A kit for detecting neuroendocrine tumor and use thereof

Abstract

The application relates to the technical field of clinical detection, and discloses a kit for detecting neuroendocrine tumors, and provides specific primers and probes for detecting neuroendocrine tumors. A plurality of pairs of primers and probes of TRMT112, PHF21A, PKD1, RSF1, APLP2, OAZ2, TXNIP, KRAS, PLD3, PQBP1, SSTR5, MORF4L2, NAP1L1, NUDT3 and RHOA targets are combined, operation time is saved, the possibility of single amplification contamination is reduced, and a fast, sensitive and specific clinical detection method is provided for the diagnosis and differential diagnosis of neuroendocrine tumors.

Description

Technical Field

[0001] This invention relates to the field of clinical testing technology, and in particular to a kit for detecting neuroendocrine tumors and its uses. Background Technology

[0002] Neuroendocrine tumors (NETs) are a type of tumor originating from neuroendocrine cells distributed throughout the body, which have both neurological and endocrine functions. NETs can occur in multiple organs, most commonly the gastrointestinal tract (such as the pancreas, stomach, and small intestine) and the lungs, but can also occur in other locations, such as the thyroid gland and adrenal glands.

[0003] Physiologically, based on differentiation, NENs are divided into well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Based on the presence of specific germline mutations, NENs can be classified as sporadic or hereditary. Genetic syndromes caused by germline mutations in different genes (MEN1, RET, VHL, NF1, etc.) also have different characteristics. Due to the high heterogeneity and occult nature of neuroendocrine tumors, most patients are diagnosed at a poorly differentiated G2 / G3 stage, at which point they have progressed to neuroendocrine carcinoma (NEC). The tumors grow rapidly and most have metastasized. The lack of effective early screening methods and specific therapeutic targets contributes to the difficulty in treating patients diagnosed at mid-to-late stages.

[0004] With the development of high-throughput sequencing and the deepening of gene research, the relationship between cancer and gene mutations has been continuously confirmed, playing an important guiding role in the design of targeted drugs and new treatment methods. To further explore the relationship between mutations in key genes and cancer, and to deepen the application of sequencing technology in clinical treatment, targeted sequencing has emerged. By sequencing specific genes, it effectively reduces sequencing time and costs, making it more suitable for clinical applications and enabling precision medicine. Identifying the genes to be detected in targeted sequencing is a crucial prerequisite, while the preparation of detection probes is an essential means to achieve targeted sequencing.

[0005] Kits for detecting neuroendocrine tumors have emerged and play an important role in their use; however, these kits still face some problems and challenges. The following are the main problems currently existing in neuroendocrine tumor detection kits:

[0006] The detection methods are limited in scope and functionality. Currently, most tests only involve a portion of the proteins or genes being observed. For gene testing, due to limitations in gene collection capabilities and sequencing costs, the number of genes covered is relatively small, with most focusing only on sites related to targeted drug therapy, and fewer other relevant sites. This limits its function in scientific research and the discovery of new targets.

[0007] Some kits are not sensitive enough to detect low concentrations or small lesions, which may lead to false negative results.

[0008] Neuroendocrine tumors are highly heterogeneous; tumors of different locations, differentiations, and functions may express different biomarkers. This makes it difficult for a single test kit to cover all types of neuroendocrine tumors, and multiple test combinations may be required.

[0009] Therefore, there is an urgent need for a kit for detecting neuroendocrine tumors to solve the above problems. Summary of the Invention

[0010] The purpose of this invention is to provide a kit for detecting neuroendocrine tumors and its application, solving the problem of how to quickly, comprehensively and accurately detect the presence of neuroendocrine tumors in samples.

[0011] The solution of the present invention is:

[0012] A kit for detecting neuroendocrine tumors includes a detection buffer, a reactive enzyme, a primer-probe mixture, a negative control, and a positive control.

