A multiplex PCR detection kit based on microsatellite instability loci for colorectal cancer

Abstract

The present application discloses a multiplex PCR detection kit based on colorectal cancer microsatellite instability sites, the kit comprising a primer set and a PCR premix; the primer set comprises a primer for detecting at least one of the sites BAT‑25, BAT‑26, NR‑21, NR‑22, NR‑27, HSP110, BAT‑52, and BAT‑56, the primer containing two inosines (I) and a stem-loop structure (SEQ ID NO.25) to simultaneously detect different states of MSI Del and Ins; when the kit uses MSI detection to determine the microsatellite instability of group colorectal cancer for 8 combined sites and the corresponding target coverage segments, capillary electrophoresis detection and ordinary PCR and digital PCR detection can be performed simultaneously, so that the gradient between the test result values ​​is more refined, and the MSI state is accurately defined as high, low, or stable; while saving the cost of reagents and materials, the test cycle is saved, the detection of the extreme cycle is achieved, the clinical multi-sample detection needs are met, and the kit has the characteristics of high sensitivity, specificity and repeatability.

Description

Technical Field

[0001] The present application relates to the field of PCR detection, and in particular to a multiplex PCR detection kit for detecting microsatellite instability sites in colorectal cancer. Background Art

[0002] Microsatellites are short tandem repeat DNA sequences with a length of 1-6 base pairs, including single nucleotide, dinucleotide or high nucleotide repeats, with a repeat count of 10-60 times. These repeat sequences are widely present in the genome, and due to the different number of tandem repeats at the gene locus, the length of the repeat sequence often varies in different individuals.

[0003] Microsatellite instability (MSI) is a change in the length of microsatellite alleles, which is caused by the insertion or deletion of repeat units during DNA replication and the failure of the DNA mismatch repair system to correct errors. Compared with normal cells, the microsatellites in tumor cells have changes in microsatellite length due to the insertion or deletion of repeat units. A large number of studies have shown that MSI is closely related to the occurrence of various tumors, such as colorectal cancer (CRC), endometrial cancer (EC), etc., and is an important marker for Lynch syndrome screening. Microsatellite markers can be used to detect microsatellite instability (a type of genomic instability).

[0004] MSI status may change the tumor microenvironment (TME) of colorectal cancer patients in many aspects, thereby affecting the efficacy of ICIs in colorectal cancer patients. With the in-depth understanding of the factors affecting the outcome of CRC immunotherapy, patients with low tumor mutation burden (TMB; <8 mutations / 10 6 DNA bases), MSI-H CRCs have a higher TMB (>12 mutations / 10 6 In addition, MSI-H CRCs have more immune cell infiltration [especially tumor-infiltrating lymphocytes (TILs) and type I interferons], which are associated with better prognosis.

[0005] At present, the methods for detecting microsatellite instability generally include PCR and NGS gene detection. The PCR method is to first select specific primers, and then PAR amplify the microsatellite loci in the sample. The amplified products are analyzed in the display system after electrophoresis detection (such as capillary electrophoresis), and compared with normal tissues to determine whether the sequence length of the microsatellite loci has changed. NCI (National Cancer Institute) recommends 5 microsatellite loci as standard loci for MSI (microsatellite instability) detection, namely BAT-25, BAT-26, D2S123, D5S346 and D17S250, which can be used for capillary electrophoresis detection. Among them, BAT-25 and BAT-26 have mononucleotide repeat sequences, and D2S123, D5S346 and D17S250 have dinucleotide repeat sequences. The widely used commercial Promega MSI detection kit on the market can detect 5 MSI sites (BAT-25, BAT-26, MONO-27, NR-21, NR-24), all of which are single nucleotide repeat sequences. After the test, the test results of tumor and normal samples are compared. If 2 or more of the above 5 microsatellite sites change, it is called high-frequency MSI (MSI-H); if only 1 microsatellite site changes, it is called low-frequency MSI (MSI-H). MSI, MSI-L), and 5 unchanged are called microsatellite stable (Microsatellitestable, MSS). In addition, the microsatellite instability (MSI) detection kit of Paulownia gene (multiple fluorescence PCR-capillary electrophoresis method) (BAT-25, BAT-26, D2S123, D5S346 and D17S250) can also only detect 5 MSI sites. Therefore, the current detection methods / kits have the risk of misclassifying samples close to the judgment threshold as MSI-H, MSI-L, and MSS due to poor consistency of judgment results, polymorphism of detection results, and obvious gradient of detection result values. In addition, the NGS gene detection method is to directly sequence the relevant DNA, but it is difficult to popularize in clinical applications because of the high cost of gene detection and the high experimental conditions required. Summary of the invention

[0006] In response to the above technical problems, the present application proposes a multiplex PCR amplification detection kit based on colorectal cancer microsatellite instability sites to achieve accurate detection of MSI and accurate classification of the three types of MSI-H, MSI-L, and MSS.

[0007] Specifically, this application provides the following technical solutions:

[0008] A multiplex PCR amplification detection kit based on colorectal cancer microsatellite instability sites, the detection kit comprises a primer set, a plasmid covering a target gene segment of the site (a quality control product used to identify different states of MSI) and a PCR premix, wherein the primer set comprises:

[0009] 1) Upstream primers and downstream primers for sites BAT-25 (Genbank Accession L04143) and BAT-26 (Genbank Accession U41210), and gene target coverage segments for sites BAT-25 and BAT-26; and,

[0010] 2) upstream primers and downstream primers for at least three sites among NR-21 (Genbank Accession NM_012244.4), NR-22 (Genbank Accession L38961.1), NR-27 (Genbank Accession AF070674.1), HSP110 (Genbank Accession NG_053051.1), BAT-52 (Genbank Accession AL139279.7), and BAT-56 (Genbank Accession AL772178.2); and plasmids covering gene target segments corresponding to the selected at least three sites; and,

