Diagnostic kit

Abstract

The invention provides a diagnostic kit which comprises a first amplification primer or a second amplification primer, and the first amplification primer comprises a first primer and a second primer which take a first fusion gene subtype as a target spot. Two novel fusion gene subtypes of the PTPRK-RSPO3 fusion gene are found, a first amplification primer and a second amplification primer are correspondingly designed for the three fusion gene subtypes in combination with an existing fusion gene subtype, a set of comprehensive and accurate PCR detection system is constructed, the detection system adopts an innovative primer design strategy and optimized PCR reaction conditions, and the detection accuracy is improved. Various subtypes of the PTPRK-RSPO3 fusion gene can be effectively identified, including newly found subtypes, and the multiple PCR technology is combined, so that the detection range of the original detection means is expanded, and the simultaneous detection and distinguishing of the multiple subtypes are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a diagnostic kit. Background Art

[0002] Colorectal cancer (CRC) is one of the malignant tumors. In recent years, with the rapid development of molecular biology technology, people have a deeper understanding of the molecular mechanism of colorectal cancer. Among them, gene fusion, as an important cancer driver, plays a key role in the occurrence, development and prognosis of colorectal cancer. The PTPRK-RSPO3 fusion gene is an important gene fusion found in colorectal cancer in recent years. The PTPRK (Protein Tyrosine Phosphatase Receptor Type K) gene encodes a protein tyrosine phosphatase receptor that participates in cell-to-cell adhesion and signal transduction. The RSPO3 (R-spondin3) gene encodes R-spondin family proteins and is an important activator of the Wnt signaling pathway. The formation of the PTPRK-RSPO3 fusion gene leads to upregulation of RSPO3 gene expression, thereby activating the Wnt signaling pathway and promoting tumor cell proliferation and invasion.

[0003] Traditional gene fusion detection methods, such as fluorescence in situ hybridization (FISH) and RNA sequencing, have certain limitations in terms of sensitivity, specificity, cost and operational complexity. Polymerase chain reaction (PCR) technology has been widely used in gene detection and fusion gene identification due to its advantages such as high sensitivity, high specificity, simple operation and low cost.

[0004] However, due to the heterogeneity of colorectal cancer, there may be multiple subtypes of the PTPRK-RSPO3 fusion gene, which may differ in structure, function and clinical characteristics. There is currently a lack of a systematic and comprehensive PCR detection system for the various subtypes of the PTPRK-RSPO3 fusion gene. Summary of the invention

[0005] In view of this, the present invention provides a diagnostic kit, including a first amplification primer or a second amplification primer, the first amplification primer is used to detect whether it belongs to a fusion subtype group, the fusion subtype group includes a first fusion gene subtype, a second fusion gene subtype and a third fusion gene subtype, the second amplification primer is used to detect whether it is the first fusion gene subtype, the second fusion gene subtype or the third fusion gene subtype, the first amplification primer includes a first primer and a second primer targeting the first fusion gene subtype, and the first fusion gene subtype is formed by fusion of PTPRK exon 1, RSPO3 exon 2 and its downstream sequence.

[0006] Furthermore, the second fusion gene subtype is formed by the fusion of PTPRK exon 6 and its upstream sequence with RSPO3 exon 2 and its downstream sequence.

[0007] Furthermore, the first primer and the second primer are both designed within the conserved region of the target gene, the lengths of the first primer and the second primer are both 15bp to 28bp, the GC contents of the first primer and the second primer are both 45% to 55%, and there is no complementary sequence between the first primer and the second primer.

[0008] Furthermore, the first primer is designed with PTPRK exon 1 as the target gene, and the second primer is designed with RSPO3 exon 2 as the target gene.

[0009] Furthermore, the first primer is designed with the nucleotide sequence shown in SEQ ID NO.1 as the target gene, and the second primer is designed with the nucleotide sequence shown in SEQ ID NO.3 as the target gene.

[0010] Furthermore, the nucleotide sequence of the first primer is shown as SEQ ID NO.6, and the nucleotide sequence of the second primer is shown as SEQ ID NO.7.

[0011] Furthermore, the sequence of the first fusion gene subtype is shown as SEQ ID NO.4, and the sequence of the second fusion gene subtype is shown as SEQ ID NO.5.