[0013] The primer and probe mixture includes upstream and downstream primers and probes for the TRMT112 gene, the PHF21A gene, the PKD1 gene, the RSF1 gene, the APLP2 gene, the OAZ2 gene, the TXNIP gene, the KRAS gene, the PLD3 gene, the PQBP1 gene, the SSTR5 gene, the MORF4L2 gene, the NAP1L1 gene, the NUDT3 gene, and the RHOA gene.

[0014] The upstream and downstream primers of the TRMT112 gene have sequences as shown in SEQ NO.1 and SEQ NO.2, the specific probe sequence for the TRMT112 gene is shown in SEQ NO.3, and the probe for the TRMT112 gene also includes the TRMT112 molecular beacon probe, the sequence of which is shown in SEQ NO.4.

[0015] The sequences of the upstream and downstream primers for the PHF21A gene are shown in SEQ NO.5 and SEQ NO.6, the specific probe sequence for the PHF21A gene is shown in SEQ NO.7, and the probe for the PHF21A gene also includes the PHF21A molecular beacon probe, the sequence of which is shown in SEQ NO.8.

[0016] The sequences of the upstream and downstream primers for the PKD1 gene are shown in SEQ NO.9 and SEQ NO.10, and the specific probe sequence for the PKD1 gene is shown in SEQ NO.11.

[0017] The sequences of the upstream and downstream primers for the RSF1 gene are shown in SEQ NO.12 and SEQ NO.13, and the specific probe sequence for the RSF1 gene is shown in SEQ NO.14.

[0018] The sequences of the upstream and downstream primers for the APLP2 gene are shown in SEQ NO.15 and SEQ NO.16, and the specific probe sequence for the APLP2 gene is shown in SEQ NO.17.

[0019] The upstream and downstream primers for the OAZ2 gene are shown in SEQ NO.18 and SEQ NO.19. The specific probes for the OAZ2 gene include a type A OAZ2 gene-specific probe and a type B OAZ2 gene-specific probe. The sequence of the type A OAZ2 gene-specific probe is shown in SEQ NO.20, and the sequence of the type B OAZ2 gene-specific probe is shown in SEQ NO.21.

[0020] The sequences of the upstream and downstream primers for the TXNIP gene are shown in SEQ NO.22 and SEQ NO.23, and the specific probe sequence for the TXNIP gene is shown in SEQ NO.24.

[0021] The sequences of the upstream and downstream primers for the KRAS gene are shown in SEQ NO.25 and SEQ NO.26, and the specific probe sequence for the KRAS gene is shown in SEQ NO.27.

[0022] The sequences of the upstream and downstream primers for the PLD3 gene are shown in SEQ NO.28 and SEQ NO.29, and the specific probe sequence for the PLD3 gene is shown in SEQ NO.30.

[0023] The sequences of the upstream and downstream primers for the PQBP1 gene are shown in SEQ NO.31 and SEQ NO.32, and the specific probe sequence for the PQBP1 gene is shown in SEQ NO.33.

[0024] The sequences of the upstream and downstream primers for the SSTR5 gene are shown in SEQ NO.34 and SEQ NO.35, and the specific probe sequence for the SSTR5 gene is shown in SEQ NO.36.

[0025] The sequences of the upstream and downstream primers for the MORF4L2 gene are shown in SEQ NO.37 and SEQ NO.38, and the specific probe sequence for the MORF4L2 gene is shown in SEQ NO.39.

[0026] The sequences of the upstream and downstream primers for the NAP1L1 gene are shown in SEQ NO.40 and SEQ NO.41, and the specific probe sequence for the NAP1L1 gene is shown in SEQ NO.42.

[0027] The sequences of the upstream and downstream primers for the NUDT3 gene are shown in SEQ NO.43 and SEQ NO.44, and the specific probe sequence for the NUDT3 gene is shown in SEQ NO.45.

[0028] The sequences of the upstream and downstream primers targeting the RHOA gene are shown in SEQ NO.46 and SEQ NO.47, and the specific probe sequence targeting the RHOA gene is shown in SEQ NO.48.