[0011] Each primer in the primer set contains an inosine-stem-loop structure sequence, which consists of two inosines (also known as inosine nucleosides, with a molecular formula of C 10 H 12 N 4 O 5 , whose structural formula is shown in I and represented by I in the sequence table) and a sequence connected to inosine as shown in SEQ ID NO.25, which is composed of a stem-loop structure sequence (CGCTCACTTCGAGCG), inosine is close to the 5' end of the primer, and the stem-loop structure is close to the 3' end of the primer, so as to simultaneously detect different states of MSI Del and Ins,

[0012]

[0013] In a specific implementation, inosine is connected to the stem-loop structure, and inosine (I) is arranged at about 10-25bp away from the 5' end of the primer to prevent the formation of primer dimers, prevent the primer's own extension amplification and non-specific amplification between primers; the stem-loop structure is about 2-8bp away from the 3' end of the primer; in general, the inosine-stem-loop structure is located in the middle of the primer sequence, biased towards the 3' end, to reduce non-specific binding and amplification, achieve specific binding to the target target sequence, reduce the efficiency of binding to other non-specific targets, and achieve multiple super-multiple PCR detection.

[0014] Furthermore, the above reaction PCR premix is ​​a conventional premix of a PCR detection kit, such as the commercially available product ddPCR TM Supermix for Probes (without dUTP), PCR amplification buffer, polymerase, metal cations, glycerol, detergents, surfactants, dyes, fluorescent molecules, etc.; in an embodiment of the present application, the PCR premix includes a commercially available PCR reaction solution and an enzyme mixture.

[0015] Further, the primers for the better MSIPCR detection respectively target 8 sets of primers for 8 gene detection at microsatellite loci, 2 loci BAT-25, BAT-26 mandatory loci, combined with at least 4 combination loci of NR-21, NR-22, NR-27, HSP110, BAT-52, BAT-56, and perform MSI PCR detection. The primers for the better MSI PCR detection respectively target 6 sets of primers for 6 gene detection at microsatellite loci, 2 loci BAT-25, BAT-26 mandatory loci, combined with 4 combination loci of NR-22, NR-27, HSP110, BAT-56, and a total of 6 combination loci for MSI Del and Ins different states detection.

[0016] Furthermore, in order to ensure that the specificity of the primer design and the synthetic purity meet the requirements of PCR amplification accuracy, the primers prepared in the present application are all double purified, and DHPLC is selected for purification. Two inosines and a stem-loop structure (SEQ ID NO.25) are innovatively added in the primer design to enhance the binding of the primer to the target sequence while reducing the efficiency of binding to other non-specific targets, thereby realizing multiple PCR detection. At the same time, the primers with two inosines and a stem-loop structure have lower background and higher detection sensitivity for detection, are compatible with longer sequences, eliminate homologous interference, prevent nonspecific amplification, and can improve the stability of the primers and target molecules. In the case of shorter lengths, a very high Tm value can be maintained, thereby realizing accurate detection of different states of MSI DeI and Ins.

[0017] When the kit provided by the present application uses MSI detection to determine the microsatellite instability of group colorectal cancer for a total of 8 combination sites of 2+6 and the corresponding target coverage segment, capillary electrophoresis detection and PCR detection (including digital PCR and ordinary PCR detection) can be performed simultaneously to make the gradient between the test result values ​​more refined. In addition, when the present application uses MSI detection to detect a total of 6 combination sites of 2+4, while saving the cost of reagent materials, it can also save the detection cycle, achieve the detection of the extreme cycle, and meet the clinical multi-sample detection needs. At the same time, the MSI state can be accurately defined as high, low, or stable, and has the characteristics of high sensitivity, specificity and repeatability compared to existing analysis methods, and can also achieve the same detection accuracy as the Promega kit and the microsatellite instability (MSI) detection kit of the Paulownia gene.

[0018] The 2+6 combined sites and corresponding target coverage segments selected in this application are based on the existing 5 mononucleotide repeat sites. On the basis of the existing 2 required site detections of BAT-25 and BAT-26, other sites (including NR-21, NR-22, NR-27, HSP110, BAT-52, BAT-56) are added to target colorectal cancer and long mononucleotide repeat sites. Therefore, it is not limited to the existing capillary electrophoresis detection, and the different states of MSIDel and Ins can be detected by PCR technology. At the same time, single-base variations can be identified to improve the sensitivity and specificity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the clinical test results of colorectal cancer MSS at the NR-21 and BAT-26 sites (tumor samples);

[0020] Figure 2 The results of clinical detection of MSS of colorectal cancer at BAT-25 and NR-27 sites (tumor samples);

[0021] Figure 3 These are the clinical test results of colorectal cancer MSS at NR-21 and BAT-26 sites (normal samples);

[0022] Figure 4 These are the clinical test results of colorectal cancer MSS at BAT-25 and NR-27 sites (normal samples);

[0023] Figure 5 The clinical test results of MSI-L in colorectal cancer at the NR-21 and BAT-26 loci (tumor samples);

[0024] Figure 6 The clinical test results of MSI-L in colorectal cancer at BAT-25 and NR-27 sites (tumor samples);

[0025] Figure 7 The clinical test results of MSI-L in colorectal cancer at NR-21 and BAT-26 sites (normal samples);

[0026] Figure 8 The clinical test results of MSI-L in colorectal cancer at BAT-25 and NR-27 sites (normal samples);

[0027] Fig. 9 The clinical test results of MSI-H in colorectal cancer at the NR-21 and BAT-26 loci (tumor samples);

[0028] Fig.10 The clinical test results of MSI-H in colorectal cancer at BAT-25 and NR-27 sites (tumor samples);

