Kit for left colonic carcinoma detection

A kit and technology for colon cancer, applied in biological testing, measuring devices, material inspection products, etc., to achieve the effects of low cost, easy preparation, and easy storage

Active Publication Date: 2017-03-22
汉氏联合(天津)干细胞研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the existing detection methods can meet this requirement. Therefore, it is particularly important to develop a means that can distinguish the three types of cancer.

Method used

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  • Kit for left colonic carcinoma detection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1, screening and identification of rectal cancer markers

[0014] 1. Sample collection

[0015] 5mL peripheral blood samples were collected from 15 patients with rectal cancer clinically identified, 13 patients with right colon cancer and 10 patients with left colon cancer, anticoagulated and stored at -80°C. The patients mentioned above were all from the Cancer Hospital of Tianjin Medical University, and signed informed consent with the patients, which also met the ethical requirements.

[0016] 2. RNA extraction

[0017] Total RNA was extracted from colorectal cancer tissues and normal control tissues according to the operation instructions of the Trizol Reagent kit, dissolved in DEPC-treated water, and the concentration and purity of total RNA were measured by Nanodrop-1000 nucleic acid and protein analyzer, and stored at -80°C. Through detection, the RNA concentration is generally between 100ug-250ug / mL, which can be used for subsequent library establishme...

Embodiment 2

[0025] Screening and preparation of embodiment 2 KLK6 protein nucleic acid aptamer

[0026] Design a random nucleic acid library comprising approximately 20 nucleotides at both ends and 36 nucleotides in the middle as follows:

[0027] 5'-TCAAGCATGAATTGCAGAATGAC(N36)AGCAAGTGAGTAGCCAGTAAGCA-3'; N36 represents 36 random nucleotides.

[0028] (1) Firstly, the original random oligonucleotide library is amplified to increase its copy number to facilitate screening. The original random oligonucleotide library was amplified by PCR with upstream primers and biotin-modified downstream primers, using a PCR kit purchased from Shanghai Sangong Company. The reaction system is as follows:

[0029] Upstream primer 100pmol (5'-TCAAGCATGAATTGCAGAATGAC-3')

[0030] Downstream primer 100pmol (5,-biotin-TGCTTACTGGCTACTCACTTGCT-3,)

[0031] Template DNA 10μg (5'-TCAAGCATGAATTGCAGAATGAC(N36)AGCAAGTGAGTAGCCAGTAAGCA-3')

[0032] Wherein, the downstream primer is a biotin-modified downstream prim...

Embodiment 3

[0038] Example 3 The performance measurement of protein binding suitable gametes

[0039] Based on the property of graphene oxide oxidation to adsorb single-stranded DNA, a method for verifying the affinity of oligonucleotide aptamers was constructed. A fixed concentration of the KLK6 protein target (1 μM) was incubated with a series of corresponding candidate oligonucleotide aptamers of different concentrations (10, 25, 50, 75, 100, 150, 200 uM) in a total volume of 300 uL, Incubate at 37°C in the dark for 2 hours, and use BB buffer instead of the target as a negative control group. After incubation and binding, GO with the optimal dosage ratio was added to adsorb the aptamer that was not bound to the target. After centrifugation, the fluorescence intensity emitted by the supernatant at 520nm under 490nm excitation was measured with an F-7000 fluorescence photometer. Use dark treatment. Taking the fluorescence intensity of the experimental group relative to the negative con...

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Abstract

The invention discloses a kit for left colonic carcinoma detection. The kit comprises an aptamer capable of detecting KLK6 protein and can be used for screening left colonic carcinoma. The kit has the advantages of being high in sensitivity, low in cost and suitable for large-scale screening.

Description

technical field [0001] The invention relates to a kit for detecting left colon cancer. Background technique [0002] Colorectal cancer is the third most common cancer in the world after lung and breast cancer. It is at a relatively high level in developed countries and regions. Since the 1970s, with the development of society and economy, changes in the living habits and diet structure of Chinese residents, and the acceleration of population aging, the incidence and mortality of colorectal cancer in my country have been increasing. Gradually rising trend, it has become one of the main malignant tumors that endanger the health of our residents, causing a heavy burden on the social economy and seriously affecting the healthy development of our country's social economy. [0003] Colorectal cancer is one of the digestive system tumors that can be well prevented and cured. The screening method is safe and effective, and the clinical treatment effect is better. Studies have shown...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/574C12N15/115
CPCC12N15/115C12N2310/16G01N33/57446G01N33/57488G01N33/68
Inventor 郎秀玲
Owner 汉氏联合(天津)干细胞研究院有限公司
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