[0029] As a preferred technical solution, each probe in the TRMT112 gene-specific probe is labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other; the TRMT112 gene-specific probe exists in a mixed form, including type A TRMT112 gene-specific probe and type B TRMT112 gene-specific probe. The 5' end of the type A TRMT112 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB; the 5' end of the type B TRMT112 gene-specific probe sequence is modified with ROX and the 3' end is modified with MGB.

[0030] The 5' end of the specific probe sequence of the PHF21A gene is modified with HEX, and the 3' end is modified with MGB.

[0031] The 5' end of the specific probe sequence of the PKD1 gene is modified with ROX, and the 3' end is modified with BHQ2.

[0032] The 5' end of the specific probe sequence of the RSF1 gene is modified with CY5, and the 3' end is modified with BHQ2;

[0033] The APLP2 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The APLP2 gene-specific probes exist in a mixed form, including type A APLP2 gene-specific probes and type B APLP2 gene-specific probes. The 5' end of the type A APLP2 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the type B APLP2 gene-specific probe sequence is modified with HEX and the 3' end is modified with MEG.

[0034] The OAZ2 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The OAZ2 gene-specific probes exist in a mixed form, including type A OAZ2 gene-specific probes and type B OAZ2 gene-specific probes. The 5' end of the type A OAZ2 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the type B OAZ2 gene-specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2.

[0035] The TXNIP gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The TXNIP gene-specific probes exist in a mixed form, including type A TXNIP gene-specific probes and type B TXNIP gene-specific probes. The 5' end of the type A TXNIP gene-specific probe sequence is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the type B TXNIP gene-specific probe sequence is modified with CY5, and the 3' end is modified with BHQ2.

[0036] The KRAS gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The KRAS gene-specific probes exist in a mixed form, including type A KRAS gene-specific probes and type B V gene-specific probes. The 5' end of the type A KRAS gene-specific probe sequence is modified with FAM and the 3' end is modified with BHQ1. The 5' end of the type B KRAS gene-specific probe sequence is modified with Q705 and the 3' end is modified with BHQ3.

[0037] The specific probes for the PLD3 gene are each labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other. The specific probes for the PLD3 gene exist in a mixed form, including type A PLD3 gene specific probes and type B PLD3 gene specific probes. The 5' end of the type A PLD3 gene specific probe sequence is modified with HEX and the 3' end is modified with BHQ1; the 5' end of the type B PLD3 gene specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2.

[0038] The specific probes for the PQBP1 gene are each labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other. The specific probes for the PQBP1 gene exist in a mixed form, including type A PQBP1 gene specific probes and type B PQBP1 gene specific probes. The 5' end of the type A PQBP1 gene specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2; the 5' end of the type B PQBP1 gene specific probe sequence is modified with Q705 and the 3' end is modified with BHQ3.

[0039] The SSTR5 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The SSTR5 gene-specific probes exist in a mixed form, including type A SSTR5 gene-specific probes and type B SSTR5 gene-specific probes. The 5' end of the type A SSTR5 gene-specific probe sequence is modified with Q705 and the 3' end is modified with BHQ3. The 5' end of the type B SSTR5 gene-specific probe sequence is modified with HEX and the 3' end is modified with BHQ1.

[0040] The 5' end of the specific probe sequence of the MORF4L2 gene is modified with FAM, and the 3' end is modified with MGB.

[0041] The NAP1L1 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The NAP1L1 gene-specific probes exist in a mixed form, including type A NAP1L1 gene-specific probes and type B NAP1L1 gene-specific probes. The 5' end of the type A NAP1L1 gene-specific probe sequence is modified with HEX and the 3' end is modified with MGB. The 5' end of the type B NAP1L1 gene-specific probe sequence is modified with ROX and the 3' end is modified with MGB.

[0042] The NUDT3 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The NUDT3 gene-specific probes exist in a mixed form, including type A NUDT3 gene-specific probes and type B NUDT3 gene-specific probes. The 5' end of the type A NUDT3 gene-specific probe sequence is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the type B NUDT3 gene-specific probe sequence is modified with Q705, and the 3' end is modified with BHQ3.

[0043] The 5' end of the RHOA gene-specific probe sequence is modified with Q705, and the 3' end is modified with BHQ3.

[0044] As a preferred technical solution, the qPCR reaction system is a 30 μl system as follows:

[0045] Prepare 10.0 μl of detection buffer, 0.5 μl of reactive enzyme, 5 μl of primer-probe mixture, and 5–15 μl of detection template. Add double-distilled water to a final volume of 30 μl.

[0046] As a preferred technical solution, the detection buffer comprises Tris-SO4, magnesium sulfate, potassium chloride and dNTPs, and the reaction enzyme is Taq enzyme.

[0047] As a preferred technical solution, the qPCR reaction program is 50℃ for 5 min; 95℃ for 3 min; then enter the cycling stage: denaturation at 95℃ for 15 s, annealing and extension at 60℃ for 60 s, for a total of 45 cycles.

[0048] The present invention also discloses the use of a kit for detecting neuroendocrine tumors.

[0049] Advantages of this invention:

[0050] Combining multiple primer pairs and probes of TRMT112, PHF21A, PKD1, RSF1, APLP2, OAZ2, TXNIP, KRAS, PLD3, PQBP1, SSTR5, MORF4L2, NAP1L1, and NUDT3 with the RHOA target saves operation time and reduces the possibility of contamination from individual amplification.

[0051] Primers and probes were designed specifically for the target sequences of neuroendocrine tumors to rapidly detect the presence of neuroendocrine tumor DNA in samples.

[0052] The reagents in this kit exhibit high specificity, enabling the detection of neuroendocrine tumor DNA. This kit provides etiological monitoring of neuroendocrine tumors and offers experimental evidence for clinical diagnosis. The probes in the kit specifically detect high-risk genes, enabling more efficient detection of gene mutations that are significant for diagnosis and treatment. It also reduces the cost of gene testing for patients and the operational difficulty for lab technicians. Furthermore, it may help some neuroendocrine tumor patients participate in clinical trials, providing important guidance for clinical diagnosis and the discovery of targeted therapies. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the prior art and the embodiments will be briefly introduced below.

[0054] Figure 1 This is a scatter plot of one dimension of the FAM detection channel in an embodiment of the present invention;

[0055] Figure 2 This is a scatter plot of one dimension of the HEX detection channel in an embodiment of the present invention;

[0056] Figure 3 This is a scatter plot of one dimension of the ROX detection channel in an embodiment of the present invention;

[0057] Figure 4 This is a scatter plot of one dimension of the Cy5 detection channel in an embodiment of the present invention;

[0058] Figure 5 This is a scatter plot of the Q705 detection channel in an embodiment of the present invention. Detailed Implementation

[0059] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0060] Example:

[0061] The testing process is as follows:

[0062] 1. Nucleic acid extraction

[0063] 2. Consistent preparation and quantification of cDNA

[0064] 2.1. cDNA Preparation

[0065] 2.2. Quantitative Consistency

[0066] Take 2 μL of cDNA sample and test its concentration using a UV spectrophotometer. Select ssDNA mode for the spectrophotometer test mode. After the test, dilute the cDNA template with pure water to adjust it to 14–40 ng / μL to facilitate a loading volume of 5–15 μL when the sample loading amount is 200 ng.

[0067] 3. Preparation of digital PCR reaction system

[0068] 3.1. Main Reagents

[0069]

[0070] The PCR detection system includes the following reaction buffers: 80 mM Tris-SO4 at pH 9.2, 2.5 mM magnesium sulfate, 75 mM potassium chloride, and 0.2 mM dNTPs; the reaction enzyme: 2.5 U / 30 μL Taq enzyme; the detection primers are all at concentrations of 200–400 nM, and the probes are all at concentrations of 100–400 nM.

[0071] 3.2. System Preparation

[0072] a. Remove each component and allow it to fully dissolve at room temperature. Mix thoroughly and briefly centrifuge to collect the reagents at the bottom of the reagent tube.

[0073] b. Prepare a digital PCR detection system for multiple respiratory pathogens according to the table below.

