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Kit for detecting free colorectal cancer cell markers in blood

A technology of colorectal cancer cells and markers, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of non-specificity of tumor cells, high detection cost, and inaccurate detection methods

Active Publication Date: 2014-04-16
牛刚 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, the chemical and physical means adopted have no therapeutic specificity for tumor cells
But this detection method is very inaccurate
We know that the increase in the content of tumor markers may be due to the secretion of tumor cells, or other factors such as stress responses may cause the increase in the protein content of the marker, so even if the increase in the content of tumor markers is detected, it cannot be accurately judged whether the tumor markers are in the blood. Tumor cells or cancer stem cells really exist
Faced with these problems, the usual practice of those skilled in the art is to combine multiple tumor markers to increase the accuracy of detection, but the cost of detection is very high, and it is difficult for ordinary patients to accept
In addition, even if combined with detection, it cannot be concluded that the detection must be accurate

Method used

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  • Kit for detecting free colorectal cancer cell markers in blood
  • Kit for detecting free colorectal cancer cell markers in blood
  • Kit for detecting free colorectal cancer cell markers in blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of Magnetic Beads

[0031]Preparation of antibody immunomagnetic beads: the antibodies used were anti-BerEP4 monoclonal antibody, anti-cytokeratin monoclonal antibody and anti-CK20 monoclonal antibody. This kit uses the above three antibodies to be labeled separately and then mixed for use.

[0032] Methods: Dynabeads Antibody Coupling Kit produced by Invitrogen was used. The labeling method was carried out in strict accordance with the manufacturer's instructions.

Embodiment 2

[0033] Example 2 Separation of colorectal cancer cells

[0034] 1. Sample collection:

[0035] Collect 5-7.5 ml of peripheral blood from patients with colorectal cancer, anticoagulate with EDTA, and use it within 4 hours (or within 48 hours after storage at 4 degrees).

[0036] 2. Select magnetic beads:

[0037] 2.1 Handling of magnetic beads:

[0038] · Mix the magnetic beads evenly, use a pipette to blow gently, and do not use a vortexer;

[0039] Use PBS buffer solution to wash, and do not touch the microspheres when washing;

[0040] · Pipette more magnetic beads than the required sample volume into a 1.5 ml centrifuge tube;

[0041] Put on the magnet for 1 minute, discard the supernatant;

[0042] · Wash 3 times with 1ml PBS to remove preservatives;

[0043] · Remove the supernatant;

[0044] · Aspirate 100ul microspheres containing PBS with the same volume as the original one.

[0045] 2.2 Selection of tumor cells

[0046] Put 5ml of peripheral blood into a 15...

Embodiment 3

[0065] Example 3 Detection of colorectal cancer cell tumor markers

[0066] 3.1 Kit preparation

[0067] · Keep the test tube at room temperature;

[0068] Put the RNase-free water in the kit at room temperature;

[0069] · Place the centrifuge tube above the tube containing the supernatant on ice;

[0070] · Prepare magnetic beads.

[0071] 3.2 Preparation of oligonucleotides

[0072] Take an appropriate amount of magnetic beads with oligonucleotide Oligo(dT) (it is recommended not to mix with a suspension apparatus, but to suspend by hand);

[0073] · Transfer to 1.5ml tube;

[0074] · Wash 2 times with Lysis / Binding Buffer.

[0075] 3.3 Binding mRNA to magnetic beads

[0076] Add 20 μl of magnetic beads to each cell lysis sample;

[0077] • Incubate for 10 minutes.

[0078] 3.4 Purification of mRNA

[0079] · Wash twice with buffer solution;

[0080] · Wash the magnetic beads twice with buffer;

[0081] · Wash again with 100 μl Tris-HCl buffer;

[0082] · Sