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Molecular markers of colorectal cancer in blood and its detection kit and method

A molecular marker, colorectal cancer technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as influence detection, poor specificity, and large influence of diet

Active Publication Date: 2021-11-30
ANCHORDX MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But it has a series of disadvantages: First, its specificity is poor and it is greatly affected by diet
Although colonoscopy is currently the gold standard for colorectal cancer, it also has its own disadvantages: First, it requires pre-examination preparations. For colonoscopy, you need to start eating a liquid or semi-liquid diet 3 days before the examination. On an empty stomach, start taking mannitol and other cathartics to clean the intestines the day before the examination. The amount of liquid taken is usually up to 2L. Clean the enema to ensure the cleanliness of the intestines, and no food can be eaten afterwards
However, these current methods also have certain problems: For stool samples, there are many kinds of enzymes in the stool, which can digest and degrade the DNA in the stool. In addition, the components in the stool are complex and there are too many interfering components. The processing of the sample puts forward relatively high requirements, and the improper processing of the sample will easily affect the downstream detection. The most important disadvantage is that since the feces contain a large amount of microbial DNA, which accounts for an overwhelming proportion, the detection of human DNA The sensitivity and specificity of the method put forward a very high requirement, and corresponding processing will also increase the cost of the detection method and the complexity of the operation
[0006] However, for early tumor samples, the content of cfDNA is lower, which is a great challenge to develop a detection method that meets the throughput of multiple markers and requires detection methods with sufficient sensitivity. There are certain technical challenges in terms of optimization and stability

Method used

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  • Molecular markers of colorectal cancer in blood and its detection kit and method
  • Molecular markers of colorectal cancer in blood and its detection kit and method
  • Molecular markers of colorectal cancer in blood and its detection kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] A method for detecting molecular markers of colorectal cancer in blood, comprising the steps of:

[0066] 1. Whole Blood Processing

[0067] 1.1 Use EDTAK2 anticoagulant vacuum blood collection tube (BD, Cat#367525) to collect 10mL whole blood, mix well,

[0068] Avoid hemolysis and process whole blood for plasmapheresis within 4-6 hours.

[0069] 1.2 Whole blood was centrifuged in a low-speed centrifuge at 4°C, 1600g, for 15 minutes, and the upper layer of plasma was carefully absorbed to avoid absorbing the buffy coat in the middle. The obtained plasma was centrifuged again in a high-speed centrifuge at 4°C, 16000g, for 10 minutes to obtain Sample plasma.

[0070] 2. Extraction of cfDNA from plasma

[0071] Specific method: The specific operation steps of plasma DNA extraction follow the MagMAX of Thermo Fisher Company TM Cell-Free DNA Isolation Kit instructions.

[0072] 3. Sulphite conversion of extracted cfDNA

[0073] The extracted DNA is subjected to bisul...

Embodiment 2

[0098] Using the experimental method of Example 1, the plasma samples of 46 normal people without colorectal cancer and 175 patients with colorectal cancer were tested. The specific detection kits, experimental methods and data judgment and processing were consistent with those in Example 1. Combinations of primers and probes are also preferably used in Example 1. 10 markers (TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, ZNF132) were selected for modeling and analysis using the logistic regression model, and 100 repetitions were performed according to the division of 6:4. At 90% specificity, the detection sensitivity of the test set was 78 ± 12% for Phase 1, 77 ± 10% for Phase 2, 82 ± 11% for Phase 3, and 88% for Phase 4 ±5%, for all colorectal cancer samples, the overall sensitivity of detection was 85±5%. The overall AUC was 0.91 ± 0.03. It shows that these markers are highly correlated with colorectal cancer, and through this method, colorectal ca...

Embodiment 3

[0100] The detection kit and experimental method of Example 1 were used to detect the plasma samples of 46 normal people without colorectal cancer and 175 patients with colorectal cancer. The specific experiments and data judgment and processing were consistent with those in Example 1. The primers The combination with the probe is also preferably used in Example 1. Select 4 markers (TWIST1, C9orf50, KCNJ12, ZNF132) to use the logistic regression model for modeling analysis, according to the 6:4 segmentation, 100 repetitions, under 90% specificity, the detection of the first phase of the test set The sensitivity was 78 ± 12%, the detection sensitivity of phase 2 was 76 ± 10%, the detection sensitivity of phase 3 was 81 ± 11%, and the detection sensitivity of phase 4 was 88 ± 5%. For all colorectal cancer samples, the detected The overall sensitivity was 82±5%. The overall AUC was 0.91 ± 0.03. It shows that these markers are highly correlated with colorectal cancer, and throug...

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Abstract

The present invention relates to a molecular marker, a kit and a detection method that can be used to detect colorectal cancer. Three pairs were designed for each marker ST8SIA4, ZNF132, TAF3, Twist1, VAV3‑AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, THBD, ZNF304, EVC, S1PR1, ZFHX4‑AS1, EMID1, FAM19A4, NEUROD1 Primers and three probes. The detection method includes multiplex PCR and then qPCR quantitative detection. The sensitivity and specificity of the kit of the present invention and the detection method meet the clinical needs. For the detection scheme of these multiple markers, the diagnosis of colorectal cancer in the first stage achieves a specificity of 90%. , while having a sensitivity of 83%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker of colorectal cancer in blood, a detection kit and a method thereof. Background technique [0002] There are several commonly used techniques for colorectal cancer screening. One is a fecal occult blood test. Usually, the occurrence and evolution of bowel cancer can occur without any symptoms in the early stage. Cancer cells can grow on the wall of the large intestine for decades and then metastasize to other parts. discharge. The fecal occult blood test is to detect the blood components in the stool (the detection object is hemoglobin). If multiple and continuous positive reactions indicate gastrointestinal bleeding, further examination should be done to guard against the occurrence of intestinal tumors. The main advantage of the fecal occult blood test is that the results are displayed quickly and clearly, and the results can be semi-quantitative. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/154C12Q2531/113C12Q2537/143C12Q2523/125C12Q2563/107
Inventor 许林浩王洪陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD
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