Prostate cancer multi-target joint detection kit, detection system and application

Abstract

The invention provides a prostate cancer multi-target joint detection kit, a detection system and application, multiple targets in the kit are selected from an MSMB gene, an AMACR gene, a PCGEM1 gene, an MALAT1 gene and an MIR4435 gene, and the kit further comprises a primer and probe combination aiming at each marker. According to the method, the diagnosis accuracy can be improved, the weight interpretation model can be constructed, the clinical decision can be optimized, and a non-invasive and high-compliance auxiliary diagnosis tool is provided for the problems of traumatic detection and gray areas in the prior art.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, molecular genetics and detection technology, and in particular to detection technology, detection products and applications for the detection of human prostate cancer. Background Technology

[0002] Prostate cancer (PCa) is a malignant tumor that seriously threatens men's health. It ranks second in global cancer incidence and fifth in mortality, and is the fastest-growing male malignant tumor. Although the overall incidence and mortality rates of PCa in China are lower than the global average, its ranking among cancers is steadily rising, and its disease burden is showing a trend of shifting towards younger populations. In the early stages of prostate cancer, due to its insidious onset and slow growth, most patients are asymptomatic. As the tumor grows, prostate cancer can manifest as lower urinary tract obstruction symptoms, such as urinary frequency, urgency, slow urination, difficulty urinating, and even urinary retention or incontinence, severely impacting health and quality of life.

[0003] The most widely used auxiliary diagnostic methods for prostate cancer (PCa) in clinical practice include digital rectal examination (DRE), serum prostate-specific antigen (PSA) testing, and fusion detection using transrectal ultrasound of the prostate (TRUS) / magnetic resonance imaging (MRI). However, these methods each have limitations in sensitivity and specificity. Since most primary PCa occurs in the peripheral region of the gland, DRE can be used to examine local lesions within the gland. However, when the lesion is less than 0.2 mL or located within the gland, it cannot be detected by digital rectal examination. Richie's statistics show that the detection rate of prostate cancer in patients with abnormal DRE results is only 18%. The low sensitivity of this method as a single test leads to a high rate of missed diagnoses for PCa. Therefore, DRE must be used in conjunction with other diagnostic methods to fully realize its clinical value.

[0004] Currently, the diagnosis of prostate cancer mainly relies on serum PSA testing, combined with MRI imaging and biopsy techniques. However, firstly, the so-called "gold standard"—biopsy—usually requires about 12 needle pricks, a procedure that is not only painful but also carries a high risk of complications. Furthermore, while serum PSA testing and TRUS / MRI fusion testing have high sensitivity, they suffer from low specificity. Serum PSA is a substance secreted by the male prostate gland. Under normal circumstances, only extremely low levels of PSA are present in the blood. An increase in serum PSA concentration indicates pathological changes or trauma to the prostate. However, because PSA is organ-specific and not specific to prostate cancer (PCa), various causes such as prostatitis and benign prostatic hyperplasia (BPH) can cause elevated serum PSA levels. Serum PSA has low diagnostic specificity for PCa; in its gray zone (4-10 ng / mL, most suspected prostate cancer patients), the positive rate of biopsy is only 10-25%. MRI-TRUS fusion imaging provides MRI information from TRUS images to guide puncture. This technique combines the superior visibility of suspicious lesions by MRI with real-time guidance by TRUS, increasing the positive detection rate to 29.5%, but still very low.

[0005] In addition, although there are dual-target detection kits for PCA3 and PSA in the existing technology, they still need to be used in conjunction with MRI images and RT-qPCR technology to detect a single marker separately. The overall interpretation logic is not strong, and there is still much room for improvement in diagnostic accuracy. Moreover, the sample type is urine sample after digital rectal examination. The sample collection requires professional operation, which is cumbersome and has low compliance with both doctors and patients.

[0006] Therefore, there is a need for a detection method that is highly specific, accurate, user-friendly, and readily accepted by patients. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a multi-target combined detection kit, detection system and application for prostate cancer, which can improve diagnostic accuracy, build a weighted interpretation model, optimize clinical decision-making, and provide a non-invasive and highly compliant auxiliary diagnostic tool to address the invasive detection and "grey zone" problems in existing technologies.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0009] In a first aspect, the present invention provides a multi-target combined detection kit for prostate cancer, wherein the multi-targets in the kit are selected from the following biomarkers and combinations thereof:

[0010] The MSMB gene, whose selected sequence is shown in SEQ NO.1 of the sequence listing,

[0011] SEQ NO.1:

[0012] AATGTTCTCCTGGGCAGCGTTGTGATCTTTGCCACCTTCGTGACTTTATGCAATGCATCATGCTATTTCATACCTAATGAGGGAGTTCCAGGAGATTCAACCAGGAAATGCATGGATCTCAAAGGAAACAAACACCCAATAAACTCGGAGTGGCAGACTGACAACTGTGAGACATGCACTTGCTACGAAACAGAAATTTCATGTTGCACCCT;

[0013] The AMACR gene, the selected sequence of which is shown as SEQ NO.2 in the sequence list,

[0014] SEQ NO.2:

[0015] AGCCGCTTGGGCCGGGGCAAGCGCTCGCTAGTGCTGGACCTGAAGCAGCCGCGGGGAGCCGCCGTGCTGCGGCGTCTGTGCAAGCGGTCGGATGTGCTGCTGGAGCCCTTCCGCCGCGGTGTCATGGAGAAACTCCAGCTGGGCCCAGAGATTCTGCAGCGGGAAAATCCAAGGCTTATTTATGCCAGGCTGAGTGGATTTGGCCAGTCAGGAAGCTTCTGCCGGTTAGCTGGCCACGATATCAACTATTTGGCTTTGTCAGGTGTTCTCTCAAAAATTGGCAGAAGTGGTGAGAATCCGTATGCCCCGCTGAATCTCCTGGCTGACTTTGCTGGTGGTGGCCTTATGTGTGCACTGGGCATTATAATGGCTCTTTTTGACCGCACACGCACTGGCAAGGGTCAGGTCATTGATGCAAATATG;

[0016] The PCGEM1 gene, the selected sequence of which is shown as SEQ NO.3 in the sequence list,

[0017] SEQ NO.3:

[0018] ATGGAGTCTTGCCCTGTCTCCAAGGCTGGAGCCCAATGGTGTGATCTTGGCTCACTGCAACCTCCACCTCCCAGGTTCAAACGTTTCTCCTGCCTCAGCCTCCCAAGTAACTGGGATTACAGCAGGCTTGGTGCATTTGACACTTCATGATATCAGCCAAAGTGGAACTAAAAACAGCTCCTGGAAGAGGACTATGACATCATCAGGTTGGGAGTCTCCAGGGACAGCGGACCCTTTGGAAAAGGACTAGAAAGTGTGAAATCTATTAGTCTTCGATATGAAATTCTCTGTCTCTGTAAAAGCATTTCATATTTACAAGACACAGGCCTA;

[0019] The MALAT1 gene, whose selected sequence is shown as SEQ NO.4 in the sequence list,

[0020] SEQ NO.4:

