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Colorectal cancer detection primer, method and kit

A technology for detection kits and detection primers, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem of invasiveness, easy complications, limited application, and inability to adapt to screening of high-risk groups and diagnosis, to achieve the effect of convenient and flexible sample collection

Inactive Publication Date: 2016-06-01
SHENZHEN RES INST THE CHINESE UNIV OF HONG KONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Endoscopic examination is currently the best method for colorectal cancer screening, but its application in general screening is limited due to the invasiveness and complications of this method
Therefore, the current diagnosis of colon cancer prognosis is based on clinical, pathological and imaging information, and is still in the stage of empirical judgment, which is far from being suitable for the screening of high-risk groups with a risk of colorectal cancer recurrence. and diagnostic needs

Method used

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  • Colorectal cancer detection primer, method and kit
  • Colorectal cancer detection primer, method and kit
  • Colorectal cancer detection primer, method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Down-regulation of CA4 expression in colorectal cancer:

[0041] The total cellular RNA was extracted by Trizol method and reverse-transcribed into cDNA by Applied Biosystems cDNAReverseTranscription kit, and the operations were carried out according to the method recommended by the kit. Primers CA4-F1 and CA4-R1 for semi-quantitative PCR were designed according to the human CA4 nucleotide sequence; wherein,

[0042] Primer CA4-F1: 5'-CCGGCTCAGAGGACTCTT-3',

[0043] Primer CA4-R1: 5'-GTTGGAGGACTCGGCTTGAA-3';

[0044] The mRNA expression level of CA4 in 9 intestinal cancer cell lines: CaCO2, DLD-1, HCT116, HT29, LOVO, LS180, SW480, SW620 and SW1116 was detected by semi-quantitative PCR. The results are as follows: figure 1 as shown, figure 1 To detect the expression level of CA4 mRNA in colon cancer cell lines in Example 1. from figure 1 It can be seen from the results that the mRNA levels of CA4 in these 9 intestinal cancer cells were lower than those in normal int...

Embodiment 2

[0056] In vivo test of CA4 on tumor growth in nude mice:

[0057] The control cell lines HCT116 / vector or HCT116 / CA4 were randomly injected into the dorsal side of nude mice, and the tumor growth patterns were compared. Tumor volumes were measured every 3 days for approximately three weeks. Tumor volume (mm3) was calculated by measuring the longest and shortest tumor diameter lengths (volume formula = 0.5 x length x 2 x width). Eighteen days after inoculation, the experiment was terminated and the mice were sacrificed. Tumor growth curves in nude mice Figure 4 shown. The average tumor size of nude mice injected with HCT116 / CA4 was significantly lower than that of control nude mice injected with HCT116 / vector (P image 3 in Part B). like image 3 In part B, the histograms represent the tumor weights of the HCT116 / CA4 and HCT116 / vector groups respectively (P value less than 0.01, t test). Immunostaining with specific antibodies confirmed the expression of CA4 protein in tu...

Embodiment 3

[0059] Intestinal cancer cell CA4 expression level up-regulated after 5-aza-2'-deoxycytidine treatment to identify CA4 methylation as a potential colorectal cancer biomarker test: On the basis of Example 1, in order to determine the role of CA4 in intestinal cancer cells Whether the low expression in is caused by the methylation of the promoter region, we treated 9 kinds of intestinal cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine: CaCO2, DLD-1, HCT116, HT29 , LOVO, LS180, SW480, SW620 and SW1116; and then the expression of CA4mRNA in cells was detected. Specifically, 1×106 cells were seeded in a 100 mm culture dish and cultured for 24 hours. Experiment with the following groups:

[0060] The control group was the cells without drug addition in the same period;

[0061] The experimental group was cells treated with 2μM 5-Aza-dC for 96h,

[0062] The treated experimental group changed the medium every 24h.

[0063] The experimental results show...

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Abstract

The present invention provides a CA4 gene detection primer, the CA4 gene detection primer comprises a methylation amplification primer, wherein the methylation amplification primer comprises upstream primer CA4-MF having sequence table SEQ. ID. No. 1 base sequence and downstream primer CA4-MR having sequence table SEQ. ID. No. 2 base sequence; and / or, the CA4 gene detection primer comprises a BGS primer, wherein the BGS primer comprises upstream primer CA4-BF having sequence table SEQ. ID. No. 5 base sequence and downstream primer CA4-BR having sequence table SEQ. ID. No. 6 base sequence. The CA4 gene detection primer is based on CA4 gene as a biomarker, after extracting and amplifying of the CA4 gene, CA4 gene methylation status is detected, and then the CA4 gene detection primer can be further used for assisted detection of the possibility of colorectal cancer and recurrence; and compared with detection methods in the prior art, the detection method has the characteristics of being non-invasive, convenient and flexible in sample collection, and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a CA4 gene detection primer, a kit and a method for using the kit. Background technique [0002] Colon cancer is a common malignant tumor of the digestive system, and its morbidity and mortality both rank third in the cause of cancer death. Clinically, the main reason for the failure of surgical treatment of colon cancer is postoperative recurrence and metastasis, especially liver metastasis. Therefore, the analysis of the prognostic factors of colon cancer is helpful to identify high-risk individuals who may have postoperative recurrence, screen independent indicators affecting postoperative recurrence of colon cancer patients, and select more appropriate treatment options for clinical practice. [0003] Traditional postoperative screening methods include fecal occult blood test, fecal immune test, colonic barium enema, sigmoidoscopy and colonoscopy, etc. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 于君沈祖尧张静婉
Owner SHENZHEN RES INST THE CHINESE UNIV OF HONG KONG
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