Microsatellite instability site combination based on ngs technology and application and detection method thereof

Abstract

The application discloses a microsatellite instability site combination based on an NGS technology, application and detection method thereof, the microsatellite instability site combination comprises at least 1800 combinations of microsatellite sites numbered as MS-1 to MS-3200 shown in table 1, the position of the microsatellite site in the genome is shown in table 1; the detection method comprises: using reagents for detecting the microsatellite site combination to carry out high-throughput sequencing on a tumor sample to be detected, obtaining sample targeted capture sequencing data; processing the sample targeted capture sequencing data, and inputting the processed numerical value into an MSI model to obtain an MSI final value y, so as to determine the genomic microsatellite instability state. The microsatellite instability site combination and the detection method provided by the application can obtain efficient and accurate results in various cancers; meanwhile, the application also has the advantages of high-throughput, objectivity and simple operation, and is suitable for clinical promotion and application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a microsatellite unstable site combination based on NGS technology, its application, and detection method. Background Technology

[0002] Microsatellite instability (MSI) refers to the change in microsatellite allele length during DNA replication caused by abnormal insertion or removal of repetitive sequences. This change cannot be corrected by the DNA mismatch repair system (MMR) due to various reasons (such as promoter methylation inhibiting gene expression or inactivation / truncation mutations in genes involved in DNA mismatch repair mechanisms). Specifically, MSI manifests as the instability and length variation of microsatellite sites, which have a relatively stable number of repetitive units in normal tissues, in abnormal tissues.

[0003] The phenomenon of interstitial colorectal cancer (MSI) was first discovered in 1993 by Aaltonen et al. in hereditary nonpolyposis colorectal cancer (HNPCC, also known as Lynch syndrome). Common mechanisms leading to Lynch syndrome include germline inactivation or truncation mutations in genes such as MLH1, MSH2, MSH6, PMS2, or EPCAM. Subsequently, researchers have found sporadic MSI in lung cancer, gastrointestinal cancer, endometrial cancer, and ovarian cancer. The incidence of MSI varies significantly among different cancer types; for example, approximately 15% of colorectal cancers are MSI tumors, with an MSI incidence rate reaching 30% in early-onset colorectal cancer, while the proportion of MSI tumors in HNPCC is as high as 90%. Clinical studies have shown that patients with stage II / III colorectal cancer exhibiting high-frequency MSI (MSI-H) have a better prognosis and do not benefit from adjuvant chemotherapy with fluorouracil-based drugs (such as 5-FU). Therefore, the detection of MSI has multiple clinicopathological significances.

[0004] Currently, most diagnostic methods for tumor microsatellite instability (MSI) on the market are limited to detecting single or a few sites in a single tumor. These methods all have different drawbacks: immunohistochemistry has low specificity and reproducibility, requires high sample quality, and is relatively complex to operate; PCR typically selects 5-11 single nucleotide repeat sites, about 25 bp in length, and determines the microsatellite instability state of the sample by measuring the length distribution range after PCR amplification using capillary electrophoresis, which is currently the gold standard detection method. However, it requires additional test samples and normal tissue from the patient as a control for state determination, making it inconvenient from an operational perspective. In addition, neither of the above two detection methods can meet the requirements of large sample size, multiple detection sites, wide distribution, and high detection accuracy.

[0005] With next-generation sequencing (NGS) technology becoming the mainstream technology in oncology, MSI detection methods based on NGS are emerging. However, existing research and reports show that this method still exhibits bias towards different cancer types, leading to inaccurate test results and failing to meet clinical application requirements. Possible reasons for this phenomenon include: a limited number of detection sites (8-100) or unsuitable detection sites.

[0006] Therefore, a combination of sites and a detection method that can simultaneously and accurately detect microsatellite instability in different cancer types is a pressing practical need in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a microsatellite unstable site combination based on NGS technology, its application, and detection method to solve the problem that existing technologies have biases in different cancer types, leading to inaccurate detection results. At the same time, the detection method provided by this invention also has the advantages of high throughput, high sensitivity, high specificity, objectivity, and simple operation, making it suitable for clinical promotion and application.

[0008] To achieve the purpose of this invention, this invention first provides a combination of sites for detecting microsatellite instability, including a combination of at least 1800 of the microsatellite sites numbered MS-1 to MS-3200 shown in Table 1. The 1800 sites included in each cancer type may vary slightly. The locations of the microsatellite sites numbered MS-1 to MS-3200 in the genome are shown in Table 1.

[0009] Preferably, it comprises a combination of the 3200 microsatellite sites shown in Table 1.

[0010] In another aspect, the present invention provides the application of the site combination for detecting microsatellite instability as described in any of the preceding claims in the preparation of microsatellite instability detection products.

[0011] Preferably, the product includes testing reagents, testing kits, testing devices, and testing systems.

[0012] In another aspect, the present invention provides a reagent based on NGS technology for detecting microsatellite instability, the reagent comprising a capture probe for the site combination described in any of the preceding claims.

[0013] Preferably, the kit includes the reagents described above.

[0014] In another aspect, the present invention provides a method for detecting microsatellite instability, comprising the following steps:

[0015] S1. High-throughput sequencing of the tumor sample to be tested is performed using the reagents or kits described above to obtain sample targeted capture sequencing data.

[0016] S2, process the sample targeted capture sequencing data obtained in step S1, and input the processed values ​​into the MSI model to obtain the final MSI value y, so as to determine the microsatellite instability state of the genome;

[0017] The MSI model mentioned above is:

[0018]

[0019] Where a1 = 65.94706936, a2 = 36.30939656, a3 = -49.91115649.

[0020] If the final value of MSI y ≥ 0.7, it is judged as a highly unstable microsatellite MSI-H; if the final value of MSI y < 0.7, it is judged as a stable microsatellite MSS.

[0021] Preferably, in step S2, the specific steps for processing the sample targeted capture sequencing data obtained in step S1 are as follows: perform a chi-square test on each site to obtain the first set of MSI values ​​x1 of the sample, and perform Euclidean distance calculation on each site to obtain the second set of MSI values ​​x2 of the sample.

[0022] Preferably, the tumor includes colorectal cancer, endometrial cancer, and gastric cancer.

[0023] In another aspect, the present invention provides the use of the site combination for detecting microsatellite instability as described in any of the preceding claims in the preparation of a product for assessing the pairing of tumor samples or blood cfDNA with normal control samples.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) Compared with existing MSI detection technologies, the detection products and methods based on 3200 microsatellite loci (numbered MS-1 to MS-3200) provided by this invention can achieve efficient and accurate results in a variety of cancer types. At the same time, this invention also has the advantages of high throughput, high sensitivity, high specificity, objectivity and ease of operation, and is suitable for clinical promotion and application.

[0026] (2) This invention uses the currently mature second-generation sequencing technology to obtain the genetic information of the tumor sample of the test subject, and can perform single tumor tissue analysis without relying on control samples.

[0027] (3) The panel designed using 3200 MSI sites in this invention can also assess whether tumor tissue or cfDNA is paired with normal controls. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to embodiments. However, those skilled in the art will readily understand that the content described in the embodiments is only for illustration and explanation of the present invention and is not intended to limit the present invention as described in detail in the claims. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional methods, and the experimental materials used can be obtained from commercial companies unless otherwise specified.

[0029] Terminology Explanation

[0030] In this invention, "MSI-H" refers to highly unstable microsatellites; "MSS" refers to stable microsatellites.

[0031] As mentioned earlier, the most common methods for MSI detection are MSI-PCR or IHC analysis of tumor tissue samples. However, both methods require paired detection of tumor and normal samples, limiting their applicability and leading to problems such as low sensitivity and specificity, and poor reproducibility due to the limited number of microsatellite loci detected. Next-generation sequencing (NGS) can detect more microsatellite loci, but existing NGS-based MSI detection products or studies suffer from bias in some cancer types due to the limited number of loci detected (8-100) or unsuitable loci, resulting in inaccurate results. Therefore, the applicant of this invention, through extensive screening and research, identified an optimized combination of 3200 microsatellite loci suitable for MSI status assessment and developed a method for detecting and analyzing sample MSI status using NGS technology based on this combination. This method allows for rapid, automated, high-throughput, high-sensitivity, and high-specificity assessment of the stable or unstable microsatellite status of samples from different cancer types, providing meaningful theoretical and clinical guidance for subsequent treatment of related symptoms.