[0074] dPCR reaction system

[0075]

[0076] C. Mix thoroughly and briefly centrifuge to enrich the liquid at the bottom of the tube. Dispense 15 μL into each of the matching 12-tube sets. Then add 5–15 μL of sample nucleic acid, 5 μL of negative control, and 5 μL of positive control in sequence. Make up to 30 μL with nuclease-free water. Mix by pipetting and then briefly centrifuge to remove air bubbles.

[0077] 3.3. Preparation process of the primer-probe MIX

[0078] 3.3.1. All primers and probes were sent to the synthesis company (Bailige Biotechnology (Shanghai) Co., Ltd., address: Building 23, No. 168 Shuya Road, Songjiang District, Shanghai). The probes were purified by HPLC and the primers were purified by PAGE. The synthesized products were obtained in dry powder form.

[0079] Its sequence is shown in Table 1 below:

[0080]

[0081]

[0082] 3.3.2. Dissolution of primers and probes:

[0083] The dry powder tubes were centrifuged at 12000×g for 5 minutes to concentrate all the dry powder at the bottom of the reagent tubes.

[0084] The label on the dry powder tube indicates that the primers should be dissolved in 100 μM ddH2O. Only half the labeled volume needs to be added. The dissolving solution is LOW (EDTA)TE. Vortex for 10 seconds to mix thoroughly, then centrifuge for 2 seconds to collect all liquid at the bottom of the tube. Let stand at room temperature for 10 minutes to allow the Oligo to fully dissolve and disperse. This step is labeled as Mother Solution A.

[0085] 3.3.3. Primer and probe concentration setting:

[0086] Take 2 μL of each primer and probe stock solution A and measure the ssDNA concentration using a UV spectrophotometer. Then, convert the stock solution A concentration to the actual molar concentration (μM) using the following formula.

[0087]

[0088] Note: N b = Number of base pairs (ssDNA), ssDNA is single-stranded DNA, N b To express the quantity of nucleotides, pmol (picomolar), μg (microgram), and pg (picogram) are all units.

[0089] 3.3.4. Primer and probe concentration uniformity

[0090] Adjust the concentration of all primers and probes to 100 μM and let them stand at room temperature for later use.

[0091] 3.3.5. Prepare primers and probes according to Table 2 below.

[0092]

[0093]

[0094]

[0095] 3.4. Use of dPCR instrument

[0096] a. Refer to the instrument's operating manual to complete the equipment operation (SCIDigital operation example).

[0097] b. Initialization: After the device is powered on, open the "SCIDigital" software shortcut on the computer desktop. The software will automatically connect to the device and complete the device initialization.

[0098] c. Place consumables: Open the equipment compartment door and correctly place the unused digital PCR chip, the 12-tube array with PCR reaction solution, oil phase A and oil phase B, the injection tip, and the waste box into the corresponding workstation of the equipment in sequence.

[0099] d. Configure the reaction program in the software settings interface as follows:

[0100]

[0101] e. Detection channels to select: FAM, HEX, ROX, Cy5, and Q705.

[0102] f. Run the experiment, wait for the program to finish, and then view the analysis data.

[0103] 4. Data Collection and Result Interpretation

[0104] Once the equipment operation is complete, data acquisition is finished. First, adjust the threshold line in the one-dimensional scatter plot. The threshold line can be set automatically by software or manually: it should just exceed the highest point of the negative droplet. By comparing the coding information of different target points and the fluorescence signals of the detection results, the type of detection template is determined and the copy number is counted. Based on the positive rate of positive points in each fluorescence channel, the co-positive rate among multiple channels, the inclusion-exclusion principle, and the Poisson distribution principle, the copy number of each coded target point is calculated, and the detection results are interpreted. Specifically:

[0105] Two-channel coding target positivity rate P 12 = 1 - (1 - T1) * (1 - T2) / (1 - T1 - T2 + T) 12 The corresponding template copy number is C12 = -ln(1-P). 12 ) / V;

[0106] Two-channel coding target positivity rate P 13 = 1 - (1 - T1) * (1 - T3) / (1 - T1 - T3 + T) 13 The corresponding template copy number is C. 13 = -ln(1-P 13 ) / V;