[0029] Fig.11 The clinical test results of MSI-H in colorectal cancer at NR-21 and BAT-26 sites (normal samples);

[0030] Fig.12 The clinical test results of MSI-H in colorectal cancer at BAT-25 and NR-27 sites (normal samples);

[0031] Fig.13 The data analysis results of MSI-H detection with different primer concentrations;

[0032] Fig.14 The data analysis results of MSI-H detection with different primer concentrations;

[0033] Fig.15 The results of the analysis of the test data of MSS clinical samples for colorectal cancer;

[0034] Fig.16 The results of the analysis of the detection data of colorectal cancer MSI-L clinical samples;

[0035] Fig.17 The results of the analysis of the detection data of colorectal cancer MSI-H clinical samples;

[0036] Fig.18 The results of the analysis of the test data of MSS clinical samples for colorectal cancer;

[0037] Fig.19 The results of the analysis of the detection data of colorectal cancer MSI-L clinical samples;

[0038] Fig. 20 These are the results of the detection data analysis of colorectal cancer MSI-H clinical samples.

[0039] Fig.21These are the test data analysis results for MSI WT and Ins quality control products.

[0040] Fig. 22 Schematic diagram of inosine and stem-loop structure in primers. DETAILED DESCRIPTION

[0041] The clinical samples in the following examples were provided by Nanjing Drum Tower Hospital (Drum Tower Hospital Affiliated to Nanjing University Medical School) and were approved by the hospital ethics committee.

[0042] The nucleotide sequences involved in the following examples are:

[0043] SEQ ID NO.1:5'-ATGAC ATTCT GCATT TTAA IIC GCTCA CTTCG AGCGC TATGG-3';SEQ ID NO.2:5'-CAGGT GGCAA AGGGC ATGIICG CTCAC TTCGA GCGGC TTTC-3':CTGAC TTC5 TIICTT.3 CACTT CGAGC GTCAG C-3';SEQ ID NO.4:5'-TAGCTCCTTT ATAAG CTIICGC TCACT TCGAG CGTCT TC-3';SEQ ID NO.5:5'-CAGCA GAATT CCAGCCGGIICG CTCAC TTCGA GCGAG.NO.6 TCGTT-3' CCGCA TIICGCTCACTT CGAGC GTCAC AC-3';SEQ ID NO.7:5'-CACGT CCAGC TCTGA TAIICGC TCACT TCGAGCGTCT TC-3';SEQ ID NO.8:5'-CAGAC AATTC GGATG CCAIICG' CTCAC TTCGA TTCGA NO.9:5'-CAAAG CATTG AAGTC TGCIICG CTCAC TTCGA GCGAG TTGA-3';SEQ IDNO.10:5'-CTGAG TCGAT AATAC TIICGCT CACTT CGAGC GAGCA ATG-3';SEQ ID NO.1AGCT TGAICGT TGAICCA5' CGTCA GA-3';SEQ ID NO.12:5'-TGGGA AGTGTTCATGIICGCTC ACTTC GAGCG TGCTC-3':

[0044] SEQ ID NO.13:5'-ATTGG GCAGA CACTG AACTIIC GCTCA CTTCG AGCGA GCTT-3';SEQ ID NO.14:5'-GTGGA ACCCT CTGIICG CTCAC TTCGA GCGAG CCAGA-3':

[0045] SEQ ID NO.15:5'-CGGAA GCTGC AGTAIIC GCTCA CTTCG AGCGA GCCGA G-3';SEQID NO.16:5'-CTGCC ACTTA GCAAGII CGCTC ACTTC GAGCG TCCAT CT-3';SEQ ID NO.17:

[0046] 5'-TACATAGAAAGAGATGTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTAGAAGACTTGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCTCCAAGAATGTAGTGGGAGTGATTCTAAAGTTTTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCAAGAAT TGAGAACAGAGCATTTTAGCCATAGTTAAAATGCAGAATGTCATTTAAAACAAAGTATTGGATTTTTTATAATAAGCAACACCTATAGTTAAAAAGTTAGTTTTCACTCTTTACAAGTTAAAATGAATTTAAATGGTTTTCTTTTCCTCCAACCTAATAGTTTTCACAGAGACTTGGCAGCCAA-3';

[0047] SEQ ID NO.18:

[0048] 5'-CGGTAATCAAGTTTTTGAACTCTTATCAGATGATTCCAACTTTGGACAGTTTGAACTGACTACTTTTGACTTCAGCCAGTATATGAATTGGATATTGCAGCAGTCAGAGCCCTTAACCTTTCAGGTAAAAAAAAAAAAAAAAAA AAAAAAGGGTTAAAATGTTGAATGGTTAAAAAATGTTTCATTTGACATACTGAAGAAGCTTATAAAGGAGCTAAAATATTGAAATATTTACTGGATTAGATAACTAGCTTTAAATGGCTGTATTTTTCTCTCCCCCTCC-3';

[0049] SEQ ID NO.19:

[0050] 5'-TGGTTTCTGAAGACACATCCCTTTCAGCAGAATTCCAGCCGGAGTCGCTGGCACAGTTCTATTTTTATATTTAAATGTATGTCTCCCCTGGCCTTTTTTTTTTTTTTTTTTTTTTTAGCAACA CTTTTCTTGTTTGTAAACGCGAGTGACCAGAAAGTGTGAATGCGGAGTAGGAATATTTTTCGTGTTCTCTTTTATCTGCTTGCCTTTTTTAGAGAGTAGCAGTGGTTCCTATTTCGGAAAA-3';

[0051] SEQ ID NO.20:

[0052] 5'-GATTGGGAATAAAGACTTTGAGCTTGATGTCCTGGAGGAAGGCTATACCACAGAAACATTGGCTGGTCAGGATATACAAGGTAAAGGACCTGGATAATCGAGGCTTGTCAAGGACATAAATGTCACGTCCAGCTCTGATATCTTCGCACTGAGCACATCACATTTAGGACGTTGAAGATTTTTTTTTTTTTTTTTTTTTTTTAATATGCAGTTTGTAAGAACAAAACTGGATGGCATCCGAATTGTCTGGAAGTTTTGTCTTGGGCATGATGGGCTGGGCCAAATGAAATGATTTTTATAATTCTAAACAGGTTACCAAATGAAATGTCATGG-3';SEQ ID NO.21:

[0053] 5'-CATTGAAGTCTGCAGTTGAAAAGCCCAACGTCTGTGAGATCCAGGAAACCATGCTTGCAAACCACTGGTAAAAAAAAAAAAAAAAAAAAAAAAAGCCACAGTGACTTGCTTATTGGTCATTGCTAGTATTATCGACTCAGAACCTCTTTACT-3';

[0054] SEQ ID NO.22:

[0055] 5’-TGTTAATGACTCTACTAGAGAAGTCATTTGTTAATTTTTCTTCTTTACATTATAGTATCTG AATGTTATTTTCTGCTACATACCCTTCAGTATCTTCTGAATCATGGTTCCAGATCAGAGATATTGGAAAAGGAACTGCATCTGTGACGGAAAATTCTCTAACTTTAAATGCCGGGGAAAGTATTGCACACTTAAAAAAAAAAAAAAAAATCATGAGCACATGAACACTTCCCAGTCATCTTTTAATATAAAACTCAATTCCAATGGATGGA-3’;

[0056] SEQ ID NO.23:

[0057] 5’-TTCACAACCTGCAAACCAGGAGATTCCCTCCGGTGACTACCCCATCAGGGCCCTGGGTTTCAAGCACAAAACTGGGTGGCCAATTGGGCAGACACTGAACTAGCTTCAGGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCACACTGGTGCCTGGAGACAGAACCATTTTTTTTTTTACCACACTGGTGCCAGCGAGACAGAACCATTCACTCCCTTGAAAAAGGGTTTGAAGCCAGGAAGCCAAGTGGTCTGGCTCAGAGGGTTCCACCCCAGCAGAGCCCAGG-3’;

[0058] SEQ ID NO.24:

[0059] 5'-CATGCGCCTGCAGTCCCAGCTGCTCAGGAGGCTGAGGCAGGAGGATTGCTTGAACCTGGGAGGCGGAAGCTGCAGTAAGCCGAGATTGTGCCACTGCACTCCAGCCTGGGTGACGCCTGGGCGACAGAGCGAGACTCTGT CTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCTTGCTATCCAGAATGTCATGGGAGTTAAGAGGGCAATTAAAAATGTCCCGAAAGATGGACTTGCTAAGTGGCAGCTGAGA-3';

[0060] SEQ ID NO. 25: 5'-CGCTC ACTTC GAGCG-3'.

[0061] In the above sequence, "I" stands for "inosine".

[0062] Example 1: Clinical sample detection by capillary electrophoresis

[0063] According to the experimental process of capillary electrophoresis, experimental operation, parameter setting and data reading and analysis are carried out. The main operating steps are as follows:

[0064] (1) Thaw the components of the PCR reaction solution, enzyme mixture, three clinical samples of colorectal cancer MSS, MSI-L, and MSI-H, and the MSI quality control product of the Microsatellite Instability Gene Detection Kit (fluorescence PCR-capillary electrophoresis) (National Medical Device Registration No. 20213400936) in the 8 MSI loci combined with 2 BAT-25 and BAT-26 mandatory loci, and the upstream primers and downstream primers of the 8 loci combined with NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56 at room temperature. Invert and mix all reagents and samples, centrifuge, and place them on a tube rack to prepare the reaction mixture according to the experimental plan.

[0065] The two BAT-25 and BAT-26 upstream and downstream primers in the PCR amplification reagent are required primers, wherein the nucleotide sequences of the upstream and downstream primers for the BAT-25 site are shown in SEQ ID NOs. 1 and 2, respectively; the nucleotide sequences of the upstream and downstream primers for the BAT-26 site are shown in SEQ ID NOs. 3 and 4, respectively; the combination NR-21 (the nucleotide sequences of its upstream and downstream primers are shown in SEQ ID NOs. 5 and 6, respectively), NR-22 (the nucleotide sequences of its upstream and downstream primers are shown in SEQ ID NOs. 7 and 8, respectively), NR-27 (the nucleotide sequences of its upstream and downstream primers are shown in SEQ ID NOs. 9 and 10, respectively), HSP110 (the nucleotide sequences of its upstream and downstream primers are shown in SEQ ID NOs. 11 and 12, respectively), BAT-52 (the nucleotide sequences of its upstream and downstream primers are shown in SEQ ID NOs. 13 and 14, respectively), BAT-56 (the nucleotide sequences of its upstream and downstream primers are shown in SEQ ID NOs. NO.15, 16) for a total of 8 upstream and downstream primers for the loci; in a specific implementation, the final concentration range of the upstream primer and the downstream primer is in the range of 50nmol / L-1500nmol / L, for example, 50nmol / L, 100nmol / L, 500nmol / L or 900nmol / L, etc.

[0066] In this embodiment, the final concentration of the upstream primer and downstream primer for sites BAT-25 and BAT-26 in the amplification reagent is 350 nmol / L, the final concentration of the upstream primer and downstream primer for sites NR-21, NR-22, and NR-27 is 450 nmol / L, and the final concentration of the upstream primer and downstream primer for sites HSP110, BAT-52, and BAT-56 is 600 nmol / L.

[0067] According to the experimental scheme, the following 8 groups of primers were prepared into PCR reaction mixtures for detection:

[0068] Detection of BAT-25 and BAT-26 loci PCR premix (including PCR reaction solution and enzyme mixture, both purchased from Beijing Reader Gene Technology Co., Ltd., as a component of conventional kits):

[0069] Reaction components per well Volume per well (μL) PCR reaction solution 10 Enzyme mixture 0.5 Primer upstream of BAT-25 or BAT-26 corresponding site (10 μM) 0.7 Downstream primers of BAT-25 or BAT-26 corresponding sites (10 μM) 0.7 Clinical samples (20ng / ul) or MSI quality control products 1 <![CDATA[ddH 2 The]]> 7.1 Total volume 20

[0070] PCR reaction mixture detection of NR-21, NR-22, NR-27:

[0071]

[0072]

[0073] PCR reaction mixture detection of HSP110, BAT-52, BAT-56 loci:

[0074] Reaction components per well Volume per well (μL) PCR reaction solution 10 Enzyme mixture 0.5 Upstream primers for the corresponding sites of HSP110 or BAT-52 or BAT-56 (10 μM) 1.2 HSP110 or BAT-52 or BAT-56 corresponding site downstream primers (10μM) 1.2 Clinical samples (20ng / ul) or MSI quality control products 1 <![CDATA[ddH 2 The]]> 6.1 Total volume 20

[0075] In the embodiment, the clinical samples provided by Nanjing Drum Tower Hospital (Drum Tower Hospital Affiliated to Nanjing University Medical School) are of three confirmed types of MSI status of colorectal cancer: MSS, MSI-L, and MSI-H. The sample sizes of MSS, MSI-L, and MSI-H are 62, 96, and 78, respectively. The experiment has been approved by the hospital ethics committee, and nucleic acids are extracted and stored at -20°C. It is divided into tumor tissue and normal tissue, and normal tissue is used as a control. The total amount of clinical samples input is between 10 and 250 ng, and the volume of clinical samples (20 ng / ul) and MSI quality control products is 1 ul.

[0076] (2) After the preparation is completed, the mixture is thoroughly shaken and centrifuged in a microcentrifuge (5000 rpm) for 10 seconds. The reaction plate is placed in a PCR amplification instrument and the following reaction program is set for PCR amplification.

[0077]

[0078]

[0079] (3) After the PCR reaction is completed, the samples are separated by capillary electrophoresis.

[0080] Reaction components per well Volume per well (μL) Deionized formamide HiDi 8.7 Molecular weight internal standard ROX500 0.3 PCR products 1 total 10

[0081] (4) Capillary electrophoresis was performed using ABI 3730 genetic analyzer.

[0082] (5) Use POP7 gel and select the "Fragment" electrophoresis method. For details, refer to the instruction manual of the genetic analyzer. The test data is obtained using ABI's Data analysis was performed using ELISA software (Applied Biosystems).

[0083] The test results of colorectal cancer MSS, MSI-L, and MSI-H clinical samples in this example are as follows Figure 1-Figure 12 shown.

[0084] The above test results prove that the capillary electrophoresis detection of clinical samples of colorectal cancer MSS, MSI-L and MSI-H using the two BAT-25 and BAT-26 mandatory sites designed in this embodiment, combined with NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56 primers for a total of 8 sites is consistent with the clinical sample detection results, and single base variations can be identified at the same time, with high sensitivity, accurate interpretation, and a compliance rate of 100%.

[0085] Example 2 Detection of 8 MSI sites in colorectal cancer based on PCR clinical samples

[0086] This embodiment targets the detection of 8 genes in the microsatellite locus panel combination, 2 loci BAT-25 and BAT-26 are mandatory loci, combined with 6 combination loci NR-21, NR-22, NR-27, HSP110, BAT-52, BAT-56, etc., a total of 8 combination loci are used for MSIPCR detection.

[0087] The combined amplification system and detection kit involved in this embodiment, the primers for the target sequence are manually compared in the NCBI database to determine their specificity, and non-specific primers are eliminated. At the same time, the interference between primers and the target sequence is taken into account. The primers of each combination are compared using Primer5.0 software, and the absolute value of DeltaG (ΔG) cannot exceed 9 kcal / mol. The primers are fully optimized to improve the detection sensitivity.

[0088] In this embodiment, BAT-25 and BAT-26 are the required sites, and NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56 are combined. A total of 8 MSI sites (2+6) are amplified by 8 pairs of primers. The drop-off method is used to ensure that the specificity of the primer design and the synthetic purity meet the requirements of PCR amplification accuracy. The primer purification is double purification, and HPLC is selected for purification. High-performance liquid chromatography purification is adopted. After two HPLC purifications, the product purity is extremely high, which can reach 99%. The specific steps are as follows: (1) When synthesizing high-purity DNA products, the dmtr protecting group needs to be automatically removed on the fully automatic DNA synthesizer. (2) The crude sample DNA is injected into an ion exchange high-performance liquid chromatography system with extremely strong separation performance for rough detection to confirm the main peak position, the target DNA content, etc. (3) The injection amount is reduced, and the flat head part of the old main peak is discarded. At this time, the purpose of removing impure short-chain DNA and purifying the target DNA can be achieved. In order to ensure the purity of DNA, the amount of purification is usually about 10d. (4) The waste DNA is tested for purity to confirm the reliability of purity.

[0089] At the same time, Fig. 22As shown, in this embodiment, two connected inosines (I) and a stem-loop structure (sequence is CGCTCACTTCGAGCG, SEQ ID NO.25) are innovatively added in the primer design, and the inosine is arranged before the stem-loop structure (close to the 5' end of the primer), which enhances the efficiency of the primer binding to the target sequence while reducing the efficiency of binding to other non-specific targets, thereby realizing multiplex PCR detection. At the same time, the primers with two inosines and a stem-loop structure have lower background and higher detection sensitivity for detection, are compatible with longer sequences, eliminate homologous interference, prevent non-specific amplification, can improve the stability of the primer and the target molecule, and can maintain a very high Tm value in the case of a shorter length, thereby realizing accurate detection of different states of MSI DeI and Ins.

[0090] The two sites BAT-25 and BAT-26 are mandatory sites, and the primers (same as in Example 1) and the sequences of the target coverage segments for MSIPCR detection are combined with the six combination sites NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56, a total of eight combination sites, respectively:

[0091] The primer and probe sequences and LNA modifications of the 8 MSI loci are as follows:

[0092] 1. The upstream primer BAT-25-F (SEQ ID NO.1) and the downstream primer BAT-25-R (SEQ ID NO.2) for the BAT-25 site, and the sequence of the BAT-25 target coverage segment (plasmid) are shown in SEQ ID NO.17; 2. The upstream primer BAT-26-F (SEQ ID NO.3) and the downstream primer BAT-26-R (SEQ ID NO.4) for the BAT-26 site, and the sequence of the BAT-26 target coverage segment (plasmid) are shown in SEQ ID NO.18; 3. The upstream primer NR-21-F (SEQ ID NO.5) and the downstream primer NR-21-R (SEQ ID NO.6) for the NR-21 site, and the sequence of the NR-21 target coverage segment (plasmid) are shown in SEQ ID NO.19;

[0093] 4. The upstream primer NR-22-F (SEQ ID NO.7) and the downstream primer NR-22-R (SEQ ID NO.8) for the NR-22 site, and the sequence of the NR-22 target coverage segment (plasmid) is shown in SEQ ID NO.20;

[0094] 5. The upstream primer NR-27-F (SEQ ID NO.9) and the downstream primer NR-27-R (SEQ ID NO.10) for the NR-27 site, and the sequence of the NR-27 target coverage segment (plasmid) is shown in SEQ ID NO.21; 6. The upstream primer HSP110-F (SEQ ID NO.11) and the downstream primer HSP110-R (SEQ ID NO.12) for the HSP110 site, and the sequence of the HSP110 target coverage segment (plasmid) is shown in SEQ ID NO.22; 7. The upstream primer BAT-52-F (SEQ ID NO.13) and the downstream primer BAT-52-R (SEQ ID NO.14) for the BAT-52 site, and the sequence of the BAT-52 target coverage segment (plasmid) is shown in SEQ ID NO.23; 8. The upstream primer BAT-56-F (SEQ ID NO.14) for the BAT-56 site NO.15), downstream primer BAT-56-R (SEQ ID NO.16), the sequence of the BAT-56 target coverage segment (plasmid) is shown in SEQ ID NO.24.

[0095] The above “plasmid” is used as “MSI quality control product” for PCR detection of corresponding sites.

[0096] In this embodiment, when detecting microsatellite instability, the primer concentration in the amplification reagent has a certain influence on the intensity of the fluorescence peak after electrophoresis detection of the amplification product. Taking into account the cost and detection sensitivity, the primer concentration in the amplification reagent is optimized.

[0097] In a specific implementation, the final concentrations of the upstream primers and downstream primers of the mandatory sites BAT-25 and BAT-26 in the amplification reagent, the combination of 6 sites such as NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56, and 2+6 total 8 MSI sites in the amplification reagent can all be in the range of 50nmol / L-1500nmol / L, for example, 50nmol / L, 100nmol / L, 500nmol / L or 900nmol / L.

[0098] In this example, the experimental operation, parameter setting and data reading and analysis were performed according to the BioRad Droplet Digital PCR System QX200 experimental instructions. The main operating steps are as follows:

[0099] (1) Combine BAT-25, BAT-26 with NR-21, NR-22, NR-27, HSP110, BAT-52, BAT-56 upstream primers and downstream primers and Bio-Rad ddPCR TMAll components of Supermix for Probes (Cat. No. 1863023) were thawed at room temperature. All reagents and samples were mixed by inversion, centrifuged and placed on a tube rack. PCR reaction mixtures were prepared according to the experimental scheme and tested separately:

[0100] PCR reaction mixture detection of BAT-25 and BAT-26 loci:

[0101] Reaction components per well Volume per well (μL) <![CDATA[ddPCR TM Supermix for Probes (without dUTP)]]> 10 Primer upstream of BAT-25 or BAT-26 corresponding site (10 μM) 0.7 Downstream primers of BAT-25 or BAT-26 corresponding sites (10 μM) 0.7 Clinical samples (20ng / ul) or MSI quality control products (corresponding site plasmid) 1 <![CDATA[ddH 2 The]]> 7.6 Total volume 20

[0102] PCR reaction mixture detection of NR-21, NR-22, NR-27 sites:

[0103] Reaction components per well Volume per well (μL) <![CDATA[ddPCR TM Supermix for Probes (without dUTP)]]> 10 Upstream primers corresponding to NR-21, NR-22 or NR-27 (10 μM) 0.9 NR-21 or NR-22 or NR-27 corresponding site downstream primer (10μM) 0.9 Clinical samples (20ng / ul) or MSI quality control products (corresponding site plasmid 1 <![CDATA[ddH 2 The]]> 7.2 Total volume 20

[0104] PCR reaction mixture detection of HSP110, BAT-52, BAT-56 loci:

[0105] Reaction components per well Volume per well (μL) <![CDATA[ddPCR TM Supermix for Probes (without dUTP)]]> 10 Upstream primers for the corresponding sites of HSP110 or BAT-52 or BAT-56 (10 μM) 1.2 HSP110 or BAT-52 or BAT-56 corresponding site downstream primers (10μM) 1.2 Clinical samples (20ng / ul) or MSI quality control products (corresponding site plasmid) 1 <![CDATA[ddH 2 The]]> 6.6 Total volume 20

[0106] Note: Clinical samples provided by Nanjing Drum Tower Hospital (Drum Tower Hospital affiliated to Nanjing University Medical School) have three confirmed types of MSI status for colorectal cancer: MSS, MSI-L, and MSI-H. The sample sizes for MSS, MSI-L, and MSI-H are 62, 96, and 78, respectively. This experiment has been approved by the hospital ethics committee. After nucleic acid extraction, it was stored at -20°C. It was divided into colorectal cancer tumor tissue and normal tissue. The volume of clinical samples (20ng / ul) and MSI quality control products was 1ul. Normal tissue was used as a control in the following tests.

[0107] After the reaction mixture is prepared, mix it, centrifuge it, and place it on a tube rack.

[0108] (2) Follow the droplet generation experimental steps of Bio-Rad. For specific steps, refer to QX200 TM Instruction Manual (REF 186-3007 / 12001045 / 17000034), that is, droplet generation oil: reaction mixture = 70uL: 20uL ratio to generate droplets. After the droplet generation is completed, remove the cartridge holder, remove and discard the liner. Transfer the droplets generated in the upper row of holes of the cartridge to the PCR reaction plate. Repeat the above droplet generation steps for all samples. After completion, quickly seal the PCR reaction plate with a puncturable sealing foil.

[0109] (3) After sealing, the reaction plate was placed in a PCR amplification instrument. Taking into account factors such as the Tm value and the difficult sequence with more PolyA in the LMR sequence of colorectal cancer, the following reaction program was set for PCR amplification.

[0110]

[0111] (4) The PCR reaction plate was stored away from light and placed at room temperature for 2 hours to stabilize the droplets. After completion, the data was read and analyzed.

[0112] (5) The results of the analysis of the test data of colorectal cancer MSS clinical samples are as follows:

[0113] Because it involves screening of a concentration range, this example selects 8 MSI loci with low, high and appropriate concentration primers to detect MSI-H (clinical samples with important immunotherapy significance) clinical samples.

[0114] The final concentrations of the upstream primers and downstream primers of the eight MSI sites were 50 nmol / L for BAT-25 and BAT-26, 50 nmol / L for NR-21, NR-22, and NR-27, and 100 nmol / L for HSP110, BAT-52, and BAT-56. Fig.13 shown. Fig.13 In the figure, 1)-16) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-21 test (tumor sample), NR-21 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-52 test (tumor sample), BAT-52 test (normal sample), BAT-56 test (tumor sample), BAT-56 test (normal sample).

[0115] The final concentrations of the upstream primers and downstream primers of the eight MSI sites were 1500 nmol / L for BAT-25 and BAT-26, 1500 nmol / L for NR-21, NR-22, and NR-27, and 1200 nmol / L for HSP110, BAT-52, and BAT-56. The MSI-H detection data analysis results are shown in Fig.14 shown. Fig.14In the figure, 1)-16) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-21 test (tumor sample), NR-21 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-52 test (tumor sample), BAT-52 test (normal sample), BAT-56 test (tumor sample), and BAT-56 test (normal sample), respectively.

[0116] The appropriate final concentrations of the upstream primers and downstream primers of the 8 MSI sites are 350nmol / L for BAT-25 and BAT-26, 450nmol / L for NR-21, NR-22, and NR-27, and 600nmol / L for HSP110, BAT-52, and BAT-56. The analysis results of the MSS, MSI-L, and MSI-H detection data are as follows: The detection data analysis results of colorectal cancer MSS clinical samples are as follows Fig.15 As shown in Figure 2, the analysis results of the detection data of colorectal cancer MSI-L clinical samples are as follows: Fig.16 As shown in Figure 2, the analysis results of the detection data of colorectal cancer MSI-H clinical samples are as follows: Fig.17 shown. Fig.15 In the figure, 1)-16) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-21 test (tumor sample), NR-21 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-52 test (tumor sample), BAT-52 test (normal sample), BAT-56 test (tumor sample), and BAT-56 test (normal sample), respectively. Fig.16In the figure, 1)-16) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-21 test (tumor sample), NR-21 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-52 test (tumor sample), BAT-52 test (normal sample), BAT-56 test (tumor sample), and BAT-56 test (normal sample), respectively. Fig.17 In the figure, 1)-16) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-21 test (tumor sample), NR-21 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-52 test (tumor sample), BAT-52 test (normal sample), BAT-56 test (tumor sample), BAT-56 test (normal sample).

[0117] In summary, BAT-25 and BAT-26 are mandatory sites, and the combination of NR-21, NR-22, NR-27, HSP110, BAT-52, BAT-56 and other 6 sites, 2+6 total 8 MSI sites for colorectal cancer MSS (no microsatellite sites have changed), MSI-L (only 1 microsatellite site of BAT-26 site has changed) and MSI-H (BAT-25, BAT-26 combined with NR-21, NR-22, NR-27, HSP110, BAT-52, BAT-56 8 MSI site microsatellite sites have changed) The test results are consistent with those of clinical samples. Compared with the Promega MSI detection kit which can only be used for capillary electrophoresis detection, the PCR-based detection of the present application can identify single-base variations with a 100% compliance rate, and has the characteristics of accurate interpretation, high sensitivity, specificity and repeatability.