[0021] CCCTGCAAGGCTGGGGCTCAGTTGCGTAATGGAAAGTAAAGCCCTGAACTATCACACTTTAATCTTCCTTCAAAAGGTGGTAAACTATACCTACTGTCCCTCAAGAGAACACAAGAAGTGCTTTAAGAGGCGGCGGAAGGTGATCGAATTCCGGTGATGCGAGTTGTTCTCCGTCTATAAATACGCCTCGCCCGAGCTGTGCGGTAGGCATTGAGGCAGCCAGCGCAGGGGCTTCTGCTGAGGGGGCAGGCGGAGCTTGAGGAAACCGCAGATAAGTTTTTTTCTCTTTGAAAGATAGAGATTAATACAACTACTTAAAAAATATAGTCAATAGGTTACTAAGATATTGCT;

[0022] The MIR4435-2HG gene, whose selected sequence is shown as SEQ NO.5 in the sequence list,

[0023] SEQ NO.5:

[0024] GCTCTGCCGACTTCCAGTTCTGGAACAAGATGGTTAAACTCATTTTTCCCTGCTCTGCTCCTCTAAATACAACTAAGTACCTTGGAAACTATTCAGCAGACAATGATAAAGGGCTCTGAAAGCTAGAAGAAAAGGTGTACTTGCAAGAAACCTCAGGACTTGAGTAACAGCAACA TGGAAAGGAGAAAGAGACTACCTACTGCATTTCTGTCACTCGCTGAAAAGGACACTCTGTCAGAAAATCTTCTAGCAAACTTCAAAGGGCAAAATCACCCCTTGTTACTGATAAAGCCCAGAGAGCTTCAGCAGCTAACATTCCCTGGACAGGGCACAGCAAGGATTTGAACCTA.

[0025] Furthermore, the kit also includes a first specific amplification primer and probe set targeting the MSMB gene, wherein the first specific amplification primer and probe set is selected from at least one of combination A, combination B, or combination C:

[0026] Combination A:

[0027] SEQ NO.6: MSMB-FB1: TCTTTGCCACCTTCGTGAC,

[0028] SEQ NO.7: MSMB-RB1: GCATGTCTCACAGTTGTCAGT,

[0029] SEQ NO.12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB);

[0030] Combination B:

[0031] SEQ NO.8: MSMB-FB2: GTGACTTTATGCAATGCATCAT,

[0032] SEQ NO.9: MSMB-RB2: CAGTCTGCCACTCCGAGT,

[0033] SEQ NO.12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB);

[0034] Combination C:

[0035] SEQ NO.10: MSMB-FB3: TACCTAATGAGGGAGTTCCAG,

[0036] SEQ NO.11: MSMB-RB3: TCCGAGTTTATTGGGTGTTTG,

[0037] SEQ NO. 12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB).

[0038] Furthermore, the kit also includes a second set of specific amplification primers and probes targeting the AMACR gene, wherein the second set of specific amplification primers and probes is selected from at least one of combination D, combination E, or combination F:

[0039] Combination D:

[0040] SEQ NO.13: AMACR-FB1: CTGTGCAAGCGGTCGGAT,

[0041] SEQ NO.14: AMACR-RB1: AGCCTGGCATAAATAAGCCTT,

[0042] SEQ NO.19: AMACR-IP:ATGGAGAAACTCCAGCTGG(VIC-MGB);

[0043] Combination E:

[0044] SEQ NO.15: AMACR-FB2: GTCGGATGTGCTGCTGGA,

[0045] SEQ NO.16: AMACR-RB2: GCCTGGCATAAATAAGCCT,

[0046] SEQ NO.19: AMACR-IP:ATTCAACCAGGAAATGCATG(VIC-MGB);

[0047] Combination F:

[0048] SEQ NO.17: AMACR-FB3: CTAGTGCTGGACCTGAAGCA,

[0049] SEQ NO.18: AMACR-RB3: CCTGACTGGCCAAATCCACT,

[0050] SEQ NO. 19: AMACR-IP: ATTCAACCAGGAAATGCATG (VIC-MGB).

[0051] Furthermore, the kit also includes a third set of specific amplification primers and probes targeting the PCGEM1 gene, wherein the third set of specific amplification primers and probes is selected from at least one of combination G, combination H, or combination I:

[0052] Combination G:

[0053] SEQ NO.20: PCGEM1-FB1:AACGTTTCTCCTGCCTCAG,

[0054] SEQ NO.21: PCGEM1-RB1: CCACTTTGGCTGATATCATGAAG,

[0055] SEQ NO.26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB);

[0056] Combination H:

[0057] SEQ NO.22: PCGEM1-FB2:TCCCAGGTTCAAACGTTTCTC,

[0058] SEQ NO.23: PCGEM1-RB2:ACTTTGGCTGATATCATGAAGT,

[0059] SEQ NO.26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB);

[0060] Combination I:

[0061] SEQ NO.24: PCGEM1-FB3: CTCAGCCTCCCAAGTAACTG,

[0062] SEQ NO.25: PCGEM1-RB3: GTCCTCTCCAGGAGCTGT,

[0063] SEQ NO. 26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB).

[0064] Furthermore, the kit also includes a fourth set of specific amplification primers and probes targeting the MALAT1 gene, wherein the fourth set of specific amplification primers and probes is selected from at least one of combination J, combination K, or combination L:

[0065] Combination J:

[0066] SEQ NO.27: MALAT1-FB1: GGCTCAGTTGCGTAATGGAAA,

[0067] SEQ NO.28: MALAT1-RB1:ACAACTCGCATCACCGGAAT,

[0068] SEQ NO.33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB);

[0069] Combination K:

[0070] SEQ NO.29: MALAT1-FB2: AGGTGGTAAACTATACCTACTGTC,

[0071] SEQ NO.30: MALAT1-RB2: CTCGGGCGAGGCGTATTTA,

[0072] SEQ NO.33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB);

[0073] Combination L:

[0074] SEQ NO.31: MALAT1-FB3: CTATCACACTTTAATCTTCCTTC,

[0075] SEQ NO.32: MALAT1-RB3: CGAGGCGTATTTATAGACGGA,

[0076] SEQ NO. 33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB).

[0077] Furthermore, the kit also includes a fifth set of specific amplification primers and probes targeting the MIR4435-2HG gene, wherein the fifth set of specific amplification primers and probes is selected from at least one of combination M, combination N, or combination O:

[0078] Combination M:

[0079] SEQ NO.34: MIR4435-2HG-FB1: AGGTGTACTTGCAAGAAACCT,

[0080] SEQ NO.35: MIR4435-2HG-RB1: AGCGAGTGACAGAAATGCAG,

[0081] SEQ NO.40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB);

[0082] Combination N:

[0083] SEQ NO.36: MIR4435-2HG-FB2: AGATTTTCTGACAGAGTGTCCTT,

[0084] SEQ NO.37: MIR4435-2HG-RB2: AGCTAGAAGAAAAGGTGTACTTG,

[0085] SEQ NO.40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB);

[0086] Combination O:

[0087] SEQ NO.38: MIR4435-2HG-FB3: TACTTGCAAGAAACCTCAGGACT,

[0088] SEQ NO.39: MIR4435-2HG-RB3: GTGACAGAAATGCAGTAGGTAGT,

[0089] SEQ NO. 40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB).

[0090] Preferably, the kit includes a first set of specific amplification primers and probes for the MSMB gene, a second set of specific amplification primers and probes for the AMACR gene, a third set of specific amplification primers and probes for the PCGEM1 gene, a fourth set of specific amplification primers and probes for the MALAT1 gene, and a fifth set of specific amplification primers and probes for the MIR4435-2HG gene.

