Multiplex PCR detection kit based on endometrial cancer microsatellite instability sites

Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) detection kit based on microsatellite instability sites of endometrial cancer. The kit comprises a primer group and a premixed solution, the primer group comprises a primer for detecting at least one of a site BAT-25, a site BAT-26, a site NR-24, a site MONO-27, a site CAT-25, a site BAT-59 and a site BAT-60, and the primer contains two inosine (I) and a stem-loop structure (SEQ ID NO.22) so as to simultaneously detect different states of MSI Del and Ins; according to the kit, when MSI detection is used for carrying out microsatellite instability judgment on endometrial cancer on seven combination sites and corresponding target coverage sections, capillary electrophoresis detection and common PCR (Polymerase Chain Reaction) and digital PCR detection can be carried out at the same time, so that the gradient among detection result numerical values is more refined, and the MSI state is accurately defined as high, low or stable; the kit has the advantages that reagent material cost is saved, detection period is shortened, detection in an extreme period is achieved, clinical multi-sample detection requirements are met, and the kit has the advantages of high sensitivity, specificity and repeatability.

Description

Technical Field

[0001] This application relates to the field of PCR detection, in particular to a multiplex PCR detection kit for microsatellite instability sites in endometrial cancer. Background Art

[0002] Microsatellites are short tandem repeat DNA sequences, 1-6 base pairs in length, including single nucleotide, dinucleotide or higher nucleotide repeats, with 10-60 repeats. These repetitive sequences are widely present in the genome, and due to the different number of tandem repeats at gene loci, the length of the repetitive sequences often varies among different individuals. Therefore, microsatellite markers can be used to detect microsatellite instability (a type of genomic instability).

[0003] Microsatellite Instability (MSI) is the 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 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 biomarker for Lynch syndrome screening.

[0004] Endometrial cancer (EC) is one of the three most common gynecological tumors affecting women, and there is a trend of increasing incidence year by year. Most EC patients have a low recurrence risk and can be treated by surgery alone. At the same time, the molecular subset of EC that benefits from targeted therapy (including immunotherapy) is very important. For metastatic and recurrent EC, chemotherapy and hormone therapy are the only available treatment options, but they do not significantly improve the 5-year overall survival rate, progression-free survival rate or recurrence rate. Therefore, early detection and early treatment are important means of preventing EC.

[0005] Microsatellite instability (MSI) and defective mismatch repair (dMMR) are important biomarkers for predicting the response to immune checkpoint inhibitors (ICI). PCR-based high MSI (MSI-H) and dMMR detection in endometrial cancer (EC) has produced highly consistent results. Compared with MSS / MSI-L EC, MSI-H EC has a lower tumor mutation burden (TMB), and MSI-H EC has a higher TMB. In addition, MSI-H EC has more immune cell infiltration, indicating a better prognosis.

[0006] At present, the methods for detecting microsatellite instability generally include two types: the PCR method and the NGS gene detection method. The PCR method first selects specific primers, and then amplifies the microsatellite loci in the sample by PAR. After the amplified product is detected by electrophoresis (such as capillary electrophoresis), it is analyzed in a display system and compared with normal tissues to determine whether there are changes in the sequence length of the microsatellite loci. The National Cancer Institute (NCI) of the United States has recommended 5 microsatellite loci as the standard loci for MSI detection, namely BAT-25, BAT-26, D2S123, D5S346 and D17S250, which are used for capillary electrophoresis detection. Among them, BAT-25 and BAT-26 have single nucleotide repeat sequences, and D2S123, D5S346 and D17S250 have dinucleotide repeat sequences. The commercially available Promega MSI detection kit, which is widely applicable in the market, can detect 5 MSI loci (BAT-25, BAT-26, MONO-27, NR-21, NR-24), all of which are single nucleotide repeat sequences. After the detection, the detection results of tumor and normal samples are compared. If 2 or more of the above 5 microsatellite loci change, it is called high-frequency type (High-frequency MSI, MSI-H); if only 1 microsatellite locus changes, it is called low-frequency type (Low-frequency MSI, MSI-L), and if none of the 5 change, it is called microsatellite stable type (Microsatellite stable, MSS). The microsatellite instability (MSI) detection kit (multiplex fluorescence PCR-capillary electrophoresis method) (BAT-25, BAT-26, D2S123, D5S346 and D17S250) of Tongshu Gene can also only detect 5 MSI loci. Therefore, there are risks of poor coherence of the determination results, polymorphism of the detection results, and obvious gradient of the detection result values, resulting in misclassification of MSI-H, MSI-L, and MSS for samples close to the determination threshold. The NGS gene detection method directly sequences the relevant DNA. However, due to the high cost of gene detection and the high required experimental conditions, it has not been popularized in clinical applications. Summary of the Invention

[0007] In view of the above technical problems, the present application proposes a multiplex PCR amplification detection kit based on microsatellite instability loci of endometrial cancer to achieve accurate detection of MSI and accurate classification of three types: MSI-H, MSI-L, and MSS.

