | Biomarker combination for microsatellite instability detection, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010420704.XA CN111471755B (en) | 2020-05-18 | 2020-05-18 | Biomarker combination for microsatellite instability detection, kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111471755A true CN111471755A (en) | 2020-07-31 |
| CN111471755B CN111471755B (en) | 2023-11-24 |
Family
ID=71763215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010420704.XA Active CN111471755B (en) | 2020-05-18 | 2020-05-18 | Biomarker combination for microsatellite instability detection, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111471755B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708679A (en) * | 2021-01-27 | 2021-04-27 | 厦门艾德生物医药科技股份有限公司 | Biomarker group for detecting microsatellite instability of population and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305508A (en) * | 2013-06-03 | 2013-09-18 | 中国水产科学研究院黄海水产研究所 | Marbled flounder microsatellite locus and primer |
| CN106701978A (en) * | 2017-01-24 | 2017-05-24 | 厦门艾德生物医药科技股份有限公司 | Human microsatellite instability (MSI) detection amplification primer composition and kit |
| CN107513565A (en) * | 2017-09-06 | 2017-12-26 | 南京世和基因生物技术有限公司 | A kind of microsatellite instability Sites Combination, detection kit and its application |
| CN108949757A (en) * | 2018-08-21 | 2018-12-07 | 元码基因科技(苏州)有限公司 | Primer composition, kit and method based on two generation microarray datasets detection microsatellite instability |
| CN110760599A (en) * | 2019-12-16 | 2020-02-07 | 吉林省水产科学研究院 | Cannabis harfish microsatellite molecular marker locus, polymorphism primer and application |
-
2020
- 2020-05-18 CN CN202010420704.XA patent/CN111471755B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305508A (en) * | 2013-06-03 | 2013-09-18 | 中国水产科学研究院黄海水产研究所 | Marbled flounder microsatellite locus and primer |
| CN106701978A (en) * | 2017-01-24 | 2017-05-24 | 厦门艾德生物医药科技股份有限公司 | Human microsatellite instability (MSI) detection amplification primer composition and kit |
| CN107513565A (en) * | 2017-09-06 | 2017-12-26 | 南京世和基因生物技术有限公司 | A kind of microsatellite instability Sites Combination, detection kit and its application |
| CN108949757A (en) * | 2018-08-21 | 2018-12-07 | 元码基因科技(苏州)有限公司 | Primer composition, kit and method based on two generation microarray datasets detection microsatellite instability |
| CN110760599A (en) * | 2019-12-16 | 2020-02-07 | 吉林省水产科学研究院 | Cannabis harfish microsatellite molecular marker locus, polymorphism primer and application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708679A (en) * | 2021-01-27 | 2021-04-27 | 厦门艾德生物医药科技股份有限公司 | Biomarker group for detecting microsatellite instability of population and application thereof |
| WO2022161272A1 (en) * | 2021-01-27 | 2022-08-04 | 厦门艾德生物医药科技股份有限公司 | Biomarker group for detecting population microsatellite instability, and application of biomarker group |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111471755B (en) | 2023-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108103055A (en) | A kind of method of unicellular RNA reverse transcriptions and library construction | |
| CN107699957B (en) | DNA-based fusion gene quantitative sequencing library construction, detection method and application thereof | |
| CN107893100A (en) | A kind of unicellular mRNA reverse transcriptions and the method for amplification | |
| CN113293204B (en) | Primer composition, kit and method for detecting microsatellite instability based on second-generation sequencing platform | |
| CN111690748B (en) | Probe set for detecting microsatellite instability by using high-throughput sequencing, kit and microsatellite instability detection method | |
| CN104894233A (en) | Multi-sample and multi-segment DNA methylation high-throughput sequencing method | |
| CN110791556A (en) | Primer group, detection method and kit for detecting MET gene exon14 jump mutation | |
| CN110846408A (en) | Primer combination for detecting TTN gene mutation and application thereof | |
| CN111471755B (en) | Biomarker combination for microsatellite instability detection, kit and application thereof | |
| CN110564861A (en) | Fluorescence labeling composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| CN112342289B (en) | Primer group for enriching thalassemia genes by long-fragment PCR and application thereof | |
| CN110628882B (en) | Primer and kit for PCR amplification, method for detecting MSI state and application | |
| WO2017185758A1 (en) | Primer, probe, kit, and method for microchimerism assay and individual recognition | |
| CN116463408A (en) | ABO gene amplification primer, amplification system, amplification method, sequencing library construction method and sequencing method | |
| CN108531598B (en) | ROS1 gene fusion detection primer, method and kit | |
| CN116497087A (en) | Method for selectively amplifying target sequence and application thereof | |
| CN114277114B (en) | Method for adding unique identifier in amplicon sequencing and application | |
| CN104152568B (en) | High-throughput STR sequence core repeat number detection method | |
| CN108642190B (en) | Forensic medicine composite detection kit based on 14 autosomal SNP genetic markers | |
| CN107904297B (en) | Primer group, joint group and sequencing method for microbial diversity research | |
| CN113637799B (en) | RPA detection method for chrysanthemum B virus | |
| CN117512085B (en) | Primer group and kit for detecting HLA-DPB1 genotyping | |
| CN117265091B (en) | Primer group, kit and application for HLA-DRB3/4/5 genotyping | |
| CN114480615B (en) | Primer group and kit for detecting HLA-B5101 alleles | |
| CN116445478B (en) | Primer combination for constructing IGHV gene library and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |