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Primer composition, kit and method for detecting TERT gene promoter mutation and application of primer composition

A primer composition, TERT-1R technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of complex detection and achieve high sensitivity, simple operation, and low cost.

Inactive Publication Date: 2019-03-22
BEIJING USCI MEDICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The main purpose of the present invention is to provide a primer composition, kit, method and application for detecting TERT gene promoter mutation, so as to solve the problem of complex detection in the prior art

Method used

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  • Primer composition, kit and method for detecting TERT gene promoter mutation and application of primer composition
  • Primer composition, kit and method for detecting TERT gene promoter mutation and application of primer composition
  • Primer composition, kit and method for detecting TERT gene promoter mutation and application of primer composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Detection of TERT gene promoter mutation in two thyroid biopsy samples

[0038] 1. DNA extraction

[0039] Two needles were taken from the puncture sample and extracted with Qiagen FFPE DNA kit.

[0040] 2. The first round of PCR:

[0041] The PCR reaction system shown in Table 1 was prepared on ice.

[0042] Table 1:

[0043]

[0044] Reaction conditions: 98°C for 5min; 95°C for 30s, 68°C for 30s, 72°C for 1min, 30 cycles;

[0045] 72°C, 5min; 12°C hold.

[0046] Two samples were used for the first round of PCR with two primers of different barcodes, and the first round of PCR products were detected by electrophoresis. The test results are shown in figure 1 .

[0047] 3. Purification of PCR products

[0048] Make up the volume to 50ul, add 0.5X AMP magnetic beads (incubated to room temperature in advance) (25ul), mix well, stand still for 5min, put on the magnetic stand for 2min, take the supernatant to a new tube, add 0.5X magnetic beads (still 25ul ), mix ...

Embodiment 2

[0061] Embodiment 2: sensitivity analysis

[0062] Sample DNA (mutation frequency 42%) positive for C228T was taken and diluted with human genome DNA negative for C228T so that the mutation frequencies were 5%, 2.5%, 1%, and 0.5%, respectively. Detection is carried out according to the detection method in Example 1. The test results are shown in Table 4.

[0063] Table 4:

[0064] mixed sample

[0065] The results in Table 4 show that the sensitivity of the primer composition of the present invention using the high-throughput sequencing method can detect C228T.5% mutation samples.

Embodiment 3

[0066] Example 3: TERT promoter mutation detected by different types of DNA

[0067] Select puncture samples and FFPE samples to extract DNA with poor quality (low extraction concentration, serious degradation), and perform detection according to the method in Example 1, wherein the initial amount of DNA is adjusted to a range of 3-15 ng.

[0068] The first round of PCR products was detected by electrophoresis, and the detection results are shown in Table 5 and image 3 ,From image 3 It can be seen that 3~13ngDNA can also achieve the amplification of the target fragment, and all samples can be normally constructed and sequenced.

[0069] table 5:

[0070]

[0071]

[0072] In the above examples, only puncture samples and FFPE samples were selected for research, but the detection of TERT gene promoter mutations in fresh surgical tissues, blood and body fluids with low mutation content by applying the technical solution of the present invention also falls within the sco...

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Abstract

The invention provides a primer composition, kit and method for detecting TERT gene promoter mutation and application of the primer composition. The primer composition comprises three pairs of TERT primers of SEQ ID NO: 1 to SEQ ID NO: 6; and SEQ ID NOs: 4 to 6 respectively have different barcode sequences. The primer composition furthermore comprises a general primer and three reverse primers with different indexes. By designing forward and reverse primer sequences covering the CTBT and C250T mutation sites of a TERT gene promoter, an amplification product can include the mutation sites C228Tand C250T and all other potential mutation sites. By performing a second round of PCR amplification on the amplification product of the above primers, a linker sequence is complemented, and the abovemutation is directly detected by utilizing a high-throughput sequencing method after purification, so that the advantages of being simple to operate, high in sensitivity and low in cost are provided.

Description

technical field [0001] The invention relates to the field of auxiliary diagnostic primers, in particular to a primer composition, kit, method and application for detecting mutations in the promoter of the TERT gene. Background technique [0002] Telomere is a special structure composed of repeated DNA sequences and related proteins at the end of chromosomes, which has the function of stabilizing the structure and integrity of chromosomes. Telomerase is a nucleoprotein reverse transcriptase, which consists of telomerase reverse transcriptase (TERT), telomerase RNA component (TR) and telomerase-associated protein (TEP1, etc.). Telomerase uses its own RNA as a template to synthesize telomere DNA, add telomere DNA to the end of chromosomes, and provide a genetic basis for cells to maintain division. Telomerase is inactive or inactive in normal somatic cells, but active in germ cells, stem cells and cancer cells. Telomerase reverse transcription (TERT), the catalytic part of te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
Inventor 万华方楠刘运超王建伟伍启熹刘倩刘珂弟唐宇
Owner BEIJING USCI MEDICAL LAB CO LTD
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