A kit for rapid detection of neuroendocrine tumor and use thereof

Abstract

The present application relates to the technical field of clinical detection, and discloses a kit for rapidly detecting neuroendocrine tumor, and provides specific primers and probes for detecting neuroendocrine tumor. A plurality of pairs of primers and probes of SSTR3, TPH1, C21orf7, FZD7, BNIP3L, SMARCD3, SSTR4, SSTR1, WDFY3, LEO1 and GLT8D1 targets are combined into an entirety, so that the sample, reagent and operation time are saved, and the possibility of single amplification contamination is reduced. The present application realizes the simultaneous detection of multiple targets of neuroendocrine tumor, and has good clinical practical value and application prospect.

Description

Technical Field

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

[0002] Neuroendocrine tumors (NETs) are tumors originating from neuroendocrine cells, a large class of cells in the body with neuroendocrine functions that produce a variety of hormones. They are distributed throughout the body, therefore neuroendocrine tumors can occur anywhere in the body. These tumors produce, store, and secrete various peptides and neuroamines, and can cause specific clinical manifestations. They are most common in the gastrointestinal tract and pancreas, accounting for approximately 75% of neuroendocrine tumors, and can also occur in the lungs and bronchi. The vast majority occur in adults; neuroendocrine tumors are extremely rare in children.

[0003] The clinical presentation of neuroendocrine tumors (NETs) varies depending on the tumor's origin, location, size, and the type of hormones it secretes. Some patients may be asymptomatic, while others may experience a variety of symptoms, such as indigestion, abdominal pain, diarrhea, vomiting, weight loss, hypoglycemia, hot flashes, and tachycardia. Because these symptoms resemble those of other common illnesses and are nonspecific, neuroendocrine tumors are often misdiagnosed as other gastrointestinal problems or metabolic disorders. Since most neuroendocrine tumors are asymptomatic, regular checkups are crucial for early screening of this disease.

[0004] Neuroendocrine tumor detection kits play an important role in detection, but some problems and challenges still exist.

[0005] For example, some kits lack sufficient sensitivity when detecting low concentrations or small lesions, potentially leading to false negatives. Similarly, insufficient specificity can result in false positives, especially due to cross-reactivity with other types of tumors or non-tumor diseases. This can affect early diagnosis and accurate classification.

[0006] Some tests require 24-hour urine collection, leading to poor patient compliance. Biomarkers in blood samples can be affected by various factors (such as medications, diet, and other diseases), causing instability in sample quality and impacting the reliability of test results.

[0007] 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.

[0008] Therefore, there is an urgent need for a rapid detection kit for neuroendocrine tumors to solve the above problems. Summary of the Invention

[0009] The purpose of this invention is to provide a rapid detection kit for neuroendocrine tumors and its application, solving the problem of how to quickly, comprehensively and accurately detect the presence of neuroendocrine tumors in samples.

[0010] The solution of the present invention is:

[0011] A rapid detection kit for neuroendocrine tumors, comprising detection buffer, reactive enzyme, primer-probe mixture, negative control and positive control;

[0012] The primer and probe mixture includes upstream and downstream primers and probes for the SSTR3 gene, upstream and downstream primers and probes for the TPH1 gene, upstream and downstream primers and probes for the C21orf7 gene, upstream and downstream primers and probes for the FZD7 gene, upstream and downstream primers and probes for the BNIP3L gene, upstream and downstream primers and probes for the SMARCD3 gene, upstream and downstream primers and probes for the SSTR4 gene, upstream and downstream primers and probes for the SSTR1 gene, upstream and downstream primers and probes for the WDFY3 gene, upstream and downstream primers and probes for the LEO1 gene, and upstream and downstream primers and probes for the GLT8D1 gene.

[0013] The upstream and downstream primers of the SSTR3 gene have sequences as shown in SEQ NO.1 and SEQ NO.2, the specific probe sequence for the SSTR3 gene is shown in SEQ NO.3, and the probe for the SSTR3 gene also includes the SSTR3 molecular beacon probe, the sequence of which is shown in SEQ NO.4.

[0014] The sequences of the upstream and downstream primers for the TPH1 gene are shown in SEQ NO.5 and SEQ NO.6, and the specific probe sequence for the TPH1 gene is shown in SEQ NO.7.

[0015] The sequences of the upstream and downstream primers for the C21orf7 gene are shown in SEQ NO.8 and SEQ NO.9, and the specific probe sequence for the C21orf7 gene is shown in SEQ NO.10.

[0016] The sequences of the upstream and downstream primers for the FZD7 gene are shown in SEQ NO.11 and SEQ NO.12, and the specific probe sequence for the FZD7 gene is shown in SEQ NO.13.

[0017] The sequences of the upstream and downstream primers for the BNIP3L gene are shown in SEQ NO.14 and SEQ NO.15, and the specific probe sequence for the BNIP3L gene is shown in SEQ NO.16.

[0018] The sequences of the upstream and downstream primers for the SMARCD3 gene are shown in SEQ NO.17 and SEQ NO.18, and the specific probe sequence for the SMARCD3 gene is shown in SEQ NO.19.

[0019] The sequences of the upstream and downstream primers for the SSTR4 gene are shown in SEQ NO.20 and SEQ NO.21, and the specific probe sequence for the SSTR4 gene is shown in SEQ NO.22.

[0020] The sequences of the upstream and downstream primers for the SSTR1 gene are shown in SEQ NO.23 and SEQ NO.24, and the specific probe sequence for the SSTR1 gene is shown in SEQ NO.25.

[0021] The sequences of the upstream and downstream primers for the WDFY3 gene are shown in SEQ NO.26 and SEQ NO.27, and the specific probe sequence for the WDFY3 gene is shown in SEQ NO.28.

[0022] The upstream and downstream primers for the LEO1 gene are shown in SEQ NO.29 and SEQ NO.30. The specific probes for the LEO1 gene include a type A LEO1 gene-specific probe and a type B LEO1 gene-specific probe. The sequence of the type A LEO1 gene-specific probe is shown in SEQ NO.31, and the sequence of the type B LEO1 gene-specific probe is shown in SEQ NO.32.

[0023] The upstream and downstream primers for the GLT8D1 gene are shown in SEQ NO.33 and SEQ NO.34. The specific probe sequence for the GLT8D1 gene is shown in SEQ NO.35. The probes for the GLT8D1 gene also include the GLT8D1 molecular beacon probe, the sequence of which is shown in SEQ NO.36.

[0024] As a preferred technical solution, the 5' end of the specific probe sequence of the SSTR3 gene is modified with FAM, and the 3' end is modified with MGB;

[0025] The 5' end of the specific probe sequence of the TPH1 gene is modified with HEX, and the 3' end is modified with MGB.

[0026] The 5' end of the specific probe sequence of the C21orf7 gene is modified with ROX, and the 3' end is modified with BHQ2.

[0027] The 5' end of the specific probe sequence of the FZD7 gene is modified with CY5, and the 3' end is modified with BHQ2.

[0028] The 5' end of the specific probe sequence of the BNIP3L gene is modified with Q705, and the 3' end is modified with BHQ3;

[0029] The SMARCD3 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 SMARCD3 gene-specific probes exist in a mixed form, including type A SMARCD3 gene-specific probes and type B SMARCD3 gene-specific probes. The 5' end of the type A SMARCD3 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the type B SMARCD3 gene-specific probe sequence is modified with ROX and the 3' end is modified with MGB.

[0030] The SSTR4 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 SSTR4 gene-specific probes exist in a mixed form, including type A SSTR4 gene-specific probes and type B SSTR4 gene-specific probes. The 5' end of the type A SSTR4 gene-specific probe sequence is modified with HEX and the 3' end is modified with BHQ1. The 5' end of the type B SSTR4 gene-specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2.

[0031] The SSTR1 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 SSTR1 gene-specific probes exist in a mixed form, including type A SSTR1 gene-specific probes and type B SSTR1 gene-specific probes. The 5' end of the type A SSTR1 gene-specific probe sequence is modified with FAM and the 3' end is modified with BHQ1. The 5' end of the type B SSTR1 gene-specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2.

[0032] The WDFY3 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 WDFY3 gene-specific probes exist in a mixed form, including type A WDFY3 gene-specific probes and type B WDFY3 gene-specific probes. The 5' end of the type A WDFY3 gene-specific probe sequence is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the type B WDFY3 gene-specific probe sequence is modified with Cy5, and the 3' end is modified with BHQ2.

