CN111471755B - Biomarker combination for microsatellite instability detection, kit and application thereof - Google Patents

Biomarker combination for microsatellite instability detection, kit and application thereof Download PDF

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CN111471755B
CN111471755B CN202010420704.XA CN202010420704A CN111471755B CN 111471755 B CN111471755 B CN 111471755B CN 202010420704 A CN202010420704 A CN 202010420704A CN 111471755 B CN111471755 B CN 111471755B
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李夏静
王磊
许青
陈才夫
熊磊
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Shanghai 3D Medicines Co Ltd
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Abstract

The invention relates to biomarker combinations and discloses a primer composition, a method and a kit for detecting microsatellite instability based on a next generation sequencing technology. The primer composition of the present 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

Biomarker combination for microsatellite instability detection, kit and application thereof
Technical Field
The invention relates to a biomarker combination, and discloses a primer composition, a method and a kit for detecting microsatellite instability based on a next generation sequencing technology.
Background
Microsatellites are single or multiple tandem repeat nucleotide sequences widely distributed in the human genome. Consists of 1-6 nucleotides, is highly polymorphic, and most tandem repeat nucleotide sequences are typically 15-60 repeats, typically located in the gene spacer, promoter, UTR and coding region. In the normal human cell replication process, the molecular structure of the microsatellite is kept stable and unchanged with the help of a mismatch repair system, so that genetic materials are prevented from being changed, and the high fidelity of DNA replication is ensured. However, under the action of some factors, such as HNPCC (lindgy syndrome) or microsatellite DNA in cancer, the double-stranded molecule base is mismatched, inserted or deleted during replication, and the structure of the microsatellite is changed, and the microsatellite with changed structure is called microsatellite instability (Microsatellite Instability, MSI). Microsatellite instability is divided into three categories: high-uncertainty microsatellite instability (MSI-H) and low-uncertainty microsatellite instability (MSI-L) and microsatellite stability (Microsatellite stability, MSS). Clinical practice guidelines for colorectal cancer and rectal cancer issued by NCCN recommend MSI testing for all patients with a history of colorectal and rectal cancer to guide clinical medication and immunotherapy, diagnosis of the Lynch syndrome (Lynch syndrome), and thus MSI testing has become a routine clinical test.
The MSI detection technology commonly used at present mainly comprises the following two main types: DNA mismatch repair defect detection: gene mutation detection is performed on the genes involved in MSI, mainly the DNA mismatch repair system (MMR) gene, or the protein level expressed by the gene is detected by immunohistochemical method. PCR detection (MSI-PCR) by selecting specific primer and using its normal tissue as reference, in vitro PCR or multiplex fluorescence PCR amplification of microsatellite locus, gel electrophoresis imaging or Sanger fragment size analysis of amplified product to obtain the final product with mobility change. In the two types of technologies commonly used at present, the traditional gene sequencing method, such as Sanger sequencing, has the problems of high cost, low flux, low precision, high requirements on experimental conditions and the like, is not popular in clinical application, and has the defects of high requirements on sample quality, complex operation, low flux, and excessive dependence on the main opinion of a reader in the definition of results for the immunohistochemical method.
PCR has become the most effective primary screening method and has proven to be the most effective method of detection. However, the common PCR tube-sorting detection method for detecting five microsatellite genes and the gel electrophoresis method for the products have 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 between different primers is very complex, the hybridization degree of amplified products is higher, the requirements on the selection and concentration of the primers are higher, the number of the microsatellite loci which can be detected in single detection is limited, and the discrimination between MSI-H and MSS is not high.
In order to solve the problems of limited number of microsatellite loci, low sensitivity, high uncertainty of detection results and the like, the invention uses a multiplex PCR method to enrich 30 microsatellite loci with stronger differentiation, uses a next generation sequencing technology to sequence, combines a bioinformatics analysis means, and simultaneously evaluates mutation states of a plurality of microsatellite loci. The method is simple to operate, short in time consumption, high in flux, improved in detection sensitivity and accuracy, and solves the problem 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 detecting microsatellite instability based on a next generation sequencing technology.
A biomarker combination comprising a combination of 30 microsatellite loci as described below: loci_19, loci_483, loci_162, loci_62, loci_127, loci_420, loci_328, loci_231, loci_262, loci_46, loci_481, loci_376, loci_9, loci_316, loci_387, loci_245, loci_213, loci_402, loci_270, loci_140, loci_339, loci_348, loci_260, loci_203, loci_83, loci_94, loci_486, loci_430, NR24, NR27.
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 each microsatellite locus described above and each primer in the downstream primer set specifically binds downstream of each microsatellite locus described above.
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. 3,379, 20090325169A 1). Preferably, the primer composition, the upstream primer set thereof is selected from the group consisting of the sequences of SEQ ID nos: 1-30; the downstream primer group is selected from the group consisting of a primer sequence of SEQ ID No: 31-60.
A kit for detecting microsatellite status based on a second generation sequencing platform, the kit comprising the primer composition described above.
Further, detection of microsatellite status uses a second generation sequencing (Next Generation Sequencing, NGS) technique.
Further, the microsatellite status includes a high microsatellite instability (MSI-H) and a microsatellite stabilization (MSS) type.
The application of a primer composition in preparing a kit for detecting microsatellite states comprises an upstream primer set and a downstream primer set, wherein each primer of the upstream primer set and/or each primer of the downstream primer set is an rhPCR primer.
Further, the upstream primer group consists of a primer sequence shown in SEQ ID No: 1-30; the downstream primer group consists of a primer sequence shown as SEQ ID No: 31-60.
Use of a biomarker comprising 30 microsatellite loci as described above in the manufacture of a kit for detecting microsatellite status. Further, the microsatellite status includes a high microsatellite instability (MSI-H) and a microsatellite stabilization (MSS) type.
The invention uses rhPCR primer and amplification method, avoids interference between different primers, can detect more microsatellite loci simultaneously 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 loci, has no obvious preference to the microsatellite length compared with the capture method, and still maintains effective signal enrichment when the microsatellite loci are increased in length, thereby being more beneficial to distinguishing MSI-H from MSS and having high sensitivity.
The combination of 30 microsatellite loci is used for detecting microsatellite states, and has high accuracy and sensitivity, high discrimination between MSI-H and MSS and good detection stability.
Drawings
FIG. 1A is a graph of the length-depth correlation of the microsatellite loci of the present invention
FIG. 2 PCR gold standard verification verifies the reliability results of the present invention
FIG. 3 detection sensitivity of the present invention
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 sequence, and combines a bioinformatics analysis means to judge the microsatellite state.
Example 1 preference of Capture and amplification methods for microsatellite loci Length
Optionally, the same microsatellite loci are subjected to a capturing method and an amplicon method for library construction, and second generation sequencing analysis is performed.
The results are shown in FIG. 1: in the capturing method, the capturing depth gradually decreases along with the increase of the length of the microsatellite loci, and the capturing depth decreases more seriously after the length reaches 24 bp; the amplification method can relatively effectively avoid the defect of the capture method, and the longer the microsatellite loci are, the better the microsatellite loci are in the signal enrichment, so that the MSI-H and MSS can be distinguished. This is consistent with the gold-standard PCR method in which the lengths of the 5 microsatellite loci (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) are all >20bp, so that the amplification method has a stronger distinguishing capability in the aspect of microsatellite instability detection.
Example 2 selection of microsatellite loci
Microsatellite loci with better performance, which are located in an intron (intron) region, are screened from WES sequencing data, loci with better performance in the whole exon range are screened from MOSAIC paper, 108 total candidate loci are selected, performance evaluation (the evaluation method is established by the following model) is carried out on each locus, and finally the optimal 30 loci are screened for msi-value calculation.
And (3) establishing a model:
(1) Extracting repeat length distribution of each site (i);
(2) Converting the repeat length distribution into a proportional distribution;
(3) Obtaining the cumulative distribution of the (i) repeat length of the site according to the proportion distribution in the (2);
(4) For each location (i), the cumulative distributions of MSI-H and MSS samples in the training set are averaged, and then the repeat length (defining the length as the cutpoint) when the difference of the cumulative distribution averages of the two types of samples is maximum is found. Classifying according to the size relation between repeat length of reads and cut point, wherein MSI reads are defined as cut points smaller than or equal to cut point, MSS reads are defined as cut point, and the cumulative probability average value of MSS samples corresponding to cut point is p i (defined as baseline probability);
(5) Based on the training model, we assume that site (i) has N i Bar ready, baseline probability of the site pi, cutpoint C i . Finding out the MSI reads number of the site as n according to the cutpoint i The number of MSS reads is Ni-Ni stripes. We find the probability of this site as P by the following formula.
If the site cumulative probability P (X.gtoreq.n) i ) Is significantly less than 0.001, which we consider to support MSI-H. The site sensitivities were ranked according to the discrimination ability of the sites in the training samples, and the optimal 30 sites were selected (as shown in table 1).
(6) Finally, calculating MSI-value by synthesizing 30 sites, namely, the ratio of the number of sites supporting MSI-H to the total number of sites; and obtaining a 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.
Microsatellite loci screened 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 loci information
Example 3 reliability verification
The upstream primer sequences and the downstream primer sequences used for the 30 microsatellite loci shown in Table 1 are shown in tables 2 and 3, respectively. All primers used were block-cleavable rhPCR primers (from Integrated DNA Technologies, IDT company), rA, rG, rC, rU in sequence each representing one RNA base, 3SpC3 attached to the 3 'end of each sequence as a means of 3' end nucleic acid blocking (Blocker).
TABLE 2 upstream primer sequence information Table
Position of Mutation type Numbering device The 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 downstream primer sequence information Table
Position of Mutation type Numbering device 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, the primer composition in this example, the upstream primer set thereof is selected from the group consisting of the primer set having the sequence of SEQ ID No: 1-30; the downstream primer group is selected from the group consisting of a primer sequence of SEQ ID No: 31-60.
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: the paraffin-embedded samples (formalin fixed paraffin-primed, FFPE) were DNA extracted using a ReliaPrep FFPE gDNA Miniprep System (promega, cat. NO: A2352) kit.
2) Target region amplification: the 20uL reaction system was formulated according to the following table (msi_fwd and msi_rev refer to forward and reverse primer mixtures, respectively, i.e. the primer sets of tables 2, 3, both from IDT company):
TABLE 4 target region amplification reaction System
Volume of DNA X ul: the amount of DNA added into the library is 50ng, the volume of DNA added is determined as the ratio of the total amount of DNA to the concentration of DNA according to the concentration of the extracted DNA, and the added volumes of different samples are expressed by X.
The first round of amplification was performed for the target region according to the reaction procedure using a ProFlex PCR System PCR instrument (Applied biosystems by life technologies, model: proFlex);
TABLE 5 target region amplification procedure
3) Purifying target amplification products: agencourt AMPure XP beads is selected to purify the target product by 80% ethanol (which is used in the prior art).
The specific purification steps are as follows:
a. adding 30 μl of magnetic beads (1.5 x magnetic beads) into a PCR tube filled with the reaction solution, shaking and mixing, centrifuging briefly, and incubating at room temperature for 10min;
b. resting on a magnetic rack until the liquid is clear, and removing the supernatant using a pipette (note that the beads are not touched and the PCR tube is kept on the magnetic rack);
c. 200 μl of 80% ethanol was added to the PCR tube, left to stand for 30s, and the supernatant was removed;
d. repeating c;
e. sucking the liquid in the PCR tube as clean as possible by using a 10-mu l gun head, and standing at room temperature until the magnetic beads are dried;
f. adding 22 μl of nuclease-free water, removing the PCR tube from the magnetic rack, shaking, mixing, and centrifuging for a short time;
g. incubating for 5min at room temperature, placing the PCR tube on a magnetic rack, standing until liquid is clear, sucking 20 μl of supernatant, and adding into a new PCR tube;
h. adding 30 μl of magnetic beads into the PCR tube containing the supernatant, shaking and mixing, centrifuging briefly, and incubating at room temperature for 10min;
i. resting on a magnetic rack until the liquid is clear, and removing the supernatant using a pipette (note that the beads are not touched and the PCR tube is kept on the magnetic rack);
g. 200 μl of 80% ethanol was added to the PCR tube, left to stand for 30s, and the supernatant was removed;
k. repeating g;
sucking the liquid in the PCR tube as completely as possible by using a 10-mu l gun head, and standing at room temperature until the magnetic beads are dried;
and m, adding 20ul of reaction liquid prepared according to the table 5 into the dried magnetic beads, shaking and mixing uniformly, incubating for 3min at room temperature, and then placing on a PCR instrument for PCR reaction.
4) An amplification (Index PCR) reaction system for the target product was prepared as follows in table 6:
TABLE 6 amplification reaction System of target products
Amplification was performed according to the following reaction procedure;
5) Purification of the library of interest: agencourt AMPure XP beads is selected to purify target products by using 80% ethanol (used in the prior art), and the purified library is subjected to quantitative quality inspection.
The specific purification steps are as follows:
a. adding 20 μl of magnetic beads (1.0 x magnetic beads) into a PCR tube filled with the reaction solution, shaking and mixing, centrifuging briefly, and incubating at room temperature for 10min;
b. resting on a magnetic rack until the liquid is clear, and removing the supernatant using a pipette (note that the beads are not touched and the PCR tube is kept on the magnetic rack);
c. 200 μl of 80% ethanol was added to the PCR tube, left to stand for 30s, and the supernatant was removed;
d. repeating c;
e. sucking the liquid in the PCR tube as clean as possible by using a 10-mu l gun head, and standing at room temperature until the magnetic beads are dried;
f. adding 22 μl of nuclease-free water, removing the PCR tube from the magnetic rack, shaking, mixing, and centrifuging for a short time;
g. incubation was performed for 5min at room temperature, then the PCR tube was placed on a magnetic rack and left to stand until the liquid was clear, 20. Mu.l of the supernatant was aspirated and added to a new 1.5ml EP tube, thus obtaining the library.
h. The constructed library was subjected to Qubit quantification and lab chip fragment analysis.
6) And (3) sequencing the library on-machine and analyzing a letter flow.
The msi-value is calculated according to the model of example 2 and the microsatellite status of the clinical sample is determined.
Experimental results:
the results of the tests performed on 30 MSI-H and 60 MSS clinical FFPE DNA samples were compared to the results of the test using the PCR gold standard.
As a result, as shown in FIG. 2, the cutoff value is 0.4, MSS is below 0.4, MSI-H is above 0.4; the detection results of 90 samples are all consistent with the gold standard, and the consistency rate is 100%.
Example 4 sensitivity comparison
The MSI samples, which simulate 0.1%,1%,5% and 10% tumor levels respectively, were tested using the method of the present invention, assuming MSI-H cell lines (SW 48, co115, HCT-8, LS180, AN3CA) and MSS cell lines (HT 55) were mixed and the MSS cell lines were 100% tumor levels and 0% tumor levels, and MSS cell lines were diluted against MSS cell line DNA, as described in examples 2 and 3.
The comparative example uses a conventional capture method.
As a result, as shown in FIG. 3, the LOD of the method of the present invention can be detected at 5% or less, whereas the LOD of the conventional capturing method as the prior art can be detected at 10% only, and thus the sensitivity of the present invention is remarkably advantageous.
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<110> Shanghai thought Di biomedical 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 (7)