[0107] Two-channel coding target positivity rate P 14 = 1 - (1 - T1) * (1 - T4) / (1 - T1 - T4 + T) 14 The corresponding template copy number is C. 14 = -ln(1-P 14) / V;

[0108] Two-channel coding target positivity rate P 23 = 1 - (1 - T2) * (1 - T3) / (1 - T2 - T3 + T) 23 The corresponding template copy number is C.23 = -ln(1-P 23 ) / V;

[0109] Two-channel coding target positivity rate P 24 = 1 - (1 - T2) * (1 - T4) / (1 - T2 - T4 + T) 24 The corresponding template copy number is C. 24 = -ln(1-P 24 ) / V;

[0110] Two-channel coding target positivity rate P 34 = 1 - (1 - T3) * (1 - T4) / (1 - T3 - T4 + T) 34 The corresponding template copy number is C. 34 = -ln(1-P 34 ) / V;

[0111] Single-channel coding target positivity rate P1 = T1, corresponding template copy number C1 =

[0112] -ln(1-P1) / VC 12 -C 13 -C 14 ;

[0113] Single-channel coding target positivity rate P2 = T2, corresponding template copy number C2 =

[0114] -ln(1-P2) / VC 12 -C 23 -C 24 ;

[0115] Single-channel coding target positivity rate P3 = T3, corresponding template copy number C3 =

[0116] -ln(1-P3) / VC 13 -C 23 -C 34 ;

[0117] Single-channel coding target positivity rate P4 = T4, corresponding template copy number C4 =

[0118] -ln(1-P4) / VC 14 -C 24 -C 34 T represents the positive rate of the current test channel (the actual test data), the superscripts 1, 2, 3, and 4 refer to different fluorescent labels, C is the copy number concentration, and V is the volume of a single reaction chamber.

[0119] 5.15 target multi-channel encoding

[0120]

[0121] 6. Example of a positive sample test result

[0122] To verify the accuracy of the reagent detection, total RNA was extracted from cell lines, and cDNA was obtained after reverse transcription. The cDNA was then quantified using a UV spectrophotometer. The detection system was prepared according to the operating procedure and tested on a dPCR platform. The control group included both single-system and multiplex system tests. The detection results are shown in the table below.

[0123] 6.1. Chip detection results (sample amount 200ng)

[0124] 6.1.1. One-dimensional scatter plot (see...) Figures 1 to 5 )

[0125] 6.1.2. The repeatability test results are shown in Table 3 below:

[0126]

[0127] Note: Ave is the mean, STD is the standard deviation, and CV is the coefficient of variation.

[0128] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A reagent kit for detecting neuroendocrine tumors, characterized in that: Includes detection buffer, reactive enzyme, primer-probe mixture, negative control and positive control. The primer and probe mixture includes upstream and downstream primers and probes for the TRMT112 gene, the PHF21A gene, the PKD1 gene, the RSF1 gene, the APLP2 gene, the OAZ2 gene, the TXNIP gene, the KRAS gene, the PLD3 gene, the PQBP1 gene, the SSTR5 gene, the MORF4L2 gene, the NAP1L1 gene, the NUDT3 gene, and the RHOA gene. The upstream and downstream primers of the TRMT112 gene have sequences as shown in SEQ NO.1 and SEQ NO.2, the specific probe sequence for the TRMT112 gene is shown in SEQ NO.3, and the probe for the TRMT112 gene also includes the TRMT112 molecular beacon probe, the sequence of which is shown in SEQ NO.

4. The sequences of the upstream and downstream primers for the PHF21A gene are shown in SEQ NO.5 and SEQ NO.6, the specific probe sequence for the PHF21A gene is shown in SEQ NO.7, and the probe for the PHF21A gene also includes the PHF21A molecular beacon probe, the sequence of which is shown in SEQ NO.

8. The sequences of the upstream and downstream primers for the PKD1 gene are shown in SEQ NO.9 and SEQ NO.10, and the specific probe sequence for the PKD1 gene is shown in SEQ NO.