[0008] Specifically, the present application is achieved by the following technical solutions:

[0009] A multiplex PCR amplification detection kit based on microsatellite instability sites of endometrial cancer, which includes a primer set and a PCR premix;

[0010] The above primer set includes:

[0011] 1) Upstream and downstream primers for loci BAT-25 (Genbank Accession L04143) and BAT-26 (Genbank Accession U41210), and the gene target coverage segments of loci BAT-25 and BAT-26; and,

[0012] 2) Upstream and downstream primers for at least two loci among loci NR-24 (Genbank Accession X60152), MONO-27 (Genbank Accession AC007684), CAT-25 (Genbank Accession NM_032982), BAT-59 (Genbank Accession AC090424), BAT-60 (Genbank Accession AC069359); and plasmids of gene target coverage segments corresponding to the selected at least two loci; and,

[0013] Each primer in the primer set contains an inosine-stem loop structure sequence, which is composed of 2 inosines (hypoxanthine nucleoside, molecular formula C 10 H 12 N4O5, with the structural formula shown in I and represented by I in the sequence listing) and a stem loop structure sequence (CGCTCACTTCGAGCG, SEQ ID NO.22) connected to the inosine. The inosine is close to the 5' end of the primer, and the stem loop structure is close to the 3' end of the primer to simultaneously detect different states of MSIDel and Ins,

[0014]

[0015] In a specific implementation, the inosine is connected to the stem loop structure. The inosine (I) is set at about 10-25 bp from the 5' end of the primer to prevent primer dimer formation, prevent primer self-extension amplification, and non-specific amplification between primers; the stem loop structure is about 2-8 bp from the 3' end of the primer; generally speaking, the inosine-stem loop structure is located at a position in the middle and closer to the 3' end of the primer sequence to reduce non-specific binding and amplification, achieve specific binding to the target sequence, reduce the binding efficiency with other non-specific targets, and achieve multiplex and super-multiplex PCR detection.

[0016] Furthermore, the above reaction PCR premix is a conventional premix for a PCR detection kit, such as the commercially available product ddPCRTM Supermix for Probes (without dUTP), PCR amplification buffer, polymerase, metal cations, glycerol, detergents, surfactants, dyes, fluorescent molecules, etc.; in the embodiments of the present application, the PCR premix includes a commercially available PCR reaction solution and an enzyme mixture solution.

[0017] Preferably, the above primer set includes:

[0018] 1) upstream primers and downstream primers for loci BAT-25 and BAT-26, and plasmid of the gene target coverage segment for loci BAT-25 and BAT-26; and,

[0019] 2) upstream primers and downstream primers for at least three loci among loci NR-24, MONO-27, CAT-25, BAT-59, BAT-60; and plasmids of the gene target coverage segments corresponding to the selected at least two loci; and,

[0020] Each primer in the primer set contains an inosine-stem loop structure sequence.

[0021] Further, preferably, the above primers for MSI PCR detect 7-gene detection of 7 primer combinations for microsatellite loci respectively. Two loci, BAT-25 and BAT-26, are mandatory loci. Combining with the 5 combined loci of NR-24, MONO-27, CAT-25, BAT-59, BAT-60, a total of 7 combined loci are used for MSI PCR detection. That is, the above primer set includes upstream primers and downstream primers for loci BAT-25, BAT-26, NR-24, MONO-27, CAT-25, BAT-59, BAT-60, and plasmids of the gene target coverage segments of the corresponding loci; each primer in the primer set contains an inosine-stem loop structure sequence.

[0022] In a specific implementation, to ensure that the specificity and synthesis purity of the primer design meet the requirements for the accuracy of PCR amplification, the primers prepared in the present application are double-purified, and DHPLC is selected for purification. In the primer design, 2 inosines and a stem loop structure (SEQ ID NO.22) are innovatively added, which enhances the binding of the primer to the target sequence while reducing the binding efficiency with other non-specific targets, realizes multiplex PCR detection. At the same time, the primers with 2 inosines and a stem loop structure have lower background and higher detection sensitivity for detection, are compatible with longer sequences, exclude 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, thus realizing the accurate detection of different states of MSI Del and Ins.

[0023] The 7 combined loci of 2 (BAT-25, BAT-26) loci + 5 (NR-24, MONO-27, CAT-25, BAT-59, BAT-60) loci selected in this application and the corresponding target coverage segment plasmids are based on the existing 5 single nucleotide repeat loci. On the basis of detecting 2 mandatory loci of BAT-25 and BAT-26, for endometrial cancer and long single nucleotide repeat loci, other loci (including NR-24, MONO-27, CAT-25, BAT-59, BAT-60) are added. The detection kit provided in this application is not limited to capillary electrophoresis detection and MSI detection based on PCR technology (including digital PCR and ordinary PCR detection), and can also identify single base variations to improve the sensitivity and specificity of detection. Description of the Drawings

[0024] Figure 1 For the clinical detection results of MSS in endometrial cancer (tumor samples);

[0025] Among them, A is the detection result of locus BAT-25, B is the detection result of locus BAT-26, and C is the detection result of loci NR-24 and MONO-27.