1. A biomarker combination comprising a combination of 30 microsatellite Loci, loci_19, loci_483, loci_162, loci_62, loci_127, loci_420, loci_328, loci_231, loci_262, loci_46, loci_481, loci_376, loci_9, loci_316, loci_387, loci_245, loci_213, loci_402, loci_270, loci_140, loci_339, loci_348, loci_260, loci_203, loci_83, loci_94, loci_486, loci_430, NR24, NR27, the microsatellite locus information:
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 locus of claim 1 and each primer in the downstream primer set specifically binds downstream of the microsatellite locus of claim 1.
3. The primer composition of claim 2 wherein each primer of the upstream primer set and/or each primer of the downstream primer set is a rhPCR primer.
4. The primer composition of claim 3 wherein the upstream primer set consists of a primer set having the sequence of SEQ ID No: 1-30; the downstream primer group consists of a primer sequence shown as SEQ ID No: 31-60.
5. A kit for detecting microsatellite status based on a second generation sequencing platform, comprising the primer composition of any one of claims 2-4.
6. The kit of claim 5, wherein the microsatellite status comprises high microsatellite instability and microsatellite stability.
7. Use of a primer composition comprising an upstream primer set and a downstream primer set, said upstream primer set comprising a sequence of SEQ ID No: 1-30; the downstream primer group consists of a primer sequence shown as SEQ ID No: 31-60; each primer of the upstream primer set and/or each primer of the downstream primer set is a rhPCR primer; the microsatellite status includes high microsatellite instability and microsatellite stability.
CN202010420704.XA 2020-05-18 2020-05-18 Biomarker combination for microsatellite instability detection, kit and application thereof Active CN111471755B (en)

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CN112708679B (en) * 2021-01-27 2023-01-24 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)

* Cited by examiner, † Cited by third party
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
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

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