[0012] Furthermore, the second amplification primer includes a third primer and a fourth primer, the third primer is designed with PTPRK exon 1 or PTPRK exon 6 as the target gene, and the fourth primer is designed with RSPO3 exon 2 as the target gene.

[0013] Furthermore, the nucleotide sequence of the third primer is shown as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13, and the nucleotide sequence of the fourth primer is shown as SEQ ID NO.10.

[0014] Furthermore, the diagnostic kit is a colorectal cancer diagnostic kit.

[0015] Compared with the prior art, the beneficial effect of the present application lies in that, through in-depth research on the expression characteristics of the PTPRK-RSPO3 fusion gene in colorectal cancer patients, two new fusion gene subtypes of the PTPRK-RSPO3 fusion gene were discovered, namely the first fusion gene subtype and the second fusion gene subtype, and combined with an existing fusion gene subtype, namely the third fusion gene subtype, specific first amplification primers and second amplification primers were designed for the three, and a comprehensive and accurate PCR detection system was constructed. The detection system adopts innovative primer design strategies and optimized PCR reaction conditions, which can effectively identify PTPRK-RSPO3 fusion gene subtypes and clarify the types of subtypes, including newly discovered subtypes. It combines multiple PCR technology to expand the detection range of the original detection means and realize the simultaneous detection and differentiation of multiple subtypes.

[0016] Details of one or more embodiments of the present application are set forth in the following drawings and description to make other features, objects, and advantages of the present application more readily apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the fusion gene subtypes in the present invention, wherein, Figure 1 A is a schematic diagram of the first fusion gene subtype in the present invention: wherein the grid square on the left represents PTPRK exon 1 (exon1), and the shaded square on the right represents the RSPO3 exon 2 (exon2) fragment and its downstream sequence (exon3-6); Figure 1 B is a schematic diagram of the second fusion gene subtype in the present invention: wherein the grid square on the left represents PTPRK exon 6 (exon6) and its upstream sequence (exon1-5), and the shaded square on the right represents RSPO3 exon 2 (exon2) fragment and its downstream sequence (exon3-6);

[0018] Figure 2 The results of the identification of PTPRK::RSPO3 fusion gene subtypes in seven patients with colorectal cancer (CRC) by PCR in the present invention: six patients were PTPRK::RSPO3 e1-e2 type, and one patient was PTPRK::RSPO3 e6-e2 type;

[0019] Figure 3 The results of the specificity and amplification efficiency tests of five PCR amplification primers for the three PTPRK-RSPO3 fusion gene subtypes in the present invention;

[0020] Figure 4 It is the Sanger sequencing result of the PCR amplification product of the docking fragments of the three subtype fusion transcripts of PTPRK::RSPO3 e1-e2, e6-e2 and e7-e2 in the present invention, wherein, Figure 4 A is the sequencing result of the docking fragment of the e1-e2 subtype fusion transcript. Figure 4 B is the sequencing result of the docking fragment of the e6-e2 subtype fusion transcript. Figure 4 C is the sequencing result of the docking fragment of the e7-e2 subtype fusion transcript;

[0021] Figure 5 is a graph showing the agarose gel electrophoresis verification result of the cloned fragments in the present invention, wherein: Figure 5 A is the agarose gel electrophoresis verification result of the cloned fragments of PTPRK e1-e6 fragment, PTPRK e1-e7 fragment and RSPO3 e2-e5 in the present invention; Figure 5 B is the agarose gel electrophoresis verification result of the cloned fragment of the PTPRK::RSPO3 e1-e2 fusion gene subtype in the present invention; Figure 5 C is the agarose gel electrophoresis verification result of the cloned fragments of the PTPRK::RSPO3 e6-e2 and PTPRK::RSPO3 e7-e2 fusion gene subtypes in the present invention;

[0022] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0023] The embodiments of the present invention disclose a diagnostic kit, and those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The diagnostic kit described in the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the diagnostic kit described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0024] The diagnostic kit of the present invention includes a first amplification primer or a second amplification primer, the first amplification primer is used to detect whether it belongs to a fusion subtype group, the fusion subtype group includes a first fusion gene subtype, a second fusion gene subtype and a third fusion gene subtype, the second amplification primer is used to detect whether it is the first fusion gene subtype, the second fusion gene subtype or the third fusion gene subtype, the first amplification primer includes a first primer and a second primer with the first fusion gene subtype as a target, the first fusion gene subtype is formed by fusion of PTPRK exon 1, RSPO3 exon 2 and its downstream sequence, the first fusion gene subtype is PTPRK exon1-RSPO3 exon2 fusion gene subtype (abbreviated as PTPRK::RSPO3 e1-e2). The second fusion gene subtype is formed by fusion of PTPRK exon 6 and its upstream sequence, RSPO3 exon 2 and its downstream sequence, the second fusion gene subtype is PTPRK exon6-RSPO3exon2 fusion gene subtype (abbreviated as PTPRK::RSPO3 e6-e2 ) . It is now known that there is another subtype of the PTPRK-RSPO3 fusion gene, namely the third fusion gene subtype, specifically PTPRK exon 7 (exon7), which is fused with RSPO3 exon 2 (exon2) and its downstream sequence, named PTPRK exon7-RSPO3 exon2 fusion gene subtype, abbreviated as PTPRK::RSPO3e7-e2.

[0025] The mRNA code of the PTPRK gene in the GeneBank database is NM_001291981.2, and the mRNA code of the RSPO3 gene in the GeneBank database is NM_032784.5. Among them, the nucleotide sequence of the PTPRK exon 1 (exon1) is shown in SEQ ID NO.1, the nucleotide sequence of the PTPRK gene exon 6 (exon6) is shown in SEQ ID NO.2, and the nucleotide sequence of the RSPO3 gene exon 2 (exon2) is shown in SEQ ID NO.3. The sequence of the first fusion gene subtype is shown in SEQ ID NO.4, and the sequence of the second fusion gene subtype is shown in SEQ ID NO.5. The schematic diagram of the first fusion gene subtype is shown in Figure 1 As shown in A, the schematic diagram of the second fusion gene subtype is as follows Figure 1 As shown in B.

[0026] The present invention uses high-throughput sequencing technology and real-time quantitative PCR (RT-qPCR) to detect two PTPRK-RSPO3 fusion gene subtypes in seven cases whose clinical manifestations and pathologies are consistent with CRC, namely PTPRK::RSPO3 e1-e2 and PTPRK::RSPO3 e6-e2, which are variant CRC subtypes discovered for the first time. Therefore, the present invention proposes the use of the fusion gene subtype in the preparation of a diagnostic kit for related CRC using it as a detection target. In order to be able to simultaneously verify and detect patients' PTPRK::RSPO3 e1-e2, PTPRK::RSPO3 e6-e2, and PTPRK::RSPO3 e7-e2, the present invention provides the first amplification primer comprising the first primer (forward primer) and the second primer (reverse primer), the nucleotide sequence of the first primer is shown in SEQ ID NO.6, the nucleotide sequence of the second primer is shown in SEQ ID NO.7, and the detection result is shown in Figure 2 As shown. In the results, six patients were PTPRK::RSPO3 e1-e2 type, and one patient was PTPRK::RSPO3 e6-e2 type. The first primer and the second primer are both designed in the conserved region of the target gene, the length of the first primer and the second primer are both 15bp~28bp, the GC content of the first primer and the second primer are both 45%~55%, there is no complementary sequence between the first primer and the second primer, and the annealing temperature of the first primer and the second primer is the Tm value. The first primer is designed with PTPRK exon 1 as the target gene, and the second primer is designed with RSPO3 exon 2 as the target gene. Specifically, the first primer is designed with the nucleotide sequence shown in SEQ ID NO.1 as the target gene, and the second primer is designed with the nucleotide sequence shown in SEQ ID NO.3 as the target gene. The first amplification primer can be used to detect whether the patient belongs to the fusion subtype group, but on this basis, it is necessary to further determine which specific subtype the patient belongs to.

[0027] In order to construct primers that can simultaneously detect three subtypes, the present invention constructs plasmids of three subtypes of PTPRK-RSPO3 fusion genes and expands the second amplification primers on the basis of the above, that is, the second amplification primers are obtained based on the first fusion gene subtype, the second fusion gene subtype and the third fusion gene subtype. There are five second amplification primers, and the second amplification primers include a third primer (forward primer) and a fourth primer (reverse primer). The third primer is designed with PTPRK exon 1 or PTPRK exon 6 as the target gene, and the fourth primer is designed with RSPO3 exon 2 as the target gene. It can be understood that the third primer is designed with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 as the target gene, and the fourth primer is designed with the nucleotide sequence shown in SEQ ID NO.3 as the target gene.

[0028] The five second amplification primers are specifically: the nucleotide sequence of the third primer is shown in SEQ ID NO.8, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO.10; the nucleotide sequence of the third primer is shown in SEQ ID NO.9, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO.10; the nucleotide sequence of the third primer is shown in SEQ ID NO.11, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO.10; the nucleotide sequence of the third primer is shown in SEQ ID NO.12, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO.10; the nucleotide sequence of the third primer is shown in SEQ ID NO.13, and the nucleotide sequence of the fourth primer is shown in SEQ ID NO.10. In the present invention, the specificity and amplification efficiency of the five expanded second amplification primers were detected, and the detection results are as follows. Figure 3 By using the second amplification primer, it is possible to further determine whether the patient belongs to the first fusion gene subtype, the second fusion gene subtype or the third fusion gene subtype based on knowing that the patient belongs to the fusion subtype group.

[0029] In order to verify and detect the docking sequences of the three subtypes of fusion transcripts of PTPRK-RSPO3 fusion gene e1-e2, e6-e2 and e7-e2, the PCR amplification products of the docking fragments of the three subtypes of fusion transcripts were subjected to Sanger sequencing. The sequencing results are shown in Figure 4The results showed that the docking fragment of the e1-e2 subtype fusion transcript was formed by the fusion of PTPRK exon 1 and RSPO3 exon 2 and its downstream sequence, the docking fragment of the e6-e2 subtype fusion transcript was formed by the fusion of PTPRK exon 6 and its upstream sequence and RSPO3 exon 2 and its downstream sequence, and the docking fragment of the e7-e2 subtype fusion transcript was formed by the fusion of PTPRK exon 7 and its upstream sequence and RSPO3 exon 2 and its downstream sequence.

[0030] In order to subsequently study the functions of the three subtypes of PTPRK-RSPO3 fusion genes and the standards required for the PTPRK-RSPO3 fusion gene detection kit, the full-length coding sequences of the three subtypes of PTPRK::RSPO3 e1-e2, e6-e2, and e7-e2 fusion genes were cloned, among which PTPRK::RSPO3 e1-e2 was directly PCR cloned using the third amplification primer. The third amplification primer included a fifth primer and a sixth primer. The nucleotide sequence of the fifth primer (forward primer) is shown in SEQ ID NO.14, and the nucleotide sequence of the sixth primer (reverse primer) is shown in SEQ ID NO.15. The PTPRKe1-e6 fragment, PTPRK e1-e7 fragment and RSPO3 e2-e5 fragment were first cloned using the fourth amplification primer, the fifth amplification primer and the sixth amplification primer respectively. The fourth amplification primer includes the fifth primer and the seventh primer, and the nucleotide sequence of the seventh primer (reverse primer) is shown in SEQ ID NO.16. The fifth amplification primer includes the fifth primer and the eighth primer, and the nucleotide sequence of the eighth primer (reverse primer) is shown in SEQ ID NO.17. The sixth amplification primer includes the ninth primer and the sixth primer, and the nucleotide sequence of the ninth primer (forward primer) is shown in SEQ IDNO.18. Subsequently, the third amplification primer and overlapping extension PCR technology are used to obtain PTPRK::RSPO3 e6-e2 and e7-e2 subtypes; the identification results of the cloned PCR products are shown in Figure 5 shown.

[0031] Through in-depth research on the expression characteristics of the PTPRK-RSPO3 fusion gene in colorectal cancer patients, this application discovered two new fusion gene subtypes of the PTPRK-RSPO3 fusion gene, namely the first fusion gene subtype and the second fusion gene subtype, and combined with an existing fusion gene subtype, namely the third fusion gene subtype, designed specific first amplification primers and second amplification primers for the three, and constructed a comprehensive and accurate PCR detection system. This detection system adopts innovative primer design strategies and optimized PCR reaction conditions, which can effectively identify PTPRK-RSPO3 fusion gene subtypes and clarify the types of subtypes, including newly discovered subtypes. It combines multiple PCR technology to expand the detection range of the original detection means and realize the simultaneous detection and differentiation of multiple subtypes.

[0032] Example 1: Analysis and verification of clearly diagnosed cases

[0033] Patient 1: A 75-year-old female patient was admitted to the hospital due to stool bandage for more than 2 months. Routine pathology report: Specimen site: rectum; Tumor size: 3.8×2.4×0.8cm; Gross classification: Type III ulcerative type; Histological classification: Moderately differentiated adenocarcinoma; Tumor budding: low grade (Bd1); Infiltration depth: muscularis propria (T2); Vascular cancer thrombus: None; Peripheral invasion: None; Involvement of resection margins: (both resection ends, circumferential resection margins, and upper and lower resection margins) were not involved in cancer. Lymph nodes: 7 (paraintestinal lymph nodes), none of which had cancer metastasis (0 / 7). Immunohistochemistry showed: Mismatch repair proteins: MLH1 (+); MSH2 (+); MSH6 (+); PMS2 (+). Prognosis-related proteins: P53 (3+, indicating missense mutation); Ki-67 (70%+). Targeted drug-related protein: CerbB2 (0). Sanger sequencing report: Microsatellite type: microsatellite stable (MSS). This test simultaneously extracted cancer tissue and normal tissue from the same individual, and tested the above 6 sites respectively. All 12 sites were analyzed to determine the microsatellite status of the sample. The judgment criteria are as follows: MSS: none of the five microsatellite sites have changed; MSI-L: only one of the five microsatellite sites has changed; MSI-H: ≥2 of the five microsatellite sites have changed. The patient was diagnosed with rectal cancer.

[0034] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon1-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 1 (exon1) and RSPO3 exon 2 (exon2) and their downstream sequences.

[0035] Patient 2: Male patient, 79 years old, was admitted to the hospital due to intermittent abdominal pain for half a year, constipation for 1 month, and nausea for 2 days. Routine pathology report: Specimen location: right colon; Tumor size: 5×4×2cm; Gross classification: Type I raised type; Histological classification: 60% mucinous adenocarcinoma + 40% ordinary adenocarcinoma; Tumor grade: moderate to poorly differentiated; Tumor budding: high grade (Bd3); Depth of invasion: subserous or colorectal surrounding tissues not covered by peritoneum (T3); Vascular cancer embolus: yes; Peripheral invasion: yes; Resection margin involvement: (colon resection end, ileum resection end, 3cm cancer) No cancer was found in the paraintestinal lymph nodes; Lymph nodes: 15 (paraintestinal lymph nodes), of which 4 showed cancer metastasis (4 / 15); Other lesions: (appendix) chronic inflammation; Immunohistochemistry: mismatch repair protein: MLH1 (+); MSH2 (+); MSH6 (+); PMS2 (+); suggesting pMMR; Prognosis-related protein: P53 (+, mutant); ki-67 (90% +); Targeted drug-related protein: CerbB2 (0). Sanger sequencing report: Microsatellite type: microsatellite stable (MSS). This test simultaneously extracted cancer tissue and normal tissue from the same individual, and tested the above 6 sites respectively. The microsatellite status of the sample was determined by analyzing all 12 sites. The judgment criteria are as follows: MSS: none of the five microsatellite sites changed; MSI-L: only 1 of the five microsatellite sites changed; MSI-H: ≥2 of the five microsatellite sites changed. The patient was diagnosed with colon cancer.

[0036] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon1-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 1 (exon1) and RSPO3 exon 2 (exon2) and their downstream sequences.

[0037] Patient 3: A 60-year-old male patient was admitted to the hospital due to upper abdominal pain for more than half a month. Routine pathology report: Specimen location: right colon; Tumor size: 5×4×2cm; Gross classification: Type I raised type; Histological classification: 60% mucinous adenocarcinoma + 40% common adenocarcinoma; Tumor grade: moderate to poorly differentiated; Tumor budding: high grade (Bd3); Depth of invasion: subserous or colorectal surrounding tissue not covered by peritoneum (T3); Vascular cancer embolus: yes; Peripheral invasion: yes; Resection margin involvement: (colon resection end, ileum resection end, 3cm cancer) No cancer was found in the paraintestinal lymph nodes; Lymph nodes: 15 (paraintestinal lymph nodes), of which 4 showed cancer metastasis (4 / 15); Other lesions: (appendix) chronic inflammation; Immunohistochemistry: mismatch repair protein: MLH1 (+); MSH2 (+); MSH6 (+); PMS2 (+); suggesting pMMR; Prognosis-related protein: P53 (+, mutant); ki-67 (90% +); Targeted drug-related protein: CerbB2 (0). Sanger sequencing report: Microsatellite type: microsatellite stable (MSS). This test simultaneously extracted cancer tissue and normal tissue from the same individual, and tested the above 6 sites respectively. The microsatellite status of the sample was determined by analyzing all 12 sites. The judgment criteria are as follows: MSS: none of the five microsatellite sites changed; MSI-L: only 1 of the five microsatellite sites changed; MSI-H: ≥2 of the five microsatellite sites changed. The patient was diagnosed with colon cancer.