[0026] Figure 2 For the clinical detection results of MSS in endometrial cancer (normal samples);

[0027] Among them, A is the detection result of locus BAT-26, B is the detection result of locus BAT-25, and C is the detection result of loci NR-24 and MONO-27.

[0028] Figure 3 For the clinical detection results of MSI-L in endometrial cancer (tumor samples);

[0029] Among them, A is the detection result of locus BAT-26, B is the detection result of locus BAT-25, and C is the detection result of loci NR-24 and MONO-27.

[0030] Figure 4 For the clinical detection results of MSI-L in endometrial cancer (normal samples);

[0031] Among them, A is the detection result of locus BAT-26, B is the detection result of locus BAT-25, and C is the detection result of loci NR-24 and MONO-27.

[0032] Figure 5 For the clinical detection results of MSI-H in endometrial cancer (tumor samples);

[0033] Among them, A is the detection result of locus BAT-26, B is the detection result of locus BAT-25, and C is the detection result of loci NR-24 and MONO-27.

[0034] Figure 6 is the clinical detection result of MSI-H in endometrial cancer (normal sample);

[0035] Among them, A is the detection result of locus BAT-26, B is the detection result of locus BAT-25, and C is the detection result of loci NR-24 and MONO-27.

[0036] Figure 7 is the data analysis result of MSI-H detection with different primer concentrations in Example 2.

[0037] Figure 8 is the data analysis result of MSI-H detection with different primer concentrations in Example 2.

[0038] Figure 9 is the data analysis result of MSS detection with different primer concentrations in Example 2.

[0039] Figure 10 is the data analysis result of the detection of clinical samples of MSI-L in endometrial cancer.

[0040] Figure 11 is the data analysis result of the detection of clinical samples of MSI-H in endometrial cancer.

[0041] Figure 12 is the data analysis result of the detection of clinical samples of MSS in endometrial cancer.

[0042] Figure 13 is the data analysis result of the detection of clinical samples of MSI-L in endometrial cancer.

[0043] Figure 14 ]>is the data analysis result of the detection of clinical samples of MSI-H in endometrial cancer.

[0044] Figure 15 is the data analysis result of the detection of MSI WT and Ins quality control products in endometrial cancer.

[0045] Figure 16 is the schematic diagram of inosine and stem-loop structure in the primer;

[0046] Among them, A - primer region 1; B - primer region 2; I - 2 inosines; C - the hairpin (stem-loop) structure formed between primer region 1 and primer region 2. Detailed implementation manners

[0047] The clinical samples in the following examples were provided by Nanjing Drum Tower Hospital (Drum Tower Hospital Affiliated to Nanjing University School of Medicine) and have been approved by the hospital ethics committee.

[0048] The nucleotide sequences involved in the examples are as follows:

[0049] SEQ ID NO.1: ATGACATTCTGCATTTTAAIICGCTCACTTCGAGCGCTATGG;

[0050] SEQ ID NO.2: CAGGTGGCAAAGGGCATGIICGCTCACTTCGAGCGGCTTTC;

[0051] SEQ ID NO.3:

[0052] TACATAGAAAGAGATGTGACTCCCGCCATCATGGAGGATGACGAGTTGGCCCTAGACTTAGAAGACTTGCTGAGCTTTTCTTACCAGGTGGCAAAGGGCATGGCTTTCCTCGCCTCCAAGAATGTAAGTGGGAGTGATTCTCTAAAGAGTTTTGTGTTTTGTTTTTTTGATTTTTTTTTTTTTTTTTTTTTTTTTGAGAACAGAGCATTTTAGAGCCATAGTTAAAATGCAGAATGTCATTTAAAACAAAAGTATTGGATTTTTTATAATATAAGCAACACTATAGTATTAAAAAGTTAGTTTTCACTCTTTACAAGTTAAAATGAATTTAAATGGTTTTCTTTTCTCCTCCAACCTAATAGTGTATTCACAGAGACTTGGCAGCCA

[0053] SEQ ID NO.4: CTGACTACTTTTGACTIICGCTCACTTCGAGCGTCAGC

[0054] SEQ ID NO.5: TAGCTCCTTTATAAGCTIICGCTCACTTCGAGCGTCTTC

[0055] SEQ ID NO.6:

[0056] CGGTAATCAAGTTTTTAGAACTCTTATCAGATGATTCCAACTTTGGACAGTTTGAACTGACTACTTTTGACTTCAGCCAGTATATGAAATTGGATATTGCAGCAGTCAGAGCCCTTAACCTTTTTCAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTTAAAAATGTTGAATGGTTAAAAAATGTTTTCATTGACATATACTGAAGAAGCTTATAAAGGAGCTAAAATATTTTGAAATATTATTATACTTGGATTAGATAACTAGCTTTAAATGGCTGTATTTTTCTCTCCCCTCC

[0057] SEQ ID NO.7: CCATTGCTGAATTTIICGCTCACTTCGAGCGTACCTCCSEQ ID NO.8: CGTTGCAGTGAGCGGAGAIICGCTCACTTCGAGCGTTGTG

[0058] SEQ ID NO.9:

[0059] ATTATCTGAATATTTAAGGTCTGCCTTAACGTGATCCCCATTGCTGAATTTTACCTCCTGACTCCAAAAACTCTTCTCTTCCCTGGGCCCAGTCCTATTTTTTTTTTTTTTTTTTTTTTTTGTGAGACAGAGTCTCACTCTGTCACCCAGGTTGGAATGCAATGGCACAATCTCCGCTCACTGCAACGTCCGCC

[0060] SEQ ID NO.10: CAGTGAGCTGAGATIICGCTCACTTCGAGCGTGCGCCAC

[0061] SEQ ID NO.11: CAAGTTCATGATGTGGTIICGCTCACTTCGAGCGTCTGT

[0062] SEQ ID NO.12:

[0063] GCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGGAAATGGTGGGAACCCAGGGGGTGGAGATTGCAGTGAGCTGAGATTGCGCCACTGCACTCCAGCGTGGGAGACAGAGCAAGACTCTGCCTCAAAAAAAAAAAAAAAAAAAAAAAAAAATCCTGGTTTTACTTTTTTTTCTTTTTTAGTTGGCCAAGTGAAATTTGATCCACCCTTAAGAAAGGAGACAGAACCACATCATGAACTT

[0064] SEQ ID NO.13:CAGTGCCATACATGCTIICGCTCACTTCGAGCGATTGT

[0065] SEQ ID NO.14:CTTGCAGTGAGCTGAGAIICGCTCACTTCGAGCGTCGTG

[0066] SEQ ID NO.15:

[0067] ATATTATGTGGCTTTTATTTCAGTGCCATACATGCTATTGTTTTCAACCTAGAAACCTTTATCCCTGCTTATCTGAAACTTCCCAACTTCCCTGCTCTTTAAGACTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTCTGTCGCCCAGGCTGGAGGGCAGTGGCACGATCTCAGCTCACTGCAAGCTCCAACTCCC

[0068] SEQ ID NO.16:CTCCAGCCTAGGTAIICGCTCACTTCGAGCGACAGAG

[0069] SEQ ID NO.17:TACATTAAATTGCACAGIICGCTCACTTCGAGCGACCTTAA

[0070] SEQ ID NO.18:

[0071] GACATTGAGGCTGCAGTGAGCCAATATCGAGCCACTGAACTCCAGCCTAGGTAACAGAGCAAGACCTTTGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCTAATCAGACCATAGAATTAAGGTCTGTGCAATTTAATGTAGTAAATTATGTTAATAAAAATAAGGAAAAATATGAAACAAGAAAAGTCCACGCAAATCACGCAAACAT

[0072] SEQ ID NO.19: CGCCATTGCACTCCAIICGCTCACTTCGAGCGGCCTGG

[0073] SEQ ID NO.20: CCAAGTGTCGCTTGTAATIICGCTCACTTCGAGCGTCTATT

[0074] SEQ ID NO.21:

[0075] GCATGATTTTATTCTTTCGGGATGTAATCTCTTGAGTCATGCATCTCTGAGTGATGCCTATTTCATCAAAATTGAAAAGTCGATCAAATGTGTAGTCTTCTTCAAATAATTTCTGTAGAATGCCTGGGAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCCCTCAAATTTCTCCTATGCACTAATGGCTTGACCCAAAAGCTAAATACGTAGGAGATTATAGCAATGTTTGAAATCACTAAACCAGTCACTTCCACCACTCAAATGAGACCC

[0076] SEQ ID NO.22: CGCTCACTTCGAGCG

[0077] In the above sequences, "I" represents "inosine".

[0078] Example 1: Detection of Clinical Samples by Capillary Electrophoresis

[0079] Perform experimental operations, parameter settings, and data reading and analysis according to the experimental procedures of capillary electrophoresis. The main operating steps are as follows:

[0080] Thaw 7 MSI loci combinations including 2 mandatory loci of BAT-25 and BAT-26, and 7 upstream and downstream primers of NR-24, MONO-27, CAT-25, BAT-59, BAT-60, as well as the PCR reaction mixture, enzyme mixture, three clinical samples of endometrial cancer MSS, MSI-L, MSI-H, and the components of the MSI quality control product in the Yuewei Gene Microsatellite Instability Gene Detection Kit (fluorescent PCR-capillary electrophoresis method) (National Medical Device Registration No. 20213400936) at room temperature. Invert and mix all reagents and samples well, centrifuge them, and place them on the tube rack. Prepare the reaction mixture according to the experimental protocol.

[0081] In a specific implementation, the final concentrations of the upstream and downstream primers of the 2 mandatory loci of BAT-25 and BAT-26, and the 7 loci combinations of NR-24, MONO-27, CAT-25, BAT-59, BAT-60 in the amplification reagent are all 50 nmol / L - 1500 nmol / L, such as 50 nmol / L, 100 nmol / L, 500 nmol / L, or 900 nmol / L, etc.

[0082] In the amplification reagent of this example, the concentrations of the upstream and downstream primers of BAT-25 and BAT-26 are both 350 nmol / L; the concentrations of the upstream and downstream primers of NR-24, MONO-27, CAT-25 are both 560 nmol / L; the concentrations of the upstream and downstream primers of BAT-59 and BAT-60 are both 850 nmol / L. Among them, the upstream and downstream primers of locus BAT-25 are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, the upstream and downstream primers of locus BAT-26 are shown as SEQ ID NO.4 and SEQ ID NO.5 respectively, the upstream and downstream primers of locus NR-2 are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively, the upstream and downstream primers of locus MONO-27 are shown as SEQ ID NO.10 and SEQ ID NO.11 respectively, the upstream and downstream primers of locus CAT-25 are shown as SEQ ID NO.13 and SEQ ID NO.14 respectively, the upstream and downstream primers of locus BAT-59 are shown as SEQ ID NO.16 and SEQ ID NO.17 respectively, and the upstream and downstream primers of locus BAT-60 are shown as SEQ ID NO.19 and SEQ ID NO.20 respectively.

[0083] (1) Prepare the PCR reaction mixture with the following 7 groups of primers respectively according to the experimental protocol for detection, as shown in Table 1 - Table 3:

[0084] Table 1 Detection of PCR premix for BAT-25 and BAT-26 loci (including PCR reaction solution and enzyme mixture, both purchased from Beijing Yuewei Gene Technology Co., Ltd., which are components of a conventional kit):

[0085]

[0086]

[0087] Table 2 Detection of PCR reaction mixture for NR-24, MONO-27, and CAT-25:

[0088] Reaction components per well Volume per well (μL) PCR reaction solution 10 Enzyme mixture 0.5 Forward primer for the corresponding site of NR-24 or MONO-27 or CAT-25 (10 μM) 1.12 Forward primer for the corresponding site of NR-24 or MONO-27 or CAT-25 (10 μM) 1.12 Clinical sample (20 ng / ul) or MSI quality control product 1 <![CDATA[ddH2O]]> 6.26 Total volume 20

[0089] Table 3 Detection of PCR reaction mixture for BAT-59 and BAT-60 loci:

[0090] Reaction components per well Volume per well (μL) PCR reaction solution 10 Enzyme mixture 0.5 Forward primer for the corresponding site of BAT-59 or BAT-60 (10 μM) 1.7 Reverse primer for the corresponding site of BAT-59 or BAT-60 (10 μM) 1.7 [[ID=3 1 <![CDATA[ddH2O]]> 5.1 ​ 20

[0091] In the examples, the clinical samples were provided by Nanjing Drum Tower Hospital (The Affiliated Drum Tower Hospital of Nanjing University Medical School). The MSI status of endometrial cancer was determined to be three types: MSS, MSI-L, and MSI-H. The sample sizes of MSS, MSI-L, and MSI-H were 54 cases, 72 cases, and 88 cases respectively. After nucleic acid extraction, they were stored at -20°C. They were divided into tumor tissues and normal tissues, with normal tissues as controls. The total input amount of clinical samples was between 10 - 250 ng, and the volume of clinical samples and MSI quality control products was 1 μl.

[0092] (2) After preparation, mix thoroughly by shaking, centrifuge in a microcentrifuge for 10 seconds, place the reaction plate in a PCR amplifier, and perform PCR amplification. The reaction program is shown in Table 4.

[0093] Table 4 PCR amplification program

[0094]

[0095] (3) After the PCR reaction, separate the samples by capillary electrophoresis. The reaction system is shown in Table 5.

[0096] Table 5 Electrophoresis system

[0097] ​ ​ ​ 8.7 ​ 0.3 ​ 1 ​ 10

[0098] (4) Perform capillary electrophoresis detection using a 3730 genetic analyzer from ABI. Use POP7 gel and select the "Fragment" electrophoresis method. For details, refer to the instruction manual of the genetic analyzer. The detection data is analyzed using the analysis software (Applied Biosystems) from ABI.

[0099] In this example, the detection results of clinical samples of endometrial cancer with MSS, MSI-L, and MSI-H are as follows ​ shown ​ The detection results of loci BAT-25, BAT-26, NR-24, and MONO-27 are shown. Compared with the MicroRead Genetics microsatellite instability gene detection kit (fluorescent PCR-capillary electrophoresis method) used for detection, the detection loci diagram included is the same as that of the kit, and it is the detection results of 4 of the loci included in the kit

[0100] The above detection results prove that using the 2 mandatory loci BAT-25 and BAT-26 designed in this example, combined with the 7 locus primer pairs of NR-24, MONO-27, CAT-25, BAT-59, and BAT-60 for capillary electrophoresis detection of clinical samples of endometrial cancer with MSS, MSI-L, and MSI-H is consistent with the clinical sample detection results. At the same time, it can identify single-base mutations, has high sensitivity, accurate interpretation, and a coincidence rate of 100%

[0101] Example 2 Detection of 7 MSI loci of endometrial cancer in clinical samples based on PCR

[0102] In this example, 7 pairs of primer combinations for 7 genes are detected for microsatellite loci. 2 mandatory loci, BAT-25 and BAT-26, are combined with 5 combined loci such as NR-24, MONO-27, CAT-25, BAT-59, and BAT-60 for MSI PCR detection. By manually comparing the specificity of the sequences in the NCBI database, non-specific primers are eliminated. At the same time, considering the interference between primers, the primers of each combination are compared with Primer 5.0 software. Considering that the absolute value of DeltaG (ΔG) should not exceed 9 kcal / mol, the primers are fully optimized to improve the detection sensitivity

[0103] In this example, the drop off method is adopted to ensure that the specificity and synthesis purity of the primer design meet the requirements of PCR amplification accuracy. The primer purification is double purification, and DHPLC is selected for purification. 2 inosines (I) and a stem-loop structure (sequence: CGCTCACTTCGAGCG) are innovatively added in the primer design to enhance the binding of the primer to the target sequence while reducing the binding efficiency with other non-specific targets, realizing multiplex PCR detection. At the same time, the primers with 2 inosines and a stem-loop structure have lower background and higher detection sensitivity for detection, are compatible with longer sequences, exclude 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, thus realizing accurate MSI detection

[0104] The sequences of the primers and the target coverage segments for MSIPCR detection of the 7 combined loci involved in this embodiment are as follows:

[0105] The nucleotide sequences of the upstream and downstream primers of locus BAT-25 are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.3;

[0106] The nucleotides of the upstream and downstream primers of locus BAT-26 are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.6;

[0107] The nucleotide sequences of the upstream and downstream primers of locus NR-24 are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.9;

[0108] The nucleotide sequences of the upstream and downstream primers of locus MONO-27 are shown in SEQ ID NO.10 and SEQ ID NO.11 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.12;

[0109] The nucleotide sequences of the upstream and downstream primers of locus CAT-25 are shown in SEQ ID NO.13 and SEQ ID NO.14 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.15;

[0110] The nucleotide sequences of the upstream and downstream primers of locus BAT-59 are shown in SEQ ID NO.16 and SEQ ID NO.17 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.18;

[0111] The nucleotides of the upstream and downstream primers of locus BAT-60 are shown in SEQ ID NO.19 and SEQ ID NO.20 respectively, and the nucleotide sequence of the plasmid (DP&CP) of the target coverage segment of this locus is shown in SEQ ID NO.21.

[0112] The schematic diagrams of the inosine and stem-loop structures linked in the primers are as ​ shown.

[0113] 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. Considering the cost and detection sensitivity comprehensively, the primer concentration in the amplification reagent is optimized. In the amplification reagent, the final concentration of the upstream and downstream primers for a total of 7 MSI loci, namely 2 + 5, can be selected within the range of 50 nmol / L - 1500 nmol / L, such as 50 nmol / L, 100 nmol / L, 500 nmol / L, or 900 nmol / L, etc.

[0114] In the amplification reagent of this example, the mandatory loci BAT - 25 and BAT - 26, and a combination of 5 loci including NR - 24, MONO - 27, CAT - 25, BAT - 59, and BAT - 60. The final concentration of the upstream and downstream primers for a total of 7 MSI loci, namely 2 + 5, is 350 nmol / L for BAT - 25 and BAT - 26, 400 nmol / L for NR - 24, MONO - 27, and CAT - 25, and 550 nmol / L for BAT - 59 and BAT - 60.