[0091] The first specific amplification primer and probe set uses combination A:

[0092] SEQ NO.6: MSMB-FB1: TCTTTGCCACCTTCGTGAC,

[0093] SEQ NO.7: MSMB-RB1: GCATGTCTCACAGTTGTCAGT,

[0094] SEQ NO.12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB);

[0095] The second specific amplification primer and probe set is selected from combination D:

[0096] SEQ NO.13: AMACR-FB1: CTGTGCAAGCGGTCGGAT,

[0097] SEQ NO.14: AMACR-RB1: AGCCTGGCATAAATAAGCCTT,

[0098] SEQ NO.19: AMACR-IP:ATGGAGAAACTCCAGCTGG(VIC-MGB);

[0099] The third specific amplification primer and probe set uses combination H:

[0100] SEQ NO.22: PCGEM1-FB2:TCCCAGGTTCAAACGTTTCTC,

[0101] SEQ NO.23: PCGEM1-RB2:ACTTTGGCTGATATCATGAAGT,

[0102] SEQ NO.26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB);

[0103] The fourth specific amplification primer and probe set uses combination J:

[0104] SEQ NO.27: MALAT1-FB1: GGCTCAGTTGCGTAATGGAAA,

[0105] SEQ NO.28: MALAT1-RB1:ACAACTCGCATCACCGGAAT,

[0106] SEQ NO.33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB);

[0107] The fifth specific amplification primer and probe set uses combination O:

[0108] SEQ NO.38: MIR4435-2HG-FB3: TACTTGCAAGAAACCTCAGGACT,

[0109] SEQ NO. 39: MIR4435-2HG-RB3: GTGACAGAAATGCAGTAGGTAGT, SEQ NO. 40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB).

[0110] Secondly, the present invention provides a multi-target combined detection system for prostate cancer. This detection system uses the reagent kit described in the first aspect and a triple two-tube system for detection, employing an integrated model as the optimal model for positive interpretation. The two-tube system respectively contains the following biomarker combinations:

[0111] Pipe 1:

[0112] First-specific amplification primers and probes targeting the MSMB gene;

[0113] Second-specific amplification primers and probes targeting the AMACR gene;

[0114] Fifth specific amplification primer and probe set targeting the MIR4435 gene;

[0115] Pipe 2:

[0116] Third-specific amplification primers and probes targeting the PCGEM1 gene;

[0117] The fourth specific amplification primer and probe set targeting the MALAT1 gene;

[0118] Fifth specific amplification primer and probe set targeting the MIR4435 gene;

[0119] The two-tube system includes three fluorescence detection indicators;

[0120] The detection system also includes a one-step RT-PCR amplification reagent, prepared separately in two tubes. Using the one-step RT-PCR method, the system includes a reverse transcription and amplification system. The reverse transcription system contains poly(dT) primers for targeted reverse transcription of mRNA, and also includes random primers to further improve reverse transcription efficiency. The reverse transcription and qPCR processes are performed in a single system.

[0121] Preferably, the reaction concentration ratios of each primer-probe set in the detection system are as follows:

[0122] The concentration of the first specific amplification primer for the MSMB gene was 1x, and the concentration of the probe was 0.5x.

[0123] The concentration of the second specific amplification primer for the AMACR gene was 1x, and the concentration of the probe was 0.5x.

[0124] The concentration of the third specific amplification primer for the PCGEM1 gene was 1x, and the concentration of the probe was 0.5x.

[0125] The concentration of the fourth specific amplification primer for the MALAT1 gene was 1x, and the concentration of the probe was 0.5x.

[0126] The concentration of the fifth specific amplification primer for the MIR4435 gene was 0.5x, and the probe concentration was 0.25x.

[0127] Thirdly, the present invention provides the application of the prostate cancer multi-target combined detection kit described in the first aspect, or the prostate cancer multi-target combined detection system described in the second aspect, in the preparation of prostate cancer auxiliary diagnostic products.

[0128] The beneficial effects of this invention are:

[0129] 1. Compared with the detection of a single biomarker in the existing technology, the present invention adopts multi-target joint detection and a detection system with dual tubes and triple fluorescence channels. It also optimizes the multi-target joint detection interpretation model and makes full use of the information complementarity between multiple genes, which can significantly improve the diagnostic accuracy.

[0130] 2. Compared to most existing reagent kits that require low-temperature transportation and separate tube processing from sample preparation, which involves many steps and is time-consuming, this invention uses an integrated reagent kit. Samples do not need to be processed separately. After sample processing, the same sample can be tested separately and read together, saving operation steps, reducing errors caused by detection operations, and improving accuracy.

[0131] 3. Compared with existing technologies, which have weak ability to handle the "diagnostic gray area" and cannot effectively distinguish between benign and malignant lesions, leading to problems such as excessive puncture, this invention provides a non-invasive and highly compliant auxiliary diagnostic tool for the "gray area" population using morning urine samples. The detection is minimally invasive, has low operation requirements, and is highly acceptable to patients. Attached Figure Description

[0132] Figure 1 This is the overall detection flowchart of the present invention.

[0133] Figure 2 This is a display of the accuracy results of different analysis models in Embodiment 6 of the present invention.

[0134] Figure 3 This is the ROC curve of the model in Embodiment 6 of the present invention. The two ROC curves in the figure correspond to the curves of two different emphases of the optimal model. The left figure represents the positive result that patients are most concerned about, and the right figure represents the negative result that patients are most concerned about. The two figures are symmetrical about the diagonal (obtained by flipping along the axis of symmetry). In short, Figure 3These are two different representations of the ROC of the optimal model (corresponding to negative and positive, respectively). Detailed Implementation

[0135] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be described in detail, clearly, and completely below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Furthermore, based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of the present invention.

[0136] Given that this invention is based on specialized technology, in order to ensure a consistent understanding of the technical terms used in this invention, a unified explanation of the relevant technical terms is provided before introducing the embodiments. It should be noted that the explanations of terminology in the embodiments are only for assisting in understanding the invention and should not be considered as limiting the invention.

[0137] RT-qPCR: Real-time quantitative polymerase chain reaction is a technique that uses reverse transcriptase to reverse transcribe RNA into cDNA, and then uses the cDNA as a template for PCR amplification. It has been widely used in gene expression analysis and detection of RNA virus expression levels.

[0138] The diagnostic gray zone refers to a range where test values ​​fall between normal and abnormal, making a definitive judgment as either normal or abnormal. Further confirmation through other examinations or dynamic observation is required. This situation is common in the screening or diagnosis of certain diseases, such as hepatitis B markers, tumor markers, and hormone level tests, indicating uncertainty in the results and requiring careful interpretation. In this invention, the terms "diagnostic gray zone" and "gray zone" specifically refer to prostate cancer or suspected prostate cancer patients with PSA levels between 4-10 ng / mL, indicating high diagnostic uncertainty.

[0139] PI-RADS score: Prostate Imaging Reporting and Data System score, is a standardized assessment tool based on multiparameter magnetic resonance imaging (MRI) used to quantify the risk of prostate cancer. Generally, the score ranges from 1 to 5 points, with higher scores indicating a greater likelihood of clinically significant prostate cancer.

[0140] ROC analysis, short for Receiver Operating Characteristic Analysis, is a statistical method used to evaluate model performance. By plotting the true positive rate (TPR) versus the false positive rate (FPR), it quantifies the model's predictive accuracy and determines the optimal classification threshold.

[0141] Multiple detection: Detecting multiple target molecules simultaneously using the same or different indicators in the same reaction system, or detecting target molecules multiple times simultaneously using the same or different indicators.