[0038] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon1-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 1 (exon1) and RSPO3 exon 2 (exon2) and their downstream sequences.

[0039] Patient 4: A 66-year-old female patient was admitted to the hospital due to stool discharge for more than 3 months. Routine pathology report: specimen location: ascending colon near ileocecal region; tumor size: 4×3.5×1.2cm; gross classification: type III ulcerative type; histological classification: conventional adenocarcinoma; tumor grade: moderately differentiated; tumor budding: high grade (Bd3); invasion depth: subserosa or colorectal tissues not covered by peritoneum (T3); vascular cancer embolus: no; neural invasion: yes; (ileum cut end, colon cut end, 3cm beside the mass, omental tissue) no cancer involvement at the resection margin; (paraintestinal lymph nodes) 14 nodes were seen, of which 2 nodes showed cancer metastasis (2 / 14); pathological stage: pT3N1bMx; immunohistochemistry showed: P53 (-, nonsense mutation expression); Ki-67 (90%+); C-erbB-2 (0); CD31 (-); D2-40 (-). Sanger sequencing results: microsatellite type: microsatellite stable (MSS). The patient was diagnosed with colon cancer.

[0040] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon1-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 1 (exon1) and RSPO3 exon 2 (exon2) and their downstream sequences.

[0041] Patient 5: A 73-year-old female patient was admitted to the hospital due to changes in stool characteristics for more than 3 months. Routine pathology report: specimen location: rectum; tumor size: 6×5.2×0.7cm; gross classification: type III ulcerative type; histological classification: conventional adenocarcinoma; tumor grade: moderately differentiated; tumor budding: low grade (Bd1); invasion depth: serosal surface (T4a); vascular cancer embolus: yes; resection margin: no cancer involvement; neural invasion: no; lymph nodes: (paraintestinal lymph nodes) 21, of which 3 showed cancer metastasis (3 / 21), and 7 cancer nodules were found; immunohistochemistry: mismatch repair protein: MLH1(+); MSH2(+); MSH6(+); PMS2(+), suggesting pMMR; prognosis-related proteins: P53 (mutant expression); Ki-67(90%+); targeted drug-related proteins: CerbB2(0); other immunohistochemical indicators: CK(3+). Sanger sequencing: Microsatellite type: Microsatellite stable (MSS); This test extracts cancer tissue and normal tissue from the same individual at the same time, and detects the above 6 sites respectively. The microsatellite status of the sample is determined by analyzing all 12 sites. The judgment criteria are as follows: MSS: None of the five microsatellite sites have changed; MSI-L: Only one of the five microsatellite sites has changed; MSI-H: ≥2 of the five microsatellite sites have changed. The patient was diagnosed with rectal cancer.

[0042] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon1-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 1 (exon1) and RSPO3 exon 2 (exon2) and their downstream sequences.

[0043] Patient 6: A 47-year-old male patient was admitted to the hospital more than 4 months after surgery for rectal malignancy and more than 1 month after the third chemotherapy. Routine pathology report: specimen location: rectum; tumor size: 5.5×4×1.3cm; gross classification: ulcerative type; histological classification: adenocarcinoma; tumor grade: moderately differentiated; tumor budding: low grade (Bd1); invasion depth: subadventitial fibrofatty tissue; vascular cancer embolus: none; neural invasion: yes; resection margin involvement: no cancer involvement was found in both resection ends, circumferential resection margin and the lower resection margin; lymph nodes: 33 paraintestinal lymph nodes, of which 2 showed cancer metastasis (2 / 33); pathological stage: pT3N1bMx; immunohistochemistry results: mismatch repair proteins: MLH1(+); MSH2(+); MSH6(+); PMS2(+); indicating no loss of mismatch repair protein expression; prognosis-related proteins: P53 (40% strong or weak +); Ki-67 (85%+); targeted drug-related proteins: CerbB2(0). Sanger sequencing results: Microsatellite type: Microsatellite stable (MSS); This test simultaneously extracted cancer tissue and normal tissue from the same individual, tested the above 6 sites respectively, and determined the microsatellite status of the sample by analyzing all 12 sites. The judgment criteria are as follows: MSS: None of the five microsatellite sites have changed; MSI-L: Only one of the five microsatellite sites has changed; MSI-H: More than or equal to two of the five microsatellite sites have changed. The patient was diagnosed with rectal cancer.