[0115] In this example, the experiment operation, parameter setting, and data reading and analysis are carried out according to the experimental instructions of the BioRad QX200 droplet digital PCR system. The main operation steps are as follows:

[0116] (1) Thaw the upstream and downstream primers of the loci BAT - 25, BAT - 26, NR - 24, MONO - 27, CAT - 25, BAT - 59, BAT - 60, and the Bio - Rad ddPCR TM Supermix for Probes (product number 1863023) at room temperature. Invert and mix all reagents and samples well, centrifuge them, and then place them on the tube rack. Prepare the PCR reaction mixtures according to the experimental protocol for detection respectively, as shown in Table 6 - Table 8:

[0117] Table 6 Detection of the PCR reaction mixture for the BAT - 25 and BAT - 26 loci:

[0118]

[0119]

[0120] Table 7 Detection of the PCR reaction mixture for the NR - 24, MONO - 2, and CAT - 25 loci:

[0121] ​ ​ <![CDATA[ddPCR TM Supermix for Probes (without dUTP)]]> 10 ​ 0.8 ​ 0.8 ​ 1 <![CDATA[ddH2O]]> 7.4 ​ 20

[0122] Table 8 Detection of the PCR reaction mixture for the BAT - 59 and BAT - 60 loci:

[0123] ​ ​ <![CDATA[ddPCR TM Supermix for Probes (without dUTP)]]> 10 ​ 1.1 ​ 1.1 ​ 1 <![CDATA[ddH2O]]> 6.8 ​ 20

[0124] In this example, the clinical samples were provided by Nanjing Drum Tower Hospital (Drum Tower Hospital Affiliated to Nanjing University School of Medicine), and the MSI status of endometrial cancer was determined to be three types: MSS, MSI-L, and MSI-H. The sample sizes of MSS, MSI-L, and MSI-H were 54 cases, 72 cases, and 88 cases respectively. After nucleic acid extraction, they were stored at -20°C. They were divided into endometrial cancer tumor tissues and normal tissues, and the normal tissues were used as controls.

[0125] After the reaction mixture was prepared, it was mixed well, centrifuged, and then placed on a tube rack.

[0126] (2) Operate according to the droplet generation experimental procedure of Bio-Rad, that is, generate droplets at a ratio of droplet generation oil: reaction mixture = 70 μL: 20 μL. After droplet generation, take out the cartridge holder, remove and discard the gasket. 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 pierceable sealing foil.

[0127] (3) After sealing, place the reaction plate in a PCR amplifier. Considering factors such as the Tm value and the difficult sequence with more PolyA in the LMR sequence for endometrial cancer, perform PCR amplification, and the reaction program is shown in Table 9.

[0128] Table 9 PCR amplification program

[0129]

[0130] (4) Store the PCR reaction plate in the dark and place it at room temperature for 2 hours to stabilize the droplets. After that, perform data reading and analysis.

[0131] (5) The detection data analysis results of endometrial cancer MSS clinical samples are as follows:

[0132] Because it involves the screening of concentration ranges, in this example, 7 primer pairs with low, high, and appropriate concentrations at MSI loci were selected to detect clinical samples of MSI-H (samples with important immunotherapeutic significance).

[0133] The final concentrations of the upstream primers and downstream primers of the 7 MSI loci are 50 nmol / L for BAT-25 and BAT-26, 80 nmol / L for NR-24, MONO-27, and CAT-25, and 100 nmol / L for BAT-59 and BAT-60. The detection data analysis results of MSI-H are as ​ shown.

[0134] The final concentrations of the upstream and downstream primers for 7 MSI loci are 1500 nmol / L for BAT-25 and BAT-26, 1200 nmol / L for NR-24, MONO-27, and CAT-25, and 850 nmol / L for BAT-59 and BAT-60. The analysis results of the MSI-H test data are as follows ​ as shown

[0135] The appropriate final concentrations of the upstream and downstream primers for 7 MSI loci are 350 nmol / L for BAT-25 and BAT-26, 560 nmol / L for NR-24, MONO-27, and CAT-25, and 850 nmol / L for BAT-59 and BAT-60. The analysis results of the test data for clinical samples of endometrial cancer MSS are as follows ​ as shown

[0136] The analysis results of the test data for clinical samples of endometrial cancer MSI-L are as follows ​ as shown

[0137] The analysis results of the test data for clinical samples of endometrial cancer MSI-H are as follows ​ as shown

[0138] Based on the above results, it can be seen that in the test kit designed in this embodiment, BAT-25 and BAT-26 are mandatory loci, and 5 loci such as the combination of NR-24, MONO-27, CAT-25, BAT-59, and BAT-60. These 7 MSI loci in total of "2 + 5" are consistent with the test results of clinical samples for endometrial cancer MSS (no microsatellite loci have changed), MSI-L (only 1 microsatellite locus of the BAT-26 locus has changed), and MSI-H (microsatellite loci of BAT-25, BAT-26, NR-24, MONO-27, CAT-25, BAT-59, and BAT-60 microsatellite loci have changed). Compared with the Promega MSI test kit that can only be used for capillary electrophoresis detection, the PCR-based detection of this application can identify single-base variations, with a coincidence rate of 100%, and has the characteristics of accurate interpretation, high sensitivity, specificity, and repeatability.