[0142] Example 1: Design and screening of primers and probes.

[0143] The detection biomarkers selected in this invention include five genes, as detailed below:

[0144] The MSMB gene encodes microprotamine-β, a secretory protein primarily expressed in semen. This gene is located on chromosome 10 (10q11.2). It is specifically highly expressed in prostate epithelial cells, but its expression is significantly downregulated in prostate cancer tissue and high-grade prostate intraepithelial neoplasia. This reduced expression level is closely associated with the development and progression of prostate cancer, making it a potential tumor suppressor. Due to its specificity in prostate tissue and bodily fluids (such as urine), the MSMB gene is a potential non-invasive diagnostic biomarker for prostate cancer.

[0145] The AMACR gene encodes α-methylacyl-CoA racemic enzyme, a key enzyme involved in the metabolism of branched-chain fatty acids and bile acids in mitochondria and peroxisomes. This gene is located on chromosome 5 (5p13.2). Contrary to its low expression in most normal tissues, AMACR is specifically highly expressed in the vast majority of prostate cancer tissues (including high-grade PIN and malignant epithelial cells), making it a crucial biomarker for pathological diagnosis.

[0146] PCGEM1 is a prostate cancer-specific long non-coding RNA located on chromosome 2 (2q32.2). Its expression is highly tissue-specific, primarily found in prostate tissue, and is significantly overexpressed in prostate cancer, particularly androgen receptor-positive and high-risk prostate cancer. PCGEM1 promotes tumor growth by regulating cell proliferation, apoptosis resistance, and metabolic reprogramming. Its overexpression is associated with castration-resistant transformation, thus it is considered a potential prostate cancer-specific biomarker and therapeutic target.

[0147] MALAT1 gene: Metastasis-associated lung adenocarcinoma transcript 1, is a highly conserved long non-coding RNA enriched in the cell nucleus. This gene is located on chromosome 11 (11q13.1). Although initially discovered in lung cancer, subsequent studies have confirmed that MALAT1 is widely overexpressed in various malignant tumors, including prostate cancer, breast cancer, and liver cancer. In prostate cancer, its high expression is closely associated with tumor metastasis and poor prognosis. It plays a crucial role in promoting the metastatic process of prostate cancer by participating in RNA splicing, regulating gene transcription, and influencing various signaling pathways related to cell migration and invasion. Therefore, it is an important biomarker for cancer metastasis and a potential therapeutic target.

[0148] The MIR4435-2HG gene encodes a long non-coding RNA located on chromosome 2 (2p22.3). While the transcript of this gene does not encode a protein, its expression level in serum or plasma is an emerging molecular biomarker for prostate cancer detection. MIR4435-2HG regulates downstream oncogenic signaling pathways by acting as a competitive endogenous RNA (molecular sponge) to adsorb specific microRNAs, thereby promoting prostate cancer proliferation and metastasis. It can be used as a supplement to AMACR to improve the ability to identify prostate cancer.

[0149] The full mRNA sequences of each gene were found in the NCBI (National Center for Biotechnology Information) nucleic acid database.

[0150] Based on the common exon sequences or the exon sequences of the main transcript types of different genes, the selected sequence information for each gene is as follows:

[0151] MSMB gene selected sequence (5' to 3'):

[0152] SEQ NO.1:

[0153] AATGTTCTCCTGGGCAGCGTTGTGATCTTTGCCACCTTCGTGACTTTATGCAATGCATCATGCTATTTCATACCTAATGAGGGAGTTCCAGGAGATTCAACCAGGAAATGCATGGATCTCAAAGGAAACAAACACCCAATAAACTCGGAGTGGCAGACTGACAACTGTGAGACATGCACTTGCTACGAAACAGAAATTTCATGTTGCACCCT.

[0154] Selected sequence of AMACR gene (5’ to 3’):

[0155] SEQ NO.2:

[0156] AGCCGCTTGGGCCGGGGCAAGCGCTCGCTAGTGCTGGACCTGAAGCAGCCGCGGGGAGCCGCCGTGCTGCGGCGTCTGTGCAAGCGGTCGGATGTGCTGCTGGAGCCCTTCCGCCGCGGTGTCATGGAGAAACTCCAGCTGGGCCCAGAGATTCTGCAGCGGGAAAATCCAAGGCTTATTTATGCCAGGCTGAGTGGATTTGGCCAGTCAGGAAGCTTCTGCCGGTTAGCTGGCCACGATATCAACTATTTGGCTTTGTCAGGTGTTCTCTCAAAAATTGGCAGAAGTGGTGAGAATCCGTATGCCCCGCTGAATCTCCTGGCTGACTTTGCTGGTGGTGGCCTTATGTGTGCACTGGGCATTATAATGGCTCTTTTTGACCGCACACGCACTGGCAAGGGTCAGGTCATTGATGCAAATATG。

[0157] Selected sequence of PCGEM1 gene (5’ to 3’):

[0158] SEQ NO.3:

[0159] ATGGAGTCTTGCCCTGTCTCCAAGGCTGGAGCCCAATGGTGTGATCTTGGCTCACTGCAACCTCCACCTCCCAGGTTCAAACGTTTCTCCTGCCTCAGCCTCCCAAGTAACTGGGATTACAGCAGGCTTGGTGCATTTGACACTTCATGATATCAGCCAAAGTGGAACTAAAAACAGCTCCTGGAAGAGGACTATGACATCATCAGGTTGGGAGTCTCCAGGGACAGCGGACCCTTTGGAAAAGGACTAGAAAGTGTGAAATCTATTAGTCTTCGATATGAAATTCTCTGTCTCTGTAAAAGCATTTCATATTTACAAGACACAGGCCTA。

[0160] Selected sequence of MALAT1 gene (5’ to 3’):

[0161] SEQ NO.4:

[0162] CCCTGCAAGGCTGGGGCTCAGTTGCGTAATGGAAAGTAAAGCCCTGAACTATCACACTTTAATCTTCCTTCAAAAGGTGGTAAACTATACCTACTGTCCCTCAAGAGAACACAAGAAGTGCTTTAAGAGGCGGCGGAAGGTGATCGAATTCCGGTGATGCGAGTTGTTCTCCGTCTATAAATACGCCTCGCCCGAGCTGTGCGGTAGGCATTGAGGCAGCCAGCGCAGGGGCTTCTGCTGAGGGGGCAGGCGGAGCTTGAGGAAACCGCAGATAAGTTTTTTTCTCTTTGAAAGATAGAGATTAATACAACTACTTAAAAAATATAGTCAATAGGTTACTAAGATATTGCT。

[0163] Selected sequence of MIR4435 gene (5’ to 3’):

[0164] SEQ NO.5:

[0165] GCTCTGCCGACTTCCAGTTCTGGAACAAGATGGTTAAACTCATTTTTCCCTGCTCTGCTCCTCTAAATACAACTAAGTACCTTGGAAACTATTCAGCAGACAATGATAAAGGGCTCTGAAAGCTAGAAGAAAAGGTGTACTTGCAAGAAACCTCAGGACTTGAGTAACAGCAACATGGAAAGGAGAAAGAGACTACCTACTGCATTTCTGTCACTCGCTGAAAAGGACACTCTGTCAGAAAATCTTCTAGCAAACTTCAAAGGGCAAAATCACCCCTTGTTACTGATAAAGCCCAGAGAGCTTCAGCAGCTAACATTCCCTGGACAGGGCACAGCAAGGATTTGAACCTA。

[0166] Three primer systems were designed for each target (Table 1) for screening. The tests were repeated five times using morning urine samples as templates, and the specificity of the systems was verified using enzyme-free water. A one-step RT-PCR amplification reagent was used for amplification. The reaction system and conditions were prepared according to the reagent product instructions. The prepared reaction system was then placed on a real-time PCR instrument for amplification. The one-step RT-PCR amplification reagent used in this invention is 2xSensiDirect RT Premix-UNG(NA) (Probe qRT-PCR) manufactured by Zhuhai Baorui Biotechnology Co., Ltd., catalog number MD2104-NA-090801.

[0167] The quality of the system is evaluated based on the mean Ct value, detection rate, and presence or absence of nonspecific amplification, thereby selecting the primer and probe combination with the best amplification efficiency.

[0168] Table 1 Primer and probe sequences

[0169]

[0170]

[0171] In the above table,

[0172] Combination A = MSMB gene primer combination 1 + MSMB gene primer common probe;

[0173] Combination B = MSMB gene primer combination 2 + MSMB gene primer common probe;

[0174] Combination C = MSMB gene primer combination 3 + MSMB gene primer common probe;

[0175] Combination D = AMACR gene primer combination 1 + AMACR gene primer common probe; Combination E = AMACR gene primer combination 2 + AMACR gene primer common probe; Combination F = AMACR gene primer combination 3 + AMACR gene primer common probe; Combination G = PCGEM1 gene primer combination 1 + PCGEM1 gene primer common probe; Combination H = PCGEM1 gene primer combination 2 + PCGEM1 gene primer common probe;

[0176] Combination I = PCGEM1 gene primer combination 3 + PCGEM1 gene primer common probe;

[0177] Combination J = MALAT1 gene primer combination 1 + MALAT1 gene primer common probe;

[0178] Combination K = MALAT1 gene primer combination 2 + MALAT1 gene primer common probe;

[0179] Combination L = MALAT1 gene primer combination 3 + MALAT1 gene primer common probe;

[0180] Combination M = MIR4435-2HG gene primer combination 1 + MIR4435-2HG gene primer common probe;

[0181] Combination N = MIR4435-2HG gene primer combination 2 + MIR4435-2HG gene primer common probe;

[0182] Combination O = MIR4435-2HG gene primer combination 3 + MIR4435-2HG gene primer common probe.

[0183] The test results for each combination are shown in Table 2 below:

[0184] Table 2 Detection results of each system

[0185] Primers / Probes Average detection Ct Detection rate Nonspecific amplification Combination A 25.87 5 / 5 none Combination B 31.18 5 / 5 none Combination C 29.01 5 / 5 none Combination D 26.49 5 / 5 none Combination E 39.45 5 / 5 none Combination F 28.38 5 / 5 none Combination G 30.64 5 / 5 none Combination H 27.78 5 / 5 none Combination I 31.14 5 / 5 none Combination J 27.35 5 / 5 none Combination K 30.17 5 / 5 none Combination L 33.48 5 / 5 none Combination M 35.21 5 / 5 none Combination N 32.12 5 / 5 none Combination O 26.48 5 / 5 none

[0186] Based on the above results, the optimal combinations of each gene were selected: combination A for MSMB gene, combination D for AMACR, combination H for PCGEM1, combination J for MALAT1, and combination O for MIR4435-2HG.

[0187] The preferred combinations are summarized in Table 3 below:

[0188] Table 3. Finalized primer and probe sequences

[0189]

[0190]

[0191] Example 2: Optimization of the reaction system.

[0192] This embodiment further optimizes parameters such as concentration in the reaction system selected in Example 1.

[0193] 2.1 Primer and probe concentration optimization.

[0194] Based on the detection results and fluorescence intensity in Table 2, the detection triple two-tube system of the present invention is configured as shown in Table 4 below:

[0195] Table 4 Pre-packaging System Settings

[0196]

[0197] The initial baseline concentrations of primers and probes within a single system were set at 0.4 μM and 0.2 μM, respectively.

[0198] With the single-system primer and probe concentration multiplier set to 1x (1x) in Table 4, the concentration configurations for each tube are shown in Table 5 below:

[0199] Table 5 Primer and probe concentration combinations

[0200]

[0201] The templates used were RNA extracted from five morning urine samples. Different combinations of primers and probe concentrations (Table 5) were prepared and used as test reagents on a real-time PCR instrument. The results are shown in Table 6 below.

[0202] Table 6 Primer and probe concentration optimization

[0203]

[0204] The above screening determined that tubes 1 and 2 exhibited the least interference between different targets and the best detection performance under concentration combination 2. Excessive concentration of the MIR4435-2HG system in concentration combination 1 resulted in approximately 1 Ct of inhibition against MSMB and AMACR targets. The MIR4435-2HG system in concentration combination 3 showed 1–1.5 Ct of inhibition compared to other groups, while showing no significant improvement against MSMB and AMACR targets.

[0205] Based on the above results, the components and concentrations of the detection system are configured as shown in Table 7 below:

[0206] Table 7 Final primer and probe combinations and concentrations

[0207]

[0208]

[0209] 2.2 Optimization of reagent concentration for one-step RT-PCR amplification

[0210] A one-step RT-PCR method was used, comprising a reverse transcription and amplification system. The reverse transcription system included poly(dT) primers for targeted reverse transcription of mRNA, and random primers to further improve reverse transcription efficiency. Reverse transcription and qPCR were performed in a single system. The one-step RT-PCR amplification reagent used in this invention was 2xSensiDirect RT Premix-UNG(NA) (Probe qRT-PCR) manufactured by Zhuhai Baorui Biotechnology Co., Ltd., catalog number MD2104-NA-090801. The concentration of the buffer (2xSensiDirect RT Premix Buffer(NA))(dUTP)) of the one-step RT-PCR amplification reagent was kept constant. The manufacturer-recommended enzyme concentration (2xSensiDirect RTase / UNG MIX(NA)) was set at 1x, and three gradients (0.75x, 1x, and 1.25x) were configured for optimization to evaluate the effect of different concentrations of the one-step RT-PCR amplification reagent on the reaction system.

[0211] Similar to step 2.1, the template used was RNA from 5 extracted morning urine samples, and the negative control was enzyme-free water. The detection results are shown in Table 8 below:

[0212] Table 8 Optimization of reagent concentrations for one-step RT-PCR amplification

[0213]

[0214]

[0215] The results show a consistent trend in the detection data for each target. When the concentration of the one-step RT-PCR amplification reagent is 0.75x, the Ct value is 2-3 higher than other concentrations. However, when the concentration is 1.25x, the difference between the Ct result and 1x is generally within 1, indicating that the results are comparable. Therefore, considering both detection performance and cost-effectiveness, the final concentration of the PCR amplification reagent was determined to be 1x.

[0216] Example 3: Optimization of reaction conditions.

[0217] This embodiment further optimizes the reaction conditions for the reaction system finally determined in Example 2.

[0218] 3.1 Optimization of reverse transcription reaction time

[0219] Different reverse transcription reaction times were set, while the amplification procedure remained the same.

[0220] Similar to Example 2, the template used was RNA from 5 extracted morning urine samples. The prepared test reagent was placed on a real-time PCR instrument for reaction (3 different times were selected) to verify the effect of different reverse transcription times on the PCR amplification results, thereby determining the optimal reverse transcription reaction time.

