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TERT mutation detection kit and application thereof in noninvasive tumor diagnosis

A detection kit and mutation detection technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of non-early detection of patients, rising incidence of RPC, lack of early symptoms and effective diagnostic methods and other issues to achieve the effects of reducing patient pain, improving detection accuracy, high sensitivity and accuracy

Inactive Publication Date: 2020-08-21
郑成云
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the past 20 years, the incidence of RPC has increased significantly, and due to the lack of early symptoms and effective diagnostic methods, most patients are not diagnosed at an early stage.

Method used

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  • TERT mutation detection kit and application thereof in noninvasive tumor diagnosis
  • TERT mutation detection kit and application thereof in noninvasive tumor diagnosis
  • TERT mutation detection kit and application thereof in noninvasive tumor diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] In this embodiment, a ddPCR-based method for detecting the TERT promoter in tumor urine samples from bladder cancer patients is provided.

[0067] 1. ddPCR reaction conditions

[0068] I. Equipment: QX200 ddPCR system (Bio-Rad Laboratories).

[0069] II. ddPCR detection reagent

[0070] (a) Primers and probes

[0071] Primers:

[0072] 5'-CCTGCCCCTTCACCTTCCAG-3'(forward)

[0073] 5'-AGCGCTGCCTGAAACTCG-3'(reverse)

[0074] (PCR product length is 160bp).

[0075] Probe:

[0076] TERT mutation probe sequence: 5'- / 56-FAM / CCC+C+T+T+CCGG / 3IABk FQ / -3'

[0077] TERT wild-type probe sequence: 5'- / 5HEX / CCCC+C+T+CCGG / 3IABkFQ / -3'

[0078] (+: locked nucleic acid modification).

[0079] (b) Reaction system

[0080] The reaction system used in this example is shown in Table 1.

[0081] Table 1 ddPCR reaction system

[0082]

[0083] *The final concentration of primer is 900nmol / L, and the final concentration of probe is 250nmol / L.

[0084] (c) Reaction conditions

[...

Embodiment 2

[0094] In this embodiment, a ddPCR-based method for detecting the TERT promoter in the tumor and urine samples of patients with renal pelvis cancer is provided.

[0095] 1. ddPCR reaction conditions

[0096] III. Equipment: QX200 ddPCR system (Bio-Rad Laboratories).

[0097] IV. ddPCR Detection Reagent

[0098] (c) Primers and probes

[0099] Primers:

[0100] 5'-CCTGCCCCTTCACCTTCCAG-3'(forward)

[0101] 5'-AGCGCTGCCTGAAACTCG-3'(reverse)

[0102] (PCR product length is 160bp).

[0103] Probe:

[0104] TERT mutation probe sequence: 5'- / 56-FAM / CCC+C+T+T+CCGG / 3IABk FQ / -3'

[0105] TERT wild-type probe sequence: 5'- / 5HEX / CCCC+C+T+CCGG / 3IABkFQ / -3'

[0106] (+: locked nucleic acid modification).

[0107] (d) Reaction system

[0108] The reaction system used in this example is shown in Table 2.

[0109] Table 2 ddPCR reaction system

[0110]

[0111]

[0112] *The final concentration of primer is 900nmol / L, and the final concentration of probe is 250nmol / L.

[0113] ...

Embodiment 3

[0120] In this embodiment, a ddPCR-based method for detecting TERT promoter in tumor and urine samples of bladder cancer patients is provided.

[0121] 1. ddPCR reaction conditions

[0122] V. Equipment: QX200 ddPCR system (Bio-Rad Laboratories).

[0123] VI.ddPCR detection reagent

[0124] (e) Primers and probes

[0125] Primers:

[0126] 5'-CCTGCCCCTTCACCTTCCAG-3'(forward)

[0127] 5'-AGCGCTGCCTGAAACTCG-3'(reverse)

[0128] (PCR product length is 160bp).

[0129] Probe:

[0130] TERT mutation probe sequence: 5'- / 56-FAM / CCC+C+T+T+CCGG / 3IABk FQ / -3'

[0131] TERT wild-type probe sequence: 5'- / 5HEX / CCCC+C+T+CCGG / 3IABkFQ / -3'

[0132] (+: locked nucleic acid modification).

[0133] (f) Reaction system

[0134] The reaction system used in this example is shown in Table 3.

[0135] Table 3 ddPCR reaction system

[0136]

[0137] *The final concentration of primer is 900nmol / L, and the final concentration of probe is 250nmol / L.

[0138] (c) Reaction conditions

[013...

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Abstract

The invention particularly relates to a TERT mutation detection kit and an application of the TERT mutation detection kit in non-invasive tumor diagnosis. The invention provides a primer combination,a kit, a method and an application (ddPCR-TERTpm kit) for detecting TERT gene promoter mutation based on a ddPCR method, and aims to solve the problems of complex detection and low sensitivity in theprior art. The ddPCR-TERTpm kit is suitable for detection of tumor and urine mutation TERT promoters of bladder and renal pelvis cancer patients. After urine DNA is extracted, ddPCR amplification analysis is carried out, and the kit has the advantages of rapidness, simplicity, convenience, high sensitivity, specificity and the like. In addition, the detection kit or technology is also suitable fordetection of malignant melanoma, brain glioma, hepatocellular carcinoma and other tumors and blood specimens, and molecular diagnosis of thyroid tumors and DNA derived from fine needle puncture specimens.

Description

technical field [0001] The invention belongs to the technical field of TERT mutation detection, in particular to ddPCR for bladder cancer (bladder cancer, BC) and renal pelvic cancer (Renal pelvic cancer, RPC) tumor cells carrying telomerase promoter (TERTp) mutation (C228T or C250T) Detection method. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Bladder cancer (BC) is the most common malignant tumor of the urinary system in my country and the world, with an annual incidence of 350,000. Because BC is prone to relapse, patients need life-long monitoring and follow-up. Cystoscopy and urine exfoliation cytology are routine methods, but the former is expensive and invasiv...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 徐大为郑成云邢相玲袁晓天
Owner 郑成云
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