[0118] Example 3 Detection of six MSI loci in colorectal cancer based on PCR clinical samples and MSI WT and Ins quality control products

[0119] In order to reduce the detection time cycle and detection material cost, while ensuring the detection sensitivity and specificity, this embodiment targets the microsatellite locus panel combination 6 gene detection, 2 loci BAT-25, BAT-26 mandatory loci, combined with 4 combination loci such as NR-22, NR-27, HSP110, BAT-56, a total of 6 combination loci for MSIPCR detection. The invention discloses a combined amplification system and detection kit. The specificity of primers for target sequences is manually compared in the NCBI database, and non-specific primers are eliminated. Meanwhile, the interference between primers and targets is taken into consideration. The primers of each combination are compared using Primer 5.0 software. Considering that the absolute value of DeltaG (ΔG) cannot exceed 9 kcal / mol, the primers are fully optimized. Two inosines (I) and a stem-loop structure (sequence: CGCTCACTTCGAGCG) are innovatively added to the primer design to enhance the binding of primers to target sequences while reducing the efficiency of binding to other non-specific targets, thereby realizing multiplex PCR detection. Meanwhile, the primers with two inosines and a stem-loop structure have lower background and higher detection sensitivity for detection, are compatible with longer sequences, eliminate homologous interference, prevent non-specific amplification, improve the stability of primers and target molecules, and maintain a very high Tm value at a shorter length, thereby realizing accurate detection of different states of MSI DeI and Ins.

[0120] The experimental operation process and primers with 2 inosines (I) and a stem-loop structure (sequence CGCTCACTTCGAGCG) and the sequence of the target coverage segment are the same as those in Example 2. The appropriate final concentrations of the upstream primers and downstream primers of the 6 MSI sites are 350 nmol / L for BAT-25 and BAT-26, 450 nmol / L for NR-22 and NR-27, and 600 nmol / L for HSP110 and BAT-56. The results of the analysis of the MSS, MSI-L, and MSI-H detection data are as follows:

[0121] The results of the analysis of the detection data of colorectal cancer MSS clinical samples are as follows: Fig.18 As shown in Figure 2, the analysis results of the detection data of colorectal cancer MSI-L clinical samples are as follows: Fig.19 As shown in Figure 2, the analysis results of the detection data of colorectal cancer MSI-H clinical samples are as follows: Fig. 20 The test data analysis results of MSI WT and Ins quality control products are shown in Fig.21 shown. Fig.18In the figure, 1)-12) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-56 test (tumor sample), and BAT-56 test (normal sample), respectively. Fig.19 In the figure, 1)-12) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-56 test (tumor sample), and BAT-56 test (normal sample), respectively. Fig. 20 In the figure, 1)-12) are the test results of BAT-25 test (tumor sample), BAT-25 test (normal sample), BAT-26 test (tumor sample), BAT-26 test (normal sample), NR-22 test (tumor sample), NR-22 test (normal sample), NR-27 test (tumor sample), NR-27 test (normal sample), HSP110 test (tumor sample), HSP110 test (normal sample), BAT-56 test (tumor sample), and BAT-56 test (normal sample), respectively. Fig.21 In the figure, 1)-12) are the detection results of BAT-25 detection (Ins-), BAT-25 detection (WT), BAT-26 detection (Ins), BAT-26 detection (WT), NR-22 detection (Ins), NR-22 detection (WT), NR-27 detection (Ins), NR-27 detection (WT), HSP110 detection (Ins), HSP110 detection (WT), BAT-56 detection (Ins), and BAT-56 detection (WT), respectively.

[0122] This embodiment selects 6 sites for detection, which saves more reagent material costs than the detection of 8 sites selected in Example 2, and can also save detection cycles, achieve extreme cycle detection, and meet clinical multi-sample detection needs.

[0123] From the above results, it can be obtained that BAT-25 and BAT-26 are required sites, and the combination of NR-22, NR-27, HSP110, BAT-56 and other 4 sites, 2+4 total 6 MSI sites for colorectal cancer MSS, MSI-L and MSI-H have consistent detection results for clinical samples. Compared with the Promega MSI detection kit which can only be used for capillary electrophoresis detection, the PCR-based detection of the kit of this application can identify single-base variations and simultaneously detect different states of MSI DeI and Ins, with low cost, short time period, and 100% compliance rate, it has the characteristics of accurate interpretation, high sensitivity, specificity and repeatability.

Claims

1. A multiplex PCR detection kit based on colorectal cancer microsatellite instability sites, characterized in that: The detection kit includes a primer set and a PCR premix; The primer set includes primers for detecting at least one of the sites BAT-25, BAT-26, NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56; The primers in the primer set all contain an inosine-stem-loop structure, which consists of two inosines near the 5' end of the primer and a stem-loop structure near the 3' end of the primer; the nucleotide sequence of the stem-loop structure is shown in SEQ ID NO.

25.

2. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to claim 1, characterized in that: The primer set comprises: Primers for detection of loci BAT-25 and BAT-26; and, Primers for detecting at least three sites among sites NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56.

3. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to claim 1, characterized in that: The primer set comprises: 1) Primers for detection of loci BAT-25 and BAT-26; and, 2) Primers for detecting at least four sites among sites NR-21, NR-22, NR-27, HSP110, BAT-52, and BAT-56.

4. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to claim 1, characterized in that: The primer set consists of primers for detecting sites BAT-25, BAT-26, NR-22, NR-27, HSP110, and BAT-56.

5. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to any one of claims 1 to 4, characterized in that: The concentration of each primer in the detection kit is 50nmol / L-1500nmol / L.

6. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to any one of claims 1 to 4, characterized in that: The detection kit also includes a quality control product containing a target site coverage segment.

7. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to any one of claims 1 to 4, characterized in that: The PCR premix solution includes one or more of a buffer, a polymerase, a metal cation, glycerol, a detergent, a surfactant, a dye, and a fluorescent molecule.

8. The multiplex PCR detection kit based on colorectal cancer microsatellite instability loci according to any one of claims 1 to 4, characterized in that: The inosine is 10-25 bp away from the 5' end of the primer.

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