[0221] The group settings are as follows: Table 9:

[0222] Table 9 Reverse Transcription Reaction Time Settings

[0223]

[0224] The reaction results for each group are shown in Table 10 below:

[0225] Table 10 Optimization of Reverse Transcription Reaction Time

[0226]

[0227]

[0228] The results showed that when the reverse transcription time was 15 min, the detection Ct values ​​for each target were all less than those at 10 min and 20 min, with an average difference of 1–2.5. Therefore, the reverse transcription time was 15 min, and the program was: 50℃, 15 min.

[0229] 3.2 Annealing Temperature Optimization

[0230] Different annealing temperatures were set for the PCR reaction, while keeping the reverse transcription conditions constant. RNA extracted from five morning urine samples was used as a template. Enzyme-free water was used to verify the system specificity. The prepared test reagents were placed on a real-time PCR instrument for reaction to verify the effect of different annealing temperatures on the PCR amplification results, thereby determining the optimal PCR annealing temperature.

[0231] The group settings are as follows: Table 11:

[0232] Table 11 Annealing Temperature Settings

[0233]

[0234] The reaction results for each group are shown in Table 12 below:

[0235] Table 12 Annealing Temperature Optimization

[0236]

[0237]

[0238] The results showed that the detection Ct of each target was lowest when the annealing temperature was 58℃, but non-specific amplification occurred in enzyme-free water. However, when the annealing temperature was 60℃, the Ct of each target was 0–1.5 lower than that at 62℃, and the system showed good specificity. Therefore, an annealing temperature of 60℃ was selected.

[0239] The final complete procedure is shown in Table 13 below:

[0240] Table 13 Reagent and reaction conditions for one-step RT-PCR amplification

[0241]

[0242] Example 4: Pre-filled reagent kit composition of triple dual tubes.

[0243] This embodiment describes the specific form of the reagent kit piping and the verification process.

[0244] This kit uses a dual-tube (tube 1 and tube 2) / test design, both based on one-step RT-PCR amplification reagents, integrating reverse transcription and qPCR processes into a single system.

[0245] Based on the different targets determined in Example 1 above, tube 1 and tube 2 can have three different combinations, as shown in Table 14 below:

[0246] Table 14 Different Combinations of Piping

[0247]

[0248] RNA extracted from 5 morning urine samples was used as a template. The specificity of the system was verified with enzyme-free water. The prepared test reagents were placed on a real-time PCR instrument according to the above combination to verify the effect of different combinations on the PCR amplification results, so as to determine the optimal combination.

[0249] The verification results are shown in Table 15 below:

[0250] Table 15 Reagent Pre-packaging Optimization

[0251]

[0252] The results above show that non-specific amplification of AMACR and MIR4435-2HG targets occurred in combination 2, indicating interference between different targets in this combination. In combination 3, the Ct values ​​of MSMB and PCGEM1 targets were significantly higher than those of other groups, suggesting possible incomplete primer hybridization between them.

[0253] Based on the above results, combination 1 was selected as the multiplex system for this reagent, as shown in Table 16 below:

[0254] Table 16 Reagent Pre-packaging Formats

[0255]

[0256] Based on the previously validated concentrations, the final reagent formulation is shown in Table 17 below:

[0257] Table 17 Reagent Formulation

[0258]

[0259] The final reagent kit system described above has the following characteristics:

[0260] 1. Integrated process: The one-step RT-PCR reagent simplifies the operation steps and avoids the risk of contamination from opening the container.

[0261] 2. Multiple detection: Each tube can simultaneously detect 3 targets through multi-color fluorescence channels in a single run.

[0262] 3. Modular design: By combining tube 1 and tube 2, flexible detection of five unique targets, including MSMB, AMACR, PCGEM1, MALAT1, and the internal reference / quality control gene MIR4435-2HG, is achieved.

[0263] 4. Flexibility in fluorescence channel selection: Different tube sets and fluorescence channels are independently adapted for different targets, and different fluorescence channels can play different roles in different tube sets. Specifically,

[0264] FAM channel: used for MSMB in tube 1, and for PCGEM1 in tube 2.

[0265] VIC channel: Used for AMACR in tube 1 and for MALAT1 in tube 2.

[0266] Cy5 channel: used to detect MIR4435-2HG in both tubes, indicating that this target is an internal reference or quality control gene commonly included in both detection tubes.

[0267] 5. Ready-to-use: All primers, probes and core reagents are premixed and aliquoted. Users only need to add template RNA to conduct experiments, which improves the convenience and reproducibility of detection.

[0268] Example 5: Accelerated stability of the reagent at 37°C.

[0269] To verify the shelf stability of the prostate multi-target combined detection kit determined in the above embodiments, the reagent was subjected to accelerated testing in a 37°C oven. According to the Arrhenius equation, storage at 37°C for 7 days is equivalent to storage at -20°C for 1 year. RNA extracted from 5 morning urine samples was used as a template, divided into three groups, and stored at -80°C. At each time point, the template was added to the reagent and used as the test reagent in a real-time PCR instrument for reaction.

[0270] The results at each time point are summarized in Table 18 below:

[0271] Table 18 Results of accelerated stability of reagents at 37℃

[0272]

[0273]

[0274] Based on the above results, after accelerated storage at 37℃ for 0, 7, and 14 days, the Ct values ​​detected for each target fluctuated within 1 Ct, indicating that the reagent performance at each acceleration point was comparable. Preliminary verification shows that the kit can be stored at -20℃ for 24 months.

[0275] Example 6: Establishment and validation of positive judgment values ​​for prostate multi-target combined detection kit.

[0276] To confirm the interpretation rules of this invention, the prostate multi-target combined detection kit determined in the above embodiments was used to detect morning urine samples from 227 patients (98 patients with prostate cancer confirmed by puncture biopsy and 129 patients without prostate cancer) from the Second Affiliated Hospital of Zhejiang University School of Medicine. Samples had serum PSA levels of 4–10 ng / mL, or serum PSA <4 ng / mL and PI-RADS score >2. The detection procedure is as follows: Figure 1 As shown.

[0277] The detection results were analyzed using MATLAB R2022a software using eight different analytical models. This project contains eight different detection results from tube 1 (MSMB, AMACR, MIR4435-2HG) and tube 2 (PCGEM1, MALAT1, MIR4435-2HG), representing eight characteristic parameters. The sensitivity and specificity were statistically analyzed and presented as follows: Figure 2 As shown in Table 19 below:

[0278] Table 19 Statistical results of sensitivity and specificity of different analytical models

[0279] Model Number Model Name Sensitivity Specificity Overall compliance rate 1 Tree 80.41％ 84.62％ 82.82％ 2 Linear discrimination 79.44％ 89.17％ 84.58％ 3 Logistic Regression 81.82％ 86.72％ 84.58％ 4 Naive Bayes 90.82％ 79.07％ 84.14％ 5 SVM 79.59％ 81.40％ 80.62％ 6 KNN 83.67％ 79.07％ 81.06％ 7 integrated 85.71％ 85.27％ 85.46％ 8 Neural Networks 76.53％ 82.95％ 80.18％

[0280] Based on the interpretation methods established by the above eight models, the ensemble model was ultimately selected for positive interpretation. Its ROC curve is shown below. Figure 3 As shown.

[0281] The selected model was exported as a function in MATLAB and named the Prostate Cancer Multi-Target Combined Detection Kit ROC. It was then saved as a MATLAB code file (UTF-8) for later use.