[0033] The LEO1 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 LEO1 gene-specific probes exist in a mixed form, including type A LEO1 gene-specific probes and type B LEO1 gene-specific probes. The 5' end of the type A LEO1 gene-specific probe sequence is modified with ROX and the 3' end is modified with BHQ2. The 5' end of the type B LEO1 gene-specific probe sequence is modified with HEX and the 3' end is modified with MGB.

[0034] The GLT8D1 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 GLT8D1 gene-specific probes exist in a mixed form, including type A GLT8D1 gene-specific probes and type B GLT8D1 gene-specific probes. The 5' end of the type A GLT8D1 gene-specific probe sequence is modified with FAM and the 3' end is modified with BHQ1. The 5' end of the type B GLT8D1 gene-specific probe sequence is modified with HEX and the 3' end is modified with BHQ1.

[0035] As a preferred technical solution, the qPCR reaction system is a 30 μl system as follows:

[0036] 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.

[0037] As a preferred technical solution, the detection buffer comprises Tris-SO4, magnesium sulfate, potassium chloride and dNTPs, and the reaction enzyme is Taq enzyme.

[0038] 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.

[0039] The present invention also discloses the use of a kit for rapid detection of neuroendocrine tumors.

[0040] Advantages of this invention:

[0041] Combining multiple primer-probe pairs targeting SSTR3, TPH1, C21orf7, FZD7, BNIP3L, SMARCD3, SSTR4, SSTR1, WDFY3, LEO1, and GLT8D1 into a single unit saves sample and reagent costs and operation time, and also reduces the possibility of contamination from individual amplification.

[0042] The reagents provided by this invention have excellent specificity and can detect the DNA of neuroendocrine tumors.

[0043] The probe described in this invention achieves highly sensitive detection and greatly increases signal amplification efficiency; it can realize the exponential amplification of nucleic acids of various neuroendocrine tumor targets, avoid the interference of different primer types or excessively high concentrations on the amplification system, and eliminate the interference of non-specific amplification of the system.

[0044] The probe of this invention can integrate ligation reaction and amplification reaction, and the experimental method is simple and easy to perform.

[0045] This invention enables simultaneous detection of multiple targets in neuroendocrine tumors, demonstrating significant clinical value and promising application prospects. The kit provided by this invention can perform etiological monitoring of neuroendocrine tumors and provide experimental evidence for clinical diagnosis. Attached Figure Description

[0046] 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.

[0047] Figure 1 This is a scatter plot of one dimension of the FAM detection channel in an embodiment of the present invention;

[0048] Figure 2 This is a scatter plot of one dimension of the HEX detection channel in an embodiment of the present invention;

[0049] Figure 3 This is a scatter plot of one dimension of the ROX detection channel in an embodiment of the present invention;

[0050] Figure 4 This is a scatter plot of one dimension of the Cy5 detection channel in an embodiment of the present invention;

[0051] Figure 5 This is a scatter plot of the Q705 detection channel in an embodiment of the present invention. Detailed Implementation

[0052] 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.

[0053] Example:

[0054] The testing process is as follows:

[0055] 1. Nucleic acid extraction

[0056] 2. Consistent preparation and quantification of cDNA

[0057] 2.1. cDNA Preparation

[0058] 2.2. Quantitative Consistency

[0059] 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.

[0060] 3. Preparation of digital PCR reaction system

[0061] 3.1. Main Reagents

[0062]

[0063] 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.

[0064] 3.2. System Preparation

[0065] 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.

[0066] b. Prepare a digital PCR detection system for multiple respiratory pathogens according to the table below.

[0067]

[0068] 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.

[0069] 3.3. Preparation process of the primer-probe MIX

[0070] 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.

[0071] Its sequence is shown in Table 1 below:

[0072]

[0073]

[0074] 3.3.2. Dissolution of primers and probes:

[0075] 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.

[0076] 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.

[0077] 3.3.3. Primer and probe concentration setting:

[0078] 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.

[0079]

[0080] 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.

[0081] 3.3.4. Primer and probe concentration uniformity

[0082] Adjust the concentration of all primers and probes to 100 μM and let them stand at room temperature for later use.

[0083] 3.3.5. Prepare primers and probes according to Table 2 below.

[0084]

[0085]

[0086] 3.4. Use of dPCR instrument

[0087] a. Refer to the instrument's operating manual to complete the equipment operation (SCIDigital operation example).

[0088] 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.

[0089] 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.

[0090] d. Configure the reaction program in the software settings interface as follows:

[0091]

[0092] e. Detection channels to select: FAM, HEX, ROX, Cy5, and Q705.

[0093] f. Run the experiment, wait for the program to finish, and then view the analysis data.

[0094] 4. Data Collection and Result Interpretation

[0095] 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:

[0096] 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;

[0097] 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;

[0098] 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;

[0099] 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;

[0100] 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;

[0101] 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;

[0102] Single-channel coding target positivity rate P1 = T1, corresponding template copy number C1 =

[0103] -ln(1-P1) / VC 12 -C 13 -C 14 ;

[0104] Single-channel coding target positivity rate P2 = T2, corresponding template copy number C2 =

[0105] -ln(1-P2) / VC 12 -C 23 -C 24 ;

[0106] Single-channel coding target positivity rate P3 = T3, corresponding template copy number C3 =

[0107] -ln(1-P3) / VC 13 -C 23 -C 34 ;

[0108] Single-channel coding target positivity rate P4 = T4, corresponding template copy number C4 =

[0109] -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.

[0110] 5.11 target multi-channel encoding

[0111]

[0112]

[0113] 6. Example of a positive sample test result

[0114] 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.

[0115] 6.1. Chip detection results (sample amount 200ng)

[0116] 6.1.1. One-dimensional scatter plot (see...) Figures 1 to 5 )

[0117] 6.1.2. The repeatability test results are shown in Table 3 below:

[0118]

[0119] Note: Ave is the mean, STD is the standard deviation, and CV is the coefficient of variation.

[0120] 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 rapid detection kit for neuroendocrine tumors, characterized in that: This 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 SSTR3 gene, upstream and downstream primers and probes for the TPH1 gene, upstream and downstream primers and probes for the C21orf7 gene, upstream and downstream primers and probes for the FZD7 gene, upstream and downstream primers and probes for the BNIP3L gene, upstream and downstream primers and probes for the SMARCD3 gene, upstream and downstream primers and probes for the SSTR4 gene, upstream and downstream primers and probes for the SSTR1 gene, upstream and downstream primers and probes for the WDFY3 gene, upstream and downstream primers and probes for the LEO1 gene, and upstream and downstream primers and probes for the GLT8D1 gene. The upstream and downstream primers of the SSTR3 gene have sequences as shown in SEQ NO.1 and SEQ NO.2, the specific probe sequence for the SSTR3 gene is shown in SEQ NO.3, and the probe for the SSTR3 gene also includes the SSTR3 molecular beacon probe, the sequence of which is shown in SEQ NO.

4. The sequences of the upstream and downstream primers for the TPH1 gene are shown in SEQ NO.5 and SEQ NO.6, and the specific probe sequence for the TPH1 gene is shown in SEQ NO.

7. The sequences of the upstream and downstream primers for the C21orf7 gene are shown in SEQ NO.8 and SEQ NO.9, and the specific probe sequence for the C21orf7 gene is shown in SEQ NO.

10. The sequences of the upstream and downstream primers for the FZD7 gene are shown in SEQ NO.11 and SEQ NO.12, and the specific probe sequence for the FZD7 gene is shown in SEQ NO.

13. The sequences of the upstream and downstream primers for the BNIP3L gene are shown in SEQ NO.14 and SEQ NO.15, and the specific probe sequence for the BNIP3L gene is shown in SEQ NO.

16. The sequences of the upstream and downstream primers for the SMARCD3 gene are shown in SEQ NO.17 and SEQ NO.18, and the specific probe sequence for the SMARCD3 gene is shown in SEQ NO.

19. The sequences of the upstream and downstream primers for the SSTR4 gene are shown in SEQ NO.20 and SEQ NO.21, and the specific probe sequence for the SSTR4 gene is shown in SEQ NO.

