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Exosome miRNA marker for colorectal cancer diagnosis and diagnosis kit

A diagnostic kit and colorectal cancer technology, applied in the field of molecular biology, can solve the problems of limited population and low sensitivity, and achieve the effect of strong specificity and increased amplification length

Active Publication Date: 2019-03-08
北京恩泽康泰生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Endoscopic examination will bring some pain to the subjects, and some specific groups cannot tolerate it (such as patients with acute peritonitis, intestinal perforation, etc.), so the acceptable and covered groups are limited
There are also blood-based non-invasive detection methods using blood septin9 methylation detection, but the detection sensitivity is not high; therefore, it is urgent to develop a high-sensitivity non-invasive early detection method for colorectal cancer

Method used

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  • Exosome miRNA marker for colorectal cancer diagnosis and diagnosis kit
  • Exosome miRNA marker for colorectal cancer diagnosis and diagnosis kit
  • Exosome miRNA marker for colorectal cancer diagnosis and diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Screening of exosomal miRNA markers associated with colorectal cancer based on high-throughput sequencing

[0073] In order to screen for exosome markers related to the diagnosis of colorectal cancer, 15 patients with early colorectal cancer and 15 controls each took blood of no less than 10ml and separated plasma, which was used to separate exosomes in plasma by classical ultracentrifugation The RNA was extracted using the qiagen miRNeasy mini kit, and the obtained RNA was sequenced using the NEB miRNA library construction kit. The obtained data were analyzed by bioinformatics, and the differentially expressed miRNAs in early colorectal cancer patients and controls were compared, and correlated with the miRNA sequencing of tissues in TCGA, and the exosomal miRNAs derived from tumors were screened, as shown in Table 1 shown. These miRNA-level markers from exosomes can be used for early diagnosis of colorectal cancer.

[0074] Table 1

[0075]

[0076] ...

Embodiment 2

[0077] Example 2 miRNA detection system based on fluorescent quantitative PCR platform

[0078] 1. miRNA reverse transcription reaction system

[0079]miRNA reverse transcription reagents and enzymes were purchased from TAKARA, standard products were synthesized by Shanghai Yingwei Jieji, and specific reverse transcription primers were synthesized by Suzhou Synbio. Use a 20ul reverse transcription system, as follows:

[0080]

[0081] 2. PCR reaction system

[0082] The PCR reaction mixture was purchased from TAKARA, the upstream primers, probes and general downstream primers were synthesized by Suzhou Synbio, and the fluorescent quantitative PCR instrument was ABI 7500. The PCR reaction system is as follows:

[0083]

[0084] Wherein, the Premix Ex Taq comes from the kit RR390A from Takara Company. The main components of the PremixEx Taq are TaKaRa Ex Taq HS, dNTP Mixture, Mg 2+ and Tli RNaseH (heat-resistant RNaseH), etc. The main components of the Primer Mix are...

Embodiment 3

[0108] Example 3 Effect evaluation of colorectal cancer early diagnosis detection kit based on miRNA two-step detection system

[0109] 1. Sample collection

[0110] Collect 10ml of blood from patients with early stage (stage I and stage II) colorectal cancer diagnosed by the hospital, patients with benign colorectal cancer, healthy people and other control samples, and separate into plasma.

[0111] 2. Exosomal RNA extraction

[0112] Qiagen’s commercial ExoEasy kit or the company’s self-developed Exocolumn were used to separate plasma exosomes, and the isolated exosomes were extracted with Qiagen miReasy mini kit to extract miRNA in exosomes, and Agilent 2100 was used to detect the RNA concentration and mass, and record the RNA concentration.

[0113] 3. miRNA two-step detection system

[0114] Using the two-step detection system based on the PCR platform miRNA in Example 1, the plasma exosomal miRNA of 50 cases of early colorectal cancer patients and 50 cases of control ...

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Abstract

The invention provides an exosome miRNA marker for colorectal cancer diagnosis. The marker is selected from at least one of let-7b-3p, let-7b-5p, miR-150-3p, miR-145-3-, miR-139-3p, miR-139-5p, miR-15b-3p, miR-125a-5p, miR-140-5p, miR-342-3p, miR-132-5p and miR-92b-5p. The invention further provides a kit for colorectal cancer diagnosis. The invention further establishes a method for detecting themiRNA marker. A colorectal cancer diagnosis and recurrence monitoring method is developed on the basis of non-invasive detection technology, the exosome miRNA marker has high sensitivity and high specificity in early-stage colorectal cancer, AUC is up to 0.99 as multiple miRNA markers are combined, and the exosome miRNA marker has extremely excellent diagnosis performance.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an exosome miRNA marker and a diagnostic kit for the diagnosis of colorectal cancer. Background technique [0002] Colorectal cancer (carcinoma of colon and rectum) is a common malignant tumor in the gastrointestinal tract. The early symptoms are not obvious. With the increase of the cancer, symptoms such as changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain In the late stage, systemic symptoms such as anemia and weight loss are manifested. Its morbidity and mortality are second only to gastric cancer, esophageal cancer and primary liver cancer among digestive system malignant tumors. The incidence and mortality of colorectal cancer (CRC) in my country are both on the rise. The 2015 cancer statistics in China show that the incidence and mortality of colorectal cancer in my country rank fifth among all malignant...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/156
Inventor 闵力孔关义朱圣韬刘翔赵立波陈苏红张澍田
Owner 北京恩泽康泰生物科技有限公司
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