[0282] After establishing the positive cutoff value, morning urine samples from 285 patients (124 with prostate cancer confirmed by puncture biopsy and 161 without prostate cancer) from the Second Affiliated Hospital of Zhejiang University School of Medicine (samples with serum PSA between 4 and 10 ng / mL, or serum PSA < 4 ng / mL and PI-RADS score > 2) were selected to verify the positive cutoff value.

[0283] The test results were retrieved from the ROC function saved during the setup process of the prostate cancer multi-target combined detection kit. The statistical results are shown in Tables 20-22 below:

[0284] Table 20 Validation of Reference Ranges - Serum PSA (0–10 ng / mL)

[0285]

[0286] Table 21 Validation of Reference Ranges - Serum PSA (0–4 ng / mL), MRI Abnormalities

[0287]

[0288] Table 22 Validation of Reference Ranges - Serum PSA (4–10 ng / mL)

[0289]

[0290] The performance of the above three stratified statistical results is as follows:

[0291] Table 23 Validation of Reference Range - Performance Statistics

[0292]

[0293] The results of the reference value validation experiment showed that the reagent had a performance of over 80% in samples with negative serum PSA and abnormal MRI results (PI-RADS score >2), and had good detection performance in suspected prostate patients with serum PSA of 4-10 ng / mL, with an overall concordance rate of 85.04%.

[0294] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-target combined detection kit for prostate cancer, characterized in that, The multiple targets are selected from the following markers and combinations thereof: The MSMB gene, whose selected sequence is shown in SEQ NO.1 of the sequence listing, SEQ NO.1: AATGTTCTCCTGGGCAGCGTTGTGATCTTTGCCACCTTCGTGACTTTATGCAATGCATCATGCTATTTCATACCTAATGAGGGAGTTCCAGGAGATTCAACCAGGAAATGCATGGATCTCAAAGGAAACAACACCCAATAAACTCGGAGTGGCAGACTGACAACTGTGAGACATGCACTTGCTACGAAACAGAAATTTCATGTTGCACCCT; The selected sequence of the AMACR gene is shown in SEQ NO.2 of the sequence listing. SEQ NO.2: AGCCGCTTGGGCCGGGGCAAGCGCTCGCTAGTGCTGGACCTGAAGCAGCCGCGGGGAGCCGCCGTGCTGCGGCGTCTGTGCAAGCGGTCGGATGTGCTGCTGGAGCCCTTCCGCCGCGGTGTCATGGAGAAACTCCAGCTGGGCCCAGAGATTCTGCAGCGGGAAAATCCAAGGCTTATTTATGCCAGGCTGAGTGGATTTGGCCAGTCAGG AAGCTTCTGCCGGTTAGCTGGCCACGATATCAACTATTTGGCTTTGTCAGGTGTTCCTCAAAAATTGGCAGAAGTGGTGAGAATCCGTATGCCCCGCTGAATCTCCTGGCTGACTTTGCTGGTGGTGGCCTTATGTGTGCACTGGGCATTATAATGGCTCTTTTGACCGCACACGCACTGGCAAGGGTCAGGTCATTGATGCAAATATG; The selected sequence of the PCGEM1 gene is shown in SEQ NO.3 of the sequence listing. SEQ NO.3: ATGGAGTCTTGCCCTGTCTCCAAGGCTGGAGCCCAATGGTGTGATCTTGGCTCACTGCAACCTCCACCTCCCAGGTTCAAACGTTTCTCCTGCCTCAGCCTCCCAAGTAACTGGGATTACAGCAGGCTTGGTGCATTTGACACTTCATGATATCAGCCAAAGTGGAACTAAAAACAGCTCCTGGAAGAGGACTATGACATCATCAGGTTGGGAGTCTCCAGGGACAGCGGACCCTTTGGAAAAGGACTAGAAAGTGTGAAATCTATTAGTCTTCGATATGAAATTCTCTGTCTCTGTAAAAGCATTTCATATTTACAAGACACAGGCCTA; The MALAT1 gene, whose selected sequence is as shown in SEQ NO.4 in the sequence list, SEQ NO.4: CCCTGCAAGGCTGGGGCTCAGTTGCGTAATGGAAAGTAAAGCCCTGAACTATCACACTTTAATCTTCCTTCAAAAGGTGGTAAACTATACCTACTGTCCCTCAAGAGAACACAAGAAGTGCTTTAAGAGGCGGCGGAAGGTGATCGAATTCCGGTGATGCGAGTTGTTCTCCGTCTATAAATACGCCTCGCCCGAGCTGTGCGGTAGGCATTGAGGCAGCCAGCGCAGGGGCTTCTGCTGAGGGGGCAGGCGGAGCTTGAGGAAACCGCAGATAAGTTTTTTTCTCTTTGAAAGATAGAGATTAATACAACTACTTAAAAAATATAGTCAATAGGTTACTAAGATATTGCT; The MIR4435-2HG gene, whose selected sequence is as shown in SEQ NO.5 in the sequence list, SEQ NO.5: GCTCTGCCGACTTCCAGTTCTGGAACAAGATGGTTAAACTCATTTTTCCCTGCTCTGCTCCTCTAAATACAACTAAGTACCTTGGAAACTATTCAGCAGACAATGATAAAGGGCTCTGAAAGCTAGAAGAAAAGGTGTACTTGCAAGAAACCTCAGGACTTGAGTAACAGCAACA TGGAAAGGAGAAAGAGACTACCTACTGCATTTCTGTCACTCGCTGAAAAGGACACTCTGTCAGAAAATCTTCTAGCAAACTTCAAAGGGCAAAATCACCCCTTGTTACTGATAAAGCCCAGAGAGCTTCAGCAGCTAACATTCCCTGGACAGGGCACAGCAAGGATTTGAACCTA.

2. The prostate cancer multi-target combined detection kit according to claim 1, characterized in that, The kit includes a first set of specific amplification primers and probes targeting the MSMB gene, wherein the first set of specific amplification primers and probes is selected from at least one of combination A, combination B, or combination C: Combination A: SEQ NO.6: MSMB-FB1: TCTTTGCCACCTTCGTGAC, SEQ NO.7: MSMB-RB1: GCATGTCTCACAGTTGTCAGT, SEQ NO.12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB); Combination B: SEQ NO.8: MSMB-FB2: GTGACTTTATGCAATGCATCAT, SEQ NO.9: MSMB-RB2: CAGTCTGCCACTCCGAGT, SEQ NO.12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB); Combination C: SEQ NO.10: MSMB-FB3: TACCTAATGAGGGAGTTCCAG, SEQ NO.11: MSMB-RB3: TCCGAGTTTATTGGGTGTTTG, SEQ NO. 12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB).

3. The prostate cancer multi-target combined detection kit according to claim 1, characterized in that, The kit includes a second set of primers and probes specifically targeting the AMACR gene, wherein the second set of primers and probes is selected from at least one of combination D, combination E, or combination F: Combination D: SEQ NO.13: AMACR-FB1: CTGTGCAAGCGGTCGGAT, SEQ NO.14: AMACR-RB1: AGCCTGGCATAAATAAGCCTT, SEQ NO.19: AMACR-IP:ATGGAGAAACTCCAGCTGG(VIC-MGB); Combination E: SEQ NO.15: AMACR-FB2: GTCGGATGTGCTGCTGGA, SEQ NO.16: AMACR-RB2: GCCTGGCATAAATAAGCCT, SEQ NO.19: AMACR-IP:ATTCAACCAGGAAATGCATG(VIC-MGB); Combination F: SEQ NO.17: AMACR-FB3: CTAGTGCTGGACCTGAAGCA, SEQ NO.18: AMACR-RB3: CCTGACTGGCCAAATCCACT, SEQ NO. 19: AMACR-IP: ATTCAACCAGGAAATGCATG (VIC-MGB).

4. The prostate cancer multi-target combined detection kit according to claim 1, characterized in that, The kit includes a third set of specific amplification primers and probes targeting the PCGEM1 gene, wherein the third set of specific amplification primers and probes is selected from at least one of combination G, combination H, or combination I: Combination G: SEQ NO.20: PCGEM1-FB1:AACGTTTCTCCTGCCTCAG, SEQ NO.21: PCGEM1-RB1: CCACTTTGGCTGATATCATGAAG, SEQ NO.26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB); Combination H: SEQ NO.22: PCGEM1-FB2:TCCCAGGTTCAAACGTTTCTC, SEQ NO.23: PCGEM1-RB2:ACTTTGGCTGATATCATGAAGT, SEQ NO.26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB); Combination I: SEQ NO.24: PCGEM1-FB3: CTCAGCCTCCCAAGTAACTG, SEQ NO.25: PCGEM1-RB3: GTCCTCTCCAGGAGCTGT, SEQ NO. 26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB).

5. The prostate cancer multi-target combined detection kit according to claim 1, characterized in that, The kit includes a fourth set of specific amplification primers and probes targeting the MALAT1 gene, wherein the fourth set of specific amplification primers and probes is selected from at least one of combination J, combination K, or combination L: Combination J: SEQ NO.27: MALAT1-FB1: GGCTCAGTTGCGTAATGGAAA, SEQ NO.28: MALAT1-RB1:ACAACTCGCATCACCGGAAT, SEQ NO.33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB); Combination K: SEQ NO.29: MALAT1-FB2: AGGTGGTAAACTATACCTACTGTC, SEQ NO.30: MALAT1-RB2: CTCGGGCGAGGCGTATTTA, SEQ NO.33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB); Combination L: SEQ NO.31: MALAT1-FB3: CTATCACACTTTAATCTTCCTTC, SEQ NO.32: MALAT1-RB3: CGAGGCGTATTTATAGACGGA, SEQ NO. 33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB).

6. The prostate cancer multi-target combined detection kit according to claim 1, characterized in that, The kit includes a fifth set of specific amplification primers and probes targeting the MIR4435-2HG gene, wherein the fifth set of specific amplification primers and probes is selected from at least one of combination M, combination N, or combination O: Combination M: SEQ NO.34: MIR4435-2HG-FB1: AGGTGTACTTGCAAGAAACCT, SEQ NO.35: MIR4435-2HG-RB1: AGCGAGTGACAGAAATGCAG, SEQ NO.40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB); Combination N: SEQ NO.36: MIR4435-2HG-FB2: AGATTTTCTGACAGAGTGTCCTT, SEQ NO.37: MIR4435-2HG-RB2: AGCTAGAAGAAAAGGTGTACTTG, SEQ NO.40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB); Combination O: SEQ NO.38: MIR4435-2HG-FB3: TACTTGCAAGAAACCTCAGGACT, SEQ NO.39: MIR4435-2HG-RB3: GTGACAGAAATGCAGTAGGTAGT, SEQ NO. 40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB).

7. The prostate cancer multi-target combined detection kit according to claim 1, characterized in that, The kit includes a first set of specific amplification primers and probes for the MSMB gene, a second set of specific amplification primers and probes for the AMACR gene, a third set of specific amplification primers and probes for the PCGEM1 gene, a fourth set of specific amplification primers and probes for the MALAT1 gene, and a fifth set of specific amplification primers and probes for the MIR4435-2HG gene. The first specific amplification primer and probe set uses combination A: SEQ NO.6: MSMB-FB1: TCTTTGCCACCTTCGTGAC, SEQ NO.7: MSMB-RB1: GCATGTCTCACAGTTGTCAGT, SEQ NO.12: MSMB-IP:ATTCAACCAGGAAATGCATG(FAM-MGB); The second specific amplification primer and probe set is selected from combination D: SEQ NO.13: AMACR-FB1: CTGTGCAAGCGGTCGGAT, SEQ NO.14: AMACR-RB1: AGCCTGGCATAAATAAGCCTT, SEQ NO.19: AMACR-IP:ATGGAGAAACTCCAGCTGG(VIC-MGB); The third specific amplification primer and probe set uses combination H: SEQ NO.22: PCGEM1-FB2:TCCCAGGTTCAAACGTTTCTC, SEQ NO.23: PCGEM1-RB2:ACTTTGGCTGATATCATGAAGT, SEQ NO.26: PCGEM1-IP:ATTACAGCAGGCTTGGTGC(FAM-MGB); The fourth specific amplification primer and probe set uses combination J: SEQ NO.27: MALAT1-FB1: GGCTCAGTTGCGTAATGGAAA, SEQ NO.28: MALAT1-RB1:ACAACTCGCATCACCGGAAT, SEQ NO.33: MALAT1-IP: CTTTAAGAGGCGGCGGAAGG (VIC-MGB); The fifth specific amplification primer and probe set uses combination O: SEQ NO.38: MIR4435-2HG-FB3: TACTTGCAAGAAACCTCAGGACT, SEQ NO.39: MIR4435-2HG-RB3: GTGACAGAAATGCAGTAGGTAGT, SEQ NO. 40: MIR4435-2HG-IP: GAGTAACAGCAACATGGAAAGGA (CY5-MGB).

8. A multi-target combined detection system for prostate cancer, characterized in that, The detection system uses the kit described in any one of claims 1 to 7 and the triple two-tube system for detection, wherein the two-tube system respectively contains the following combinations of biomarkers: Pipe 1: First-specific amplification primers and probes targeting the MSMB gene; Second-specific amplification primers and probes targeting the AMACR gene; Fifth specific amplification primer and probe set targeting the MIR4435 gene; Pipe 2: Third-specific amplification primers and probes targeting the PCGEM1 gene; The fourth specific amplification primer and probe set targeting the MALAT1 gene; Fifth specific amplification primer and probe set targeting the MIR4435 gene; The two-tube system includes three fluorescence detection indicators; The detection system also includes one-step RT-PCR amplification reagents, which are separately prepared into two tubes.

9. The multi-target combined detection system for prostate cancer according to claim 8, characterized in that, The reaction concentration ratios of each primer-probe set in the detection system are as follows: The concentration of the first specific amplification primer for the MSMB gene was 1x, and the concentration of the probe was 0.5x. The concentration of the second specific amplification primer for the AMACR gene was 1x, and the concentration of the probe was 0.5x. The concentration of the third specific amplification primer for the PCGEM1 gene was 1x, and the concentration of the probe was 0.5x. The concentration of the fourth specific amplification primer for the MALAT1 gene was 1x, and the concentration of the probe was 0.5x. The concentration of the fifth specific amplification primer for the MIR4435 gene was 0.5x, and the probe concentration was 0.25x.

10. The application of a prostate cancer multi-target combined detection kit according to any one of claims 1 to 7, or a prostate cancer multi-target combined detection system according to any one of claims 8 or 9, in the preparation of prostate cancer auxiliary diagnostic products.

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