[0139] Example 3: PCR-based clinical sample detection of 5 MSI loci in endometrial cancer and detection of MSI WT and Ins quality control products

[0140] In order to reduce the detection time period and the cost of detecting materials, while ensuring the detection sensitivity and specificity, in this embodiment, 5 pairs of primer combinations for 5 genes at microsatellite loci are used for detection. Among them, 2 loci, BAT-25 and BAT-26, are mandatory loci, and 3 combined loci, MONO-27, CAT-25, and BAT-60, are used for MSI PCR detection, with a total of 5 combined loci. At the same time, the primers are fully optimized. Innovatively, 2 inosines (I) and a stem-loop structure (sequence: CGCTCACTTCGAGCG) are added in the primer design, which enhances the binding of the primer to the target sequence while reducing the binding efficiency with other non-specific targets, realizing multiplex PCR detection. At the same time, the primers with 2 inosines and a stem-loop structure have lower background and higher detection sensitivity for detection, are compatible with longer sequences, exclude 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, thus realizing the accurate detection of different states of MSI Del and Ins.

[0141] In this embodiment, the experimental operation process, the primers with 2 inosines (I) and a stem-loop structure (sequence: CGCTCACTTCGAGCG) used at the target loci, and the sequence (plasmid) of the target coverage section are the same as those in Example 2. The appropriate final concentrations of the upstream and downstream primers at 5 MSI loci are 350 nmol / L for BAT-25 and BAT-26, 400 nmol / L for MONO-27 and CAT-25, and 550 nmol / L for BAT-60. The analysis results of the detection data for MSS, MSI-L, and MSI-H in this embodiment are as follows ​ shown, and the analysis results of the detection data for MSI WT and Ins quality control products are as ​ shown.

[0142] In this embodiment, 5 loci are selected for detection, which saves more reagent and material costs than the detection of 7 loci in Example 2, can also save the detection period, while ensuring the detection sensitivity and specificity, realizing the detection with an extremely short period, and meeting the detection requirements of multiple clinical samples.

[0143] From the above results, it can be seen that the 2 mandatory loci, BAT-25 and BAT-26, and the 3 combined loci, MONO-27, CAT-25, and BAT-60, a total of 5 MSI loci, have consistent detection results for MSS, MSI-L, and MSI-H of endometrial cancer in clinical samples. Compared with the existing MSI detection kits that can only be used for capillary electrophoresis detection, the detection kit of this application based on PCR can identify single-base mutations and simultaneously detect different states of MSI Del and Ins, with low cost, short time period, and a coincidence rate of 100%, having the characteristics of accurate interpretation, high sensitivity, specificity, and repeatability.

Claims

1. A multiplex PCR detection kit based on microsatellite instability loci of endometrial cancer, characterized in that, The detection kit includes a primer set and a PCR premix; The primer set includes primers for detecting at least one locus among BAT-25, BAT-26, NR-24, MONO-27, CAT-25, BAT-59, and BAT-60; The primers in the primer set all contain an inosine-stem loop structure; the inosine-stem loop structure consists of 2 inosines near the 5'-end of the primer and a stem loop structure near the 3'-end of the primer, and the nucleotide sequence of the stem loop structure is as shown in SEQ ID NO.

25.

2. The multiplex PCR detection kit based on microsatellite instability sites of endometrial cancer according to claim 1, wherein The primer set includes: Primers for detecting loci BAT-25 and BAT-26; and, Primers for detecting at least two loci among NR-24, MONO-27, CAT-25, BAT-59, and BAT-60.

3. The multiplex PCR detection kit based on microsatellite instability sites of endometrial cancer according to claim 1, wherein The primer set includes: 1) Primers for detecting loci BAT-25 and BAT-26; and, 2) Primers for detecting at least three loci among NR-24, MONO-27, CAT-25, BAT-59, and BAT-60.

4. The multiplex PCR detection kit based on microsatellite instability sites of endometrial cancer according to claim 1, wherein The primer set consists of primers for detecting loci BAT-25, BAT-26, NR-24, MONO-27, CAT-25, BAT-59, and BAT-60.

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

6. The multiplex PCR detection kit based on microsatellite instability sites of endometrial cancer according to any one of claims 1-4, characterized in that The detection kit further includes a quality control product containing a target site coverage section.

7. The multiplex PCR detection kit based on microsatellite instability sites of endometrial cancer according to any one of claims 1-4, characterized in that, The PCR premix 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 microsatellite instability sites of endometrial cancer according to any one of claims 1-4, characterized in that, The inosine is 10 - 25 bp from the 5'-end of the primer.

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