[0044] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon6-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 6 (exon6) and its upstream sequence, and RSPO3 exon 2 (exon2) and its downstream sequence.

[0045] Patient 7: Male patient, 62 years old, was admitted to the hospital due to blood in stool for more than half a year. Routine pathology report: specimen site: rectum; tumor size: 3.2×3×0.5cm; gross classification: raised type; histological classification: mucinous adenocarcinoma; tumor grade: moderately differentiated; tumor budding: low grade (Bd1); invasion depth: superficial muscle layer; vascular cancer thrombus: none; nerve invasion: none; margin involvement: (including circumferential margin) no cancer involvement was found in both resection ends, patient resection margins, and upper and lower resection margins; lymph nodes: 12 paraintestinal lymph nodes, no cancer metastasis was found (0 / 12). Immunohistochemistry: mismatch repair protein: MLH1 (3+); MSH2 (3+); MSH6 (3+); PMS2 (3+); prognosis-related protein: P53 (90%+, suggesting missense mutation); ki-67 (90%+ in hotspot); targeted drug-related protein: CerbB2 (0). The patient was diagnosed with rectal cancer.

[0046] The present invention performed transcriptome sequencing on rectal tumor samples and used STAR-Fusion software to search for potential fusion genes, detected the PTPRK exon1-RSPO3 exon2 fusion gene subtype, and found that the fusion gene was formed by the fusion of PTPRK exon 1 (exon1) and RSPO3 exon 2 (exon2) and their downstream sequences.

[0047] Example 2: Amplification Experiment

[0048] For the cases in Example 1 in which transcriptome sequencing detected new fusion gene subtypes of PTPRK exon1-RSPO3 exon2 and PTPRK exon6-RSPO3 exon2, the first amplification primer and the second amplification primer designed by the present invention were used for verification.

[0049] 1. RNA extraction: When using Trizol to extract RNA from colorectal cancer tumor tissue, first grind the tissue into powder in liquid nitrogen, add 1ml Trizol reagent for every 100mg tissue, and let it stand at room temperature for 5 minutes to fully separate the nucleoprotein complex. Then add 0.2ml chloroform, shake vigorously for 15 seconds, let it stand at room temperature for 3 minutes, and centrifuge at 4℃ for 15 minutes for phase separation. Carefully pipette the upper aqueous phase into a new tube, add an equal volume of isopropanol and mix gently, and let it stand at room temperature for 10 minutes. Centrifuge at 4℃ for 10 minutes to collect RNA precipitate, wash twice with 75% ethanol, dry for 5 minutes, and then add 30μl-50μl DEPC water to dissolve. Finally, measure the RNA concentration and purity and store at -80℃. The entire process needs to be performed in an RNase-free environment.

[0050] 2. Reverse transcription PCR (RT-PCR): RNA was reverse transcribed into cDNA according to the two-step operation procedure. The PCR amplification primer was the first amplification primer or the second amplification primer. The amplification system included 0.2ul of 5u / ul Taq enzyme, 2.5μL of 10× Taq Buffer (containing MgCl 2), 0.5ul of 10mmol / L dNTP (the above reagents were purchased from Promega Biotech, USA), 0.5ul of 10umol / L upstream and downstream primers, 150ng of cDNA template, and sterile deionized water added to 25μL. The PCR amplification conditions were denaturation at 95℃ for 10min, followed by 35 cycles at 94℃ for 30s, 60℃ for 30s, and 72℃ for 30s, and finally extension at 72℃ for 7min. The PCR product was electrophoresed with 2% agarose at a voltage of 100V, stained with GelRed (purchased from Biotium), and the results were observed under ultraviolet light and sent for sequencing.