22. The sequences of the upstream and downstream primers for the SSTR1 gene are shown in SEQ NO.23 and SEQ NO.24, and the specific probe sequence for the SSTR1 gene is shown in SEQ NO.

25. The sequences of the upstream and downstream primers for the WDFY3 gene are shown in SEQ NO.26 and SEQ NO.27, and the specific probe sequence for the WDFY3 gene is shown in SEQ NO.

28. The upstream and downstream primers for the LEO1 gene are shown in SEQ NO.29 and SEQ NO.

30. The specific probes for the LEO1 gene include a type A LEO1 gene-specific probe and a type B LEO1 gene-specific probe. The sequence of the type A LEO1 gene-specific probe is shown in SEQ NO.31, and the sequence of the type B LEO1 gene-specific probe is shown in SEQ NO.

32. The upstream and downstream primers for the GLT8D1 gene are shown in SEQ NO.33 and SEQ NO.

34. The specific probe sequence for the GLT8D1 gene is shown in SEQ NO.

35. The probes for the GLT8D1 gene also include the GLT8D1 molecular beacon probe, the sequence of which is shown in SEQ NO.

36.

2. The kit for rapid detection of neuroendocrine tumors as described in claim 1, characterized in that: The 5' end of the specific probe sequence of the SSTR3 gene is modified with FAM, and the 3' end is modified with MGB. The 5' end of the specific probe sequence of the TPH1 gene is modified with HEX, and the 3' end is modified with MGB. The 5' end of the specific probe sequence of the C21orf7 gene is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the specific probe sequence of the FZD7 gene is modified with CY5, and the 3' end is modified with BHQ2. The 5' end of the specific probe sequence of the BNIP3L gene is modified with Q705, and the 3' end is modified with BHQ3; The SMARCD3 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 SMARCD3 gene-specific probes exist in a mixed form, including type A SMARCD3 gene-specific probes and type B SMARCD3 gene-specific probes. The 5' end of the type A SMARCD3 gene-specific probe sequence is modified with FAM and the 3' end is modified with MGB. The 5' end of the type B SMARCD3 gene-specific probe sequence is modified with ROX and the 3' end is modified with MGB. The SSTR4 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 SSTR4 gene-specific probes exist in a mixed form, including type A SSTR4 gene-specific probes and type B SSTR4 gene-specific probes. The 5' end of the type A SSTR4 gene-specific probe sequence is modified with HEX and the 3' end is modified with BHQ1. The 5' end of the type B SSTR4 gene-specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2. The SSTR1 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 SSTR1 gene-specific probes exist in a mixed form, including type A SSTR1 gene-specific probes and type B SSTR1 gene-specific probes. The 5' end of the type A SSTR1 gene-specific probe sequence is modified with FAM and the 3' end is modified with BHQ1. The 5' end of the type B SSTR1 gene-specific probe sequence is modified with CY5 and the 3' end is modified with BHQ2. The WDFY3 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 WDFY3 gene-specific probes exist in a mixed form, including type A WDFY3 gene-specific probes and type B WDFY3 gene-specific probes. The 5' end of the type A WDFY3 gene-specific probe sequence is modified with ROX, and the 3' end is modified with BHQ2. The 5' end of the type B WDFY3 gene-specific probe sequence is modified with Cy5, and the 3' end is modified with BHQ2. The LEO1 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 LEO1 gene-specific probes exist in a mixed form, including type A LEO1 gene-specific probes and type B LEO1 gene-specific probes. The 5' end of the type A LEO1 gene-specific probe sequence is modified with ROX and the 3' end is modified with BHQ2. The 5' end of the type B LEO1 gene-specific probe sequence is modified with HEX and the 3' end is modified with MGB. The GLT8D1 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 GLT8D1 gene-specific probes exist in a mixed form, including type A GLT8D1 gene-specific probes and type B GLT8D1 gene-specific probes. The 5' end of the type A GLT8D1 gene-specific probe sequence is modified with FAM and the 3' end is modified with BHQ1. The 5' end of the type B GLT8D1 gene-specific probe sequence is modified with HEX and the 3' end is modified with BHQ1.

3. The kit for rapid detection of 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 rapid detection of 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 kit for rapid detection of 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 rapid detection of neuroendocrine tumors as described in any one of claims 1 or 2.

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