11. The sequences of the upstream and downstream primers for the RSF1 gene are shown in SEQ NO.12 and SEQ NO.13, and the specific probe sequence for the RSF1 gene is shown in SEQ NO.

14. The sequences of the upstream and downstream primers for the APLP2 gene are shown in SEQ NO.15 and SEQ NO.16, and the specific probe sequence for the APLP2 gene is shown in SEQ NO.

17. The upstream and downstream primers for the OAZ2 gene are shown in SEQ NO.18 and SEQ NO.

19. The specific probes for the OAZ2 gene include a type A OAZ2 gene-specific probe and a type B OAZ2 gene-specific probe. The sequence of the type A OAZ2 gene-specific probe is shown in SEQ NO.20, and the sequence of the type B OAZ2 gene-specific probe is shown in SEQ NO.

21. The sequences of the upstream and downstream primers for the TXNIP gene are shown in SEQ NO.22 and SEQ NO.23, and the specific probe sequence for the TXNIP gene is shown in SEQ NO.

24. The sequences of the upstream and downstream primers for the KRAS gene are shown in SEQ NO.25 and SEQ NO.26, and the specific probe sequence for the KRAS gene is shown in SEQ NO.

27. The sequences of the upstream and downstream primers for the PLD3 gene are shown in SEQ NO.28 and SEQ NO.29, and the specific probe sequence for the PLD3 gene is shown in SEQ NO.

30. The sequences of the upstream and downstream primers for the PQBP1 gene are shown in SEQ NO.31 and SEQ NO.32, and the specific probe sequence for the PQBP1 gene is shown in SEQ NO.

33. The sequences of the upstream and downstream primers for the SSTR5 gene are shown in SEQ NO.34 and SEQ NO.35, and the specific probe sequence for the SSTR5 gene is shown in SEQ NO.

36. The sequences of the upstream and downstream primers for the MORF4L2 gene are shown in SEQ NO.37 and SEQ NO.38, and the specific probe sequence for the MORF4L2 gene is shown in SEQ NO.

39. The sequences of the upstream and downstream primers for the NAP1L1 gene are shown in SEQ NO.40 and SEQ NO.41, and the specific probe sequence for the NAP1L1 gene is shown in SEQ NO.

42. The sequences of the upstream and downstream primers for the NUDT3 gene are shown in SEQ NO.43 and SEQ NO.44, and the specific probe sequence for the NUDT3 gene is shown in SEQ NO.

45. The sequences of the upstream and downstream primers targeting the RHOA gene are shown in SEQ NO.46 and SEQ NO.47, and the specific probe sequence targeting the RHOA gene is shown in SEQ NO.

48.