[0032] The research process and application value verification of this invention will be explained in detail below through examples:

[0033] The clinical tumor tissue and blood samples used in this invention were all from cancer patients diagnosed in tertiary hospitals, and all samples had complete patient information and signed informed consent forms.

[0034] Example 1: Screening of microsatellite unstable site combinations and design and synthesis of probes

[0035] Includes the following steps:

[0036] 1. Twenty clinical tissue samples each of MSI-H and MSS colorectal cancer were obtained and high-throughput sequencing was performed (the library construction kit was the Tongshu DNA Universal Library Construction Kit, and the capture probe was WGS sequencing). After MSI analysis of the sequencing results, sites with different sequence repeat counts between tumor tissue and normal tissue in the MSI sites of the MSI-H samples and sites with clear clinical significance reported in the literature were selected. After several rounds of testing, approximately 22,698 sites were initially screened.

[0037] 2. Twenty clinical tissue samples from colorectal cancer were selected and their MSI status was determined using PCR-capillary electrophoresis. The samples were divided into two groups: one with high microsatellite instability (MSI-H) and another with microsatellite stable (MSS) (10 samples in total), for use in the next site screening test.

[0038] 3. High-throughput sequencers were used to sequence the samples from the MSI-H and MSI groups (library construction kit was from Changzhou Tongshu Biotechnology Co., Ltd., catalog number: 13006; capture probe was IDT hybridization capture reagent). After obtaining the sequencing data, the Euclidean distance method was used to calculate the value of the 22,698 loci in step 1. Then, the t-test method was used to perform an unpaired t-test on the average value of the two groups of standards, MSI-H and MSI, to calculate the p-value. 4,000 loci with p-value < 0.05 were selected for the next step of MSI status analysis of the samples.

[0039] 4. For the 4000 sites identified in step 3, probes were designed in their respective regions using probe design software. Sites with poor specificity were removed, and the probes designed for the remaining 3200 sites were combined to form the MSI Arbiter panel for clinical sample detection. In this embodiment, the MSI Arbiter panel contains a total of 3432 probes. Among them, approximately 2200 sites were found to be applicable to each cancer type, all of which are included in the 3200 sites. To facilitate the detection of multiple cancer types, a panel containing 3200 sites was designed.

[0040] The information on the 3200 microsatellite sites is shown in Table 1.

[0041] Table 1 Information on 3200 microsatellite sites

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[0079]

[0080] Example 2: Application of the present invention to the combination of 3200 unstable microsatellite sites

[0081] Based on the probe combination designed and synthesized for 3200 sites in Example 1, MSI detection and analysis were performed on 222 pairs of colorectal cancer FFPE samples (107 MSI-H samples and 115 MSS samples) from three tertiary hospitals. The performance of the 3200 sites in Table 1 for determining MSI status was evaluated. The specific steps are as follows:

[0082] 1. Sample DNA extraction

[0083] DNA was extracted from colorectal cancer tissue samples and adjacent normal control samples using a nucleic acid extraction kit (Changzhou Tongshu Biotechnology Co., Ltd., catalog number: 150091) following the instructions. Leukocyte DNA was extracted using a blood DNA mini-extraction kit (Megan, catalog number: D3111-03). Blood samples were extracted using a nucleic acid extraction kit (Changzhou Tongshu Biotechnology Co., Ltd., catalog number: 150170) and underwent quality control. The total DNA volume of both tissue and control samples should be ≥200 ng, and the total cfDNA volume of blood samples should be ≥30 ng.

[0084] It should be noted that leukocyte DNA can also be extracted from the patient's blood sample as a control. In this embodiment, the control sample is adjacent normal tissue.

[0085] 2. DNA library construction

[0086] DNA libraries were constructed using a library construction kit (Changzhou Tongshu Biotechnology Co., Ltd., catalog number: 13006).

[0087] 2.1 DNA fragmentation and magnetic bead purification (this step is not performed for cfDNA)

[0088] Remove the fragmented enzyme from the kit in advance, gently tap to mix, briefly centrifuge, and place on ice for later use; dissolve the fragmented buffer from the kit at room temperature, vortex to mix, briefly centrifuge, and place on ice for later use.

[0089] The genomic DNA extracted from tumor tissue and adjacent normal tissue in the above steps was prepared in PCR tubes according to the dosages shown in Table 2. The PCR reaction was then performed according to the fragmentation PCR procedure shown in Table 3. After the reaction was complete, the tubes were briefly centrifuged, and 5 μL of enzyme reaction stop solution was added to each tube. After vortexing and mixing, the tubes were briefly centrifuged again. The fragmented products were further purified using magnetic beads.

[0090] Table 2 Composition of DNA fragmentation reaction solution

[0091] Components Volume DNA 200 ng Nuclease-Free Water q.s. to 20 μL Fragmentation Buffer 2 μL Fragmentation Enzyme 2 μL

[0092] Table 3 Interrupted PCR Procedure

[0093] DNA Species Temperature Time Tissue DNA 37℃ 20 min White Blood Cell DNA 37℃ 25 min

[0094] 2.2 DNA fragment end repair

[0095] Remove the end-repair enzyme from the kit beforehand, gently tap to mix, briefly centrifuge, and place on ice for later use; dissolve the end-repair buffer from the kit at room temperature, vortex to mix, briefly centrifuge, and place on ice for later use.

[0096] Add the reagents as shown in Table 4 to the fragmented DNA purified in the previous step, and proceed with the reaction according to the reaction procedure shown in Table 5 (heat cap temperature is 75℃).

[0097] Table 4 Composition of DNA end repair reaction solution

[0098] Components Volume (μL) Fragmented DNA 38 End Repair Buffer 10 End Repair Enzyme 2 Total Volume 50

[0099] Table 5. Degree of DNA end repair response

[0100] Temperature Time 20℃ 15 min 72℃ 10 min 4℃ ∞

[0101] 2.3 Connector Connection and Purification

[0102] Remove the adapter from the kit beforehand and allow it to thaw at room temperature. Vortex to mix, centrifuge briefly, and then place on ice for later use.

[0103] Add the reagents shown in Table 6 sequentially to each PCR reaction tube containing the end-repair reaction product (operate on ice), vortex to mix, and place the reaction tube on an PCR instrument (do not use a hot cap) and incubate at 20°C for 15 minutes. Once the PCR instrument has cooled to 4°C, remove the PCR tubes, centrifuge, and purify using magnetic beads.

[0104] Table 6 Connection Reaction System

[0105] Components Volume (μL) Product after End Repair Reaction 50 Nuclease-Free Water 15 Adapters 5 DNA Ligase 40 Total Volume 110

[0106] 2.4 Library Amplification

[0107] Dissolve the amplification primers and amplification reaction solution in the kit at room temperature beforehand, vortex to mix, centrifuge briefly, and then place on ice for later use.

[0108] Prepare the PCR amplification reaction mixture according to the system shown in Table 7, and run the instrument for amplification according to the PCR reaction conditions shown in Table 8.

[0109] Table 7 PCR amplification reaction system

[0110]

[0111] Table 8 PCR Reaction Conditions

[0112]

[0113] 2.5 Purification and Quantification of Library Products

[0114] The library products were purified and washed using magnetic beads. The purified products were then used... The dsDNA HS AssayKit was used to quantify the library.

[0115] 3. Probe hybridization capture, enrichment, and sample delivery for sequencing.

[0116] 3.1 After preparing the system as shown in Table 9 in centrifuge tubes, place them in a concentrator and concentrate to dryness at 45°C. Add the hybridization buffer shown in Table 10 to each concentrated centrifuge tube, shake well and centrifuge briefly, then incubate at room temperature for 5-10 min. Follow the reaction procedure shown in Table 11, including incubation at 65°C for 4-16 hours.

[0117] Table 9 Hybrid Library System

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[0119]

[0120] Table 10 Hybrid buffer system

[0121] Reagent Name Amount (μL) Hybridization Buffer 8.5 Hybridization Enhancer 2.7 Nuclease-Free Water 1.8 MSI Panel 4

[0122] 3.2 On the second day, bring the M-270 magnetic beads to room temperature for equilibration for 30 minutes. Then prepare the relevant buffers and mixtures as shown in Tables 11 and 12.