[0051] 3. Results: As expected, the electrophoresis results after PCR using the primers of the present invention for detecting the PTPRK exon1-RSPO3 exon2 fusion gene subtype showed that there was a band of about 442 bp in length in colorectal cancer tissue (see Figure 2 , corresponding to patients 1, 2, 3, 4, 5, and 7). The PCR amplification products of the docking fragments of the PTPRK exon1-RSPO3 exon2 fusion transcripts were subjected to Sanger sequencing analysis to obtain the sequence of the docking fragments at the fusion site of the PTPRK exon1-RSPO3 exon2 fusion gene subtypes (see Figure 4 A), the sequence shows that the docking fragment of the fusion transcript is formed by fusion of PTPRK exon 1, RSPO3 exon 2 and its downstream sequence. The electrophoresis results after PCR using the primers of the present invention to detect the PTPRK exon6-RSPO3 exon2 fusion gene subtype showed that there was a band of about 1210 bp in colorectal cancer tissue (please refer to Figure 2 , corresponding to patient 6). The PCR amplification product of the docking fragment of the PTPRK exon6-RSPO3 exon2 fusion transcript was subjected to Sanger sequencing analysis to obtain the sequence of the docking fragment at the fusion site of the PTPRK exon6-RSPO3 exon2 fusion gene subtype (see Figure 4 B), the sequence showed that the docking fragment of the fusion transcript was formed by the fusion of PTPRK exon 6 and its upstream sequence with RSPO3 exon 2 and its downstream sequence. The PCR amplification product of the docking fragment of the PTPRK exon7-RSPO3 exon2 fusion transcript was subjected to Sanger sequencing analysis to obtain the sequence of the docking fragment at the fusion site of the PTPRK exon7-RSPO3 exon2 fusion gene subtype (see Figure 4 C).

[0052] The above description is only used to understand the method and core idea of ​​the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the rights of the present invention.

Claims

1. A diagnostic kit, characterized in that It includes a first amplification primer or a second amplification primer, the first amplification primer is used to detect whether it belongs to a fusion subtype group, the fusion subtype group includes a first fusion gene subtype, a second fusion gene subtype and a third fusion gene subtype, the second amplification primer is used to detect whether it is the first fusion gene subtype, the second fusion gene subtype or the third fusion gene subtype, the first amplification primer includes a first primer and a second primer targeting the first fusion gene subtype, the first fusion gene subtype is formed by fusion of PTPRK exon 1, RSPO3 exon 2 and its downstream sequence.

2. The diagnostic kit according to claim 1, characterized in that The second fusion gene subtype is formed by the fusion of PTPRK exon 6 and its upstream sequence with RSPO3 exon 2 and its downstream sequence.

3. The diagnostic kit according to claim 1, characterized in that The first primer and the second primer are both designed in the conserved region of the target gene, the length of the first primer and the second primer are both 15bp to 28bp, the GC content of the first primer and the second primer are both 45% to 55%, and there is no complementary sequence between the first primer and the second primer.

4. The diagnostic kit according to claim 1, characterized in that The first primer is designed with PTPRK exon 1 as the target gene, and the second primer is designed with RSPO3 exon 2 as the target gene.

5. The diagnostic kit according to claim 4, characterized in that The first primer is designed with the nucleotide sequence shown in SEQ ID NO.1 as the target gene, and the second primer is designed with the nucleotide sequence shown in SEQ ID NO.3 as the target gene.

6. The diagnostic kit according to claim 1, characterized in that The nucleotide sequence of the first primer is shown as SEQ ID NO.6, and the nucleotide sequence of the second primer is shown as SEQ ID NO.

7.

7. The diagnostic kit according to claim 2, characterized in that The sequence of the first fusion gene subtype is shown as SEQ ID NO.4, and the sequence of the second fusion gene subtype is shown as SEQ ID NO.

5.

8. The diagnostic kit according to claim 2, characterized in that The second amplification primer includes a third primer and a fourth primer, the third primer is designed with PTPRK exon 1 or PTPRK exon 6 as the target gene, and the fourth primer is designed with RSPO3 exon 2 as the target gene.

9. The diagnostic kit according to claim 8, characterized in that The nucleotide sequence of the third primer is shown as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13, and the nucleotide sequence of the fourth primer is shown as SEQ ID NO.

10.

10. The diagnostic kit according to claim 1, characterized in that The diagnostic kit is a colorectal cancer diagnostic kit.

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