2. The reagent kit for detecting neuroendocrine tumors as described in claim 1, characterized in that: The TRMT112 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The TRMT112 gene-specific probes exist in a mixed form, including type A TRMT112 gene-specific probes and type B TRMT112 gene-specific probes. The 5' end of the type A TRMT112 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the type B TRMT112 gene-specific probe sequence is modified with ROX and the 3' end is modified with MGB. The 5' end of the specific probe sequence of the PHF21A gene is modified with HEX, and the 3' end is modified with MGB. The 5' end of the specific probe sequence of the PKD1 gene is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the specific probe sequence of the RSF1 gene is modified with CY5, and the 3' end is modified with BHQ2; Each probe in the APLP2 gene-specific probe is labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other; the APLP2 gene-specific probe exists in a mixed form, including type A APLP2 gene-specific probe and type B APLP2 gene-specific probe, and the 5' end of the type A APLP2 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the B-type APLP2 gene-specific probe sequence was modified with HEX, and the 3' end was modified with MEG. The OAZ2 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The OAZ2 gene-specific probes exist in a mixed form, including type A OAZ2 gene-specific probes and type B OAZ2 gene-specific probes. The 5' end of the type A OAZ2 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the type B OAZ2 gene-specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2. The TXNIP gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The TXNIP gene-specific probes exist in a mixed form, including type A TXNIP gene-specific probes and type B TXNIP gene-specific probes. The 5' end of the type A TXNIP gene-specific probe sequence is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the type B TXNIP gene-specific probe sequence is modified with CY5, and the 3' end is modified with BHQ2. The KRAS gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The KRAS gene-specific probes exist in a mixed form, including type A KRAS gene-specific probes and type B V gene-specific probes. The 5' end of the type A KRAS gene-specific probe sequence is modified with FAM and the 3' end is modified with BHQ1. The 5' end of the type B KRAS gene-specific probe sequence is modified with Q705 and the 3' end is modified with BHQ3. The specific probes for the PLD3 gene are each labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other. The specific probes for the PLD3 gene exist in a mixed form, including type A PLD3 gene specific probes and type B PLD3 gene specific probes. The 5' end of the type A PLD3 gene specific probe sequence is modified with HEX, and the 3' end is modified with BHQ1. The 5' end of the B-type PLD3 gene-specific probe sequence was modified with CY5, and the 3' end was modified with BHQ2. The specific probes for the PQBP1 gene are each labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other. The specific probes for the PQBP1 gene exist in a mixed form, including type A PQBP1 gene specific probes and type B PQBP1 gene specific probes. The 5' end of the type A PQBP1 gene specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2; the 5' end of the type B PQBP1 gene specific probe sequence is modified with Q705 and the 3' end is modified with BHQ3. The SSTR5 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The SSTR5 gene-specific probes exist in a mixed form, including type A SSTR5 gene-specific probes and type B SSTR5 gene-specific probes. The 5' end of the type A SSTR5 gene-specific probe sequence is modified with Q705 and the 3' end is modified with BHQ3. The 5' end of the type B SSTR5 gene-specific probe sequence is modified with HEX and the 3' end is modified with BHQ1. The 5' end of the specific probe sequence of the MORF4L2 gene is modified with FAM, and the 3' end is modified with MGB. Each probe in the NAP1L1 gene-specific probe is labeled with a fluorescent group, and the fluorescent groups are different from each other and do not interfere with each other; the NAP1L1 gene-specific probe exists in a mixed form, including type A NAP1L1 gene-specific probe and type B NAP1L1 gene-specific probe, and the 5' end of the type A NAP1L1 gene-specific probe sequence is modified with HEX and the 3' end is modified with MGB. The 5' end of the B-type NAP1L1 gene-specific probe sequence was modified with ROX, and the 3' end was modified with MGB. The NUDT3 gene-specific probes are labeled with fluorescent groups, and the fluorescent groups are different from each other and do not interfere with each other. The NUDT3 gene-specific probes exist in a mixed form, including type A NUDT3 gene-specific probes and type B NUDT3 gene-specific probes. The 5' end of the type A NUDT3 gene-specific probe sequence is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the type B NUDT3 gene-specific probe sequence is modified with Q705, and the 3' end is modified with BHQ3. The 5' end of the specific probe sequence of the RHOA gene is modified with Q705, and the 3' end is modified with BHQ3.

3. A kit for detecting neuroendocrine tumors as described in claim 1 or 2, characterized in that, The qPCR reaction system is a 30 μl volume as follows: Prepare 10.0 μl of detection buffer, 0.5 μl of reactive enzyme, 5 μl of primer-probe mixture, and 5–15 μl of detection template. Add double-distilled water to a final volume of 30 μl.

4. The kit for detecting neuroendocrine tumors as described in claim 1, characterized in that: The detection buffer solution comprises Tris-SO4, magnesium sulfate, potassium chloride, and dNTPs, and the reaction enzyme is Taq polymerase.

5. The reagent kit for detecting neuroendocrine tumors as described in claim 3, characterized in that: The qPCR reaction program was 50℃ for 5 min; 95℃ for 3 min; then cycling: 95℃ denaturation for 15 s, 60℃ annealing and extension for 60 s, for a total of 45 cycles.

6. Use of a kit for detecting neuroendocrine tumors as described in any one of claims 1 or 2.

Images