[0123] Table 11 Magnetic Bead Cleaning Solution and Buffer

[0124]

[0125] Table 12 Magnetic Bead Suspension Mixture

[0126] Reagent Name Amount (μL) Hybridization Buffer 8.5 Hybridization Enhancer 2.7 Nuclease-Free Water 5.8

[0127] After the equilibration of the 3.3M-270 magnetic beads, wash the beads: Take 50 μL of each reaction solution into a 0.2 mL centrifuge tube and place it on a magnetic rack to stand. After clarification (about 5 min), remove the supernatant. Add 100 μL of magnetic bead washing solution, vortex well, and centrifuge briefly. Place it on a magnetic rack and wash twice. After washing and removing the supernatant, add 17 μL of the magnetic bead suspension mixture and vortex well. Incubate at 65℃ for 5 min for later use.

[0128] 3.4 Transfer the product incubated at 65°C in step 3.1 to the washed M-270 magnetic beads, mix quickly, and then incubate at 65°C for 45 min, taking it out and mixing quickly once every 10-12 min; after the 65°C incubation is completed, take it out and add 100 μL of 65°C preheated washing buffer 1, shake for 10 s, and then place it on a magnetic rack.

[0129] After clarification, quickly remove the supernatant and add 150 μL of preheated (65°C) washing buffer S. Vortex to mix and incubate at 65°C for 5 min. Remove and place on a magnetic rack. After clarification, remove the supernatant. Repeat this step twice.

[0130] Add 150 μL of room temperature washing buffer 1 to a centrifuge tube after removing the supernatant. Vortex for 2 min, then centrifuge briefly and place on a magnetic rack. After clarification, remove the supernatant and add 150 μL of room temperature washing buffer 2. Vortex for 2 min, then centrifuge briefly and place on a magnetic rack. After clarification, remove the supernatant and add 150 μL of room temperature washing buffer 3. Vortex for 2 min, then centrifuge briefly and place on a magnetic rack. After clarification, remove the supernatant, add 20 μL of nuclease-free water, mix well, and then use directly for PCR enrichment.

[0131] 3.5 PCR enrichment and product purification

[0132] Prepare the reaction system as shown in Table 13 and perform PCR enrichment according to the procedure shown in Table 14.

[0133] Table 13 PCR enrichment reaction system

[0134] Reagent Name Amount (μL) Purified DNA (with magnetic beads) 20 Amplification Reaction Solution 25 Amplification Primer 5

[0135] Table 14 PCR enrichment reaction procedure

[0136]

[0137] After the reaction, 75 μL of purified magnetic beads equilibrated at room temperature for 30 min was mixed with the PCR enrichment product. After vortexing, the mixture was incubated at room temperature for at least 5 min, followed by short centrifugation and placement on a magnetic rack. After clarification (at least 5 min), the supernatant was aspirated (leaving approximately 10 μL at the bottom). 200 μL of 75% ethanol was added to the magnetic beads (care should not dislodge the beads). After approximately 30 s, the supernatant was aspirated. 75% ethanol was added again, and the mixture was washed twice with 75% ethanol. After centrifugation for approximately 3 s, the mixture was placed on a magnetic rack again. After the magnetic beads were fully adsorbed, the remaining liquid was aspirated. The magnetic beads were allowed to dry at room temperature or dried in a 37°C metal bath. 22 μL of nuclease-free water was added to the dried magnetic beads. After vortexing, the mixture was incubated at room temperature for 3 min, followed by short centrifugation and placement on a magnetic rack for 1 min (or until the liquid was clear). 20 μL of the supernatant was aspirated to obtain the final purified DNA. The enrichment product was quantified using the Qubit quantitative reagent and instrument.

[0138] 3.6 The enriched library was then subjected to sequencing.

[0139] 4. After preprocessing the offline data, we use our self-developed algorithm to analyze it and obtain the ArbiterScore values ​​of paired samples to determine their MSI status.

[0140] The self-developed algorithm described is a microsatellite instability analysis model (MSI model) obtained during the same period of this application. The MSI model is as follows:

[0141]

[0142] Where y is the MSI value (Arbiter Score). If the Arbiter Score ≥ 0.7, the MSI status of the sample is determined to be "MSI-H"; if the Arbiter Score < 0.7, it is determined to be "MSS". a1 and a2 are coefficient values, and a3 is a constant term. In this embodiment, a1 = 65.94706936, a2 = 36.30939656, and a3 = -49.91115649. x1 is 57.94; x2 is 0.3159, and the calculated Arbiter Score value is 1, which is determined to be "MSI-H".

[0143] After analysis, the MSI determination results of 222 colorectal cancer samples from this example, using 3200 loci, were consistent with the results of the gold standard MSI-PCR, with both sensitivity and specificity reaching 100%. The detection results are shown in Table 15.

[0144] Table 15 Comparison of test results in Example 2

[0145]

[0146] Example 3: Application of the present invention to the combination of 3200 unstable microsatellite sites

[0147] Referring to the detection process in Example 2, this invention also detected 150 gastric cancer tumor tissue samples (40 MSI-H samples and 110 MSS samples) from two tertiary hospitals. Compared with the IHC method, the sensitivity reached 97.5% and the specificity reached 100%. The specific detection results are shown in Table 16.

[0148] MSI status was detected and analyzed in 100 endometrial cancer tumor tissue samples (61 MSI-H samples and 43 MSS samples) from two tertiary hospitals. The sensitivity was 96.7% and the specificity was 97.7%. The specific test results are shown in Table 17.

[0149] One hundred colorectal cancer cfDNA samples (69 MSI-H samples and 31 MSS samples) from two tertiary hospitals were subjected to MSI detection and analysis. Compared with tissue PCR, the sensitivity reached 98.4% and the specificity reached 100%. The specific detection results are shown in Table 18.

[0150] Table 16 Comparison of gastric cancer tumor tissue detection results in Example 3

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[0152] Table 17 Comparison of Endometrial Cancer Tissue Detection Results in Example 3

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[0154] Table 18 Comparison of Blood cfDNA Detection Results in Example 3 for Colorectal Cancer

[0155]

[0156] Example 4: Sensitivity of the present invention's combination of 3200 microsatellite unstable sites and detection method

[0157] The detection method of this invention can detect samples with a tumor content of 5% and standards with a tumor content of 3%. The 5% tumor content samples were obtained from paraffin sections from various hospitals, stained with hematoxylin and eosin (HE) to assess tumor content. The 3% standards were derived from DNA extracted from eight cell lines purchased from the Chinese Academy of Sciences. MSI status was assessed using the MSI-PCR method, resulting in two MSI-H standards and two MSS standards. Sample detection was performed using the experimental procedure described in Example 2, and the detection results were all as expected, showing 100% consistency with the MSI-PCR detection results.

[0158] Example 5: Application of the present invention's combination of 3200 microsatellite instability sites in assessing the pairing ability between tumor samples and normal control samples.

[0159] Microsatellite indeterminate detection requires paired testing of tumor samples and normal controls, followed by result interpretation. When paired samples become confused, the detection method of this invention can immediately detect the mismatch and halt subsequent analysis. Furthermore, it can identify and match paired samples within the confused pool, thus avoiding erroneous test results due to sample mismatch in clinical applications.

[0160] Paired analysis was performed on 222 colorectal cancer tumor samples and normal controls used in Example 1, and the consistency of pairwise matching with the samples identified by the PCR method pentaC was 100%.

[0161] Fifty colorectal cancer tumor samples and their normal control samples were shuffled, re-sequencing them and then the data were analyzed. All unmatched samples were identified. After re-pairing the unmatched samples according to the method of the present invention, the pairing was completely consistent with that before the numbering was shuffled, and the detection results were also completely consistent with those before the numbering was shuffled.

[0162] In summary, the detection product and method based on 3200 microsatellite loci (numbered MS-1 to MS-3200) provided by this invention can achieve efficient and accurate results in a variety of cancer types. It has very high sensitivity in distinguishing between MSI-H (microsatellite highly unstable type) and MSS (microsatellite highly stable type), with an accuracy of 100%. It can also assess the pairing between samples with 100% accuracy. At the same time, this invention also has the advantages of high throughput, objectivity, and ease of operation, making it suitable for clinical promotion and application.

[0163] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The application of reagents for detecting microsatellite locus combinations in the preparation of microsatellite instability detection products, characterized in that, The site combination includes a combination of the 3200 microsatellite sites shown in Table 1 of the specification.

2. The application as described in claim 1, characterized in that, The products include test kits, test devices, and test systems.

3. The application of reagents for detecting microsatellite locus combinations in the preparation of products for assessing the pairing between tumor samples and normal control samples, characterized in that, The site combination includes a combination of the 3200 microsatellite sites shown in Table 1 of the specification.

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