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Primer pair, probe and kit used for noninvasive polygene methylation combination detection for early stage colorectal cancer and applications thereof

A colorectal cancer, combined detection technology, applied in the determination/examination of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of limited early diagnosis and low sensitivity of CRC, and achieve intuitive results and reduced The effect of contamination, detection process optimization

Active Publication Date: 2018-06-01
上海酷乐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The PRESEPT study ended in 2013 is so far the only prospective screening using SEPT9 to detect 7941 people. It was found that the sensitivity of SEPT9 detection for CRC screening was 48%, which was compared with the previous retrospective verification. The sensitivity of the test is low
[0008] These data suggest that SEPT9 testing alone has limited early diagnosis of CRC due to low sensitivity

Method used

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  • Primer pair, probe and kit used for noninvasive polygene methylation combination detection for early stage colorectal cancer and applications thereof
  • Primer pair, probe and kit used for noninvasive polygene methylation combination detection for early stage colorectal cancer and applications thereof
  • Primer pair, probe and kit used for noninvasive polygene methylation combination detection for early stage colorectal cancer and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] This example is a primer pair and probe for non-invasive multi-gene methylation joint detection of early colorectal cancer, which includes primer pairs and probes for detecting Spetin9, NDRG4, BMP3, THBD and SDC2 gene methylation respectively. The primer pair and probe of probe and internal reference ACTB; Wherein

[0063] The primer pair used to detect the methylation of the Spetin9 gene is the sequence shown in SEQ ID No: 1 and SEQ ID No: 2, and the probe is the sequence shown in SEQ ID No: 3;

[0064] The primer pair used to detect NDRG4 gene methylation is the sequence shown in SEQ ID No: 4 and SEQ ID No: 5, and the probe is the sequence shown in SEQ ID No: 6;

[0065] The pair of primers used to detect the methylation of the BMP3 gene is the sequence shown in SEQ ID No: 7 and SEQ ID No: 8, and the probe is the sequence shown in SEQ ID No: 9;

[0066] The primer pair used to detect THBD gene methylation is the sequence shown in SEQ ID No: 10 and SEQ ID No: 11, and ...

Embodiment 2

[0071] This example is a kit for the non-invasive multi-gene methylation combined detection of early colorectal cancer containing the primer pair and probe described in Example 1.

[0072] The above kits include free DNA extraction reagents, sulfite conversion reagents, real-time fluorescent quantitative PCR amplification reaction reagents, positive control substances, and negative control substances; wherein, free DNA extraction reagents and sulfite conversion reagents are all commercially available reagents box, the free DNA extraction reagent specifically includes lysate, protease mixed solution, magnetic beads, washing solution 1, washing solution 2 and eluent, and the real-time fluorescent quantitative PCR amplification reaction reagent includes PCR amplification buffer, such as The primers and probes described in SEQ ID No: 1-SEQID No: 18, UNG enzyme and DNA polymerase, the corresponding relationship between primers and probes is specifically shown in Table 1; the positiv...

Embodiment 3

[0074] This example is a method for detecting early colorectal cancer using the kit described in Example 2.

[0075] 1. Materials, reagents, instruments

[0076] Free DNA extraction kit, sulfite conversion kit, and fluorescent quantitative PCR instrument are ABI7500.

[0077] 2. Sample Preparation

[0078] The positive control uses bovine serum albumin and human genomic DNA, and then dilutes it with DEPC water, and the negative control sample is DEPC H 2 O; The sample to be processed is the patient's peripheral blood.

[0079] 3. Extraction of cell-free DNA and sulfite conversion

[0080] Extract free DNA from 15ml samples according to the instructions of the free DNA extraction kit, which includes DNA cleavage and binding, DNA washing and elution, and the DNA obtained by eluting with 60 μl eluent at the end is the DNA to be extracted; Sulfate conversion kit performs sulfite conversion of DNA samples, which includes sulfite conversion, binding step, first wash, desulfo, se...

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Abstract

The invention relates to a primer pair and a probe used for noninvasive polygene methylation combination detection for early stage colorectal cancer, and includes the primer pair and the probe used for detecting methylation of genes Spetin9, NDRG4, BMP3, THBD and SDC2 and the primer pair and the probe for internal reference ACTB; the sequences of the primer pair and the probe are represented as the SEQ ID No.1 to the SEQ ID No.18. The invention also provides a kit containing the primer pair and the probe and applications thereof. The application method includes free DNA extraction from a plasma specimen, sulfite conversion, PCR amplification reaction, fluorescent signal detection and result determination. The kit and the method are suitable for methylation detection of the five genes Spetin9, THBD, SDC2, NDRG4 and BMP3 in human peripheral blood; compared with a conventional colorectal cancer diagnosis method, the application method fully utilizes the free DNA extraction from a plasma specimen, the DNA methylation and QPCR associated technologies, thus developing the kit having high sensitivity and specificity. The primer pair, probe and kit are used for performing early stage noninvasive screening to human colorectal cancer.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a primer pair, a probe, a kit and an application thereof for non-invasive multi-gene methylation combined detection of early colorectal cancer. Background technique [0002] Colorectal cancer is one of the most common malignant tumors in the human digestive system. According to statistics, the incidence of colorectal cancer ranks third among malignant tumors in the world. With the change of people's living habits, the incidence and mortality of colorectal cancer significantly increased, posing a serious threat to human health. The etiology of colorectal cancer is still unclear, and it is generally considered to be a multi-factorial and multi-step process. Therefore, an in-depth study of the molecular mechanism of the development of colorectal cancer is of great significance for revealing the pathogenesis of colorectal cancer, disease prevention, clear diagno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/166
Inventor 徐赛涛李杨刘荣兵
Owner 上海酷乐生物科技有限公司
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