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Primer, probe, kit and gene detection method for colorectal cancer diagnosis

A colorectal cancer, primer-probe technology, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as insufficient detection rate, inability to quantitatively compare positive results, and achieve accurate Effects of improved detection and diagnosis, sensitivity and specificity

Inactive Publication Date: 2018-09-28
深圳凌鼎基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Septin9 methylation is currently the most relevant gene marker for CRC. It has been proved in a number of clinical studies that the sensitivity is 74% and the specificity is 97%. Quantitative comparison of positive results

Method used

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  • Primer, probe, kit and gene detection method for colorectal cancer diagnosis
  • Primer, probe, kit and gene detection method for colorectal cancer diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] A primer and probe for the diagnosis of colorectal cancer, comprising:

[0050] Primer-probe combination 1 for detecting septin9 gene

[0051] Primer: SEQ ID NO: 3

[0052] Probe: SEQ ID NO: 4

[0053] Primer: SEQ ID NO: 5;

[0054] Primer-probe combination 2 for detection of SFRP2 gene

[0055] Primer: SEQ ID NO: 6

[0056] Probe: SEQ ID NO: 7

[0057] Primer: SEQ ID NO:8.

[0058] The primers and probes provided in this embodiment are used to detect the degree of methylation in at least one target region of each gene in septin9 and SFRP2; the target regions are selected from the continuous at least A fragment with a length of 18 bases, the primers and probes are identical, complementary or hybridized to the target region. Preferably, the primers and probes hybridize to the target region under moderately stringent or stringent conditions.

[0059] The primers and probes provided in this example have the following characteristics:

[0060] (1) the artificially ...

Embodiment 2

[0064] A kit for diagnosing colorectal cancer, including primers and probes, PCR Mix, bisulfite and denaturant;

[0065] PCR Mix includes Taq polymerase, DNTP and PCR Buffer;

[0066] The denaturant is diethylene glycol dimethyl ether;

[0067] Primers and probes include a primer-probe combination 1 for detecting the septin9 gene, a primer-probe combination 2 for detecting the SFRP2 gene, and a primer-probe combination 3 for detecting the internal control gene ACTB, wherein:

[0068] Primer Probe Set 1

[0069] Primer: SEQ ID NO: 3

[0070] Probe: SEQ ID NO: 4

[0071] Primer: SEQ ID NO: 5;

[0072] Primer Probe Set 2

[0073] Primer: SEQ ID NO: 6

[0074] Probe: SEQ ID NO: 7

[0075] Primer: SEQ ID NO: 8;

[0076] Primer Probe Set 3

[0077] Primer: SEQ ID NO: 9

[0078] Probe: SEQ ID NO: 10

[0079] Primer: SEQ ID NO:11.

[0080] Evaluation and analysis of the kit provided in this example for the diagnosis of colorectal cancer:

[0081] (1) Prepare samples from ...

Embodiment 3

[0090] A genetic testing method for the diagnosis of colorectal cancer, comprising:

[0091] S1. Provide samples to be tested;

[0092] S2. Extract genomic DNA from the sample to be tested, and perform pretreatment to convert unmethylated cytosine at the 5' end of genomic DNA into uracil, thymine or another base different from cytosine in hybridization ;

[0093] S3. Using the genomic DNA processed in step S2 as a template, use the primers and probes in the kit of Example 2 to perform PCR amplification to obtain the Ct values ​​of septin9, SFRP2 and ACTB of the sample to be tested;

[0094] S4. Comparing the measured Ct values ​​of septin9 and SFRP2 with the critical Ct value for judging whether to suffer from colorectal cancer, and obtaining the diagnosis result of colorectal cancer in the sample to be tested.

[0095] In this embodiment, the pretreatment described in step S2 is realized by combining bisulfite with a scavenger, and the scavenger is trihydroxybenzoic acid. ...

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Abstract

The invention provides a primer, a probe, a kit and a gene detection method for colorectal cancer diagnosis. The primer and the probe are used for detecting the methylation level of each gene in septin 9 and SFRP 2 in at least one target region; the target regions are respectively selected from a fragment with the length of consecutive at least 18 bases of SEQ ID NO: 1 and SEQ ID NO: 2; and the primer and the probe are equal to, complementary to or hybridized to each other in the target regions. By adopting the primer, the probe, the kit and the gene detection method, two target genes including the septin 9 and the SFRP 2 are combined together for colorectal cancer diagnosis, and the sensitivity and the specificity to a colorectal cancer are remarkably improved through combined use of nucleotide sequences used for detecting the methylation levels of gene fragments of the septin 9 and the SFRP 2 respectively; and in addition, through the primer, the probe, the kit and the gene detectionmethod, two-channel detection to two gene markers of the septin 9 and the SFRP 2 at the same time can be easily realized, the methylation frequency of a sample can be judged according to a fluorescence numerical value of fluorescent quantitative PCR, and the colorectal cancer can be non-invasively, rapidly and accurately detected and diagnosed.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a primer, a probe, a kit and a gene detection method for diagnosing colorectal cancer. Background technique [0002] Tumor is one of the most difficult diseases in medicine. According to the "2015 China Cancer Statistics", there were 4.29 million new cancer cases and 2.81 million deaths in my country in 2015. [0003] Colorectal cancer (CRC) is a common malignant tumor of the digestive system. Worldwide, the morbidity and mortality of CRC are on the rise, ranking third among malignant tumors. In western developed countries, the mortality rate of CRC is as high as 33%, and it is one of the most common causes of death from malignant tumors. The lifetime risk of CRC in the American population is as high as 5%. Due to changes in living habits, the incidence of colorectal cancer in our country has been increasing year by year, and it has ranked the top 3-4 in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/154C12Q2563/107C12Q2545/114
Inventor 何轩陈文魏斌
Owner 深圳凌鼎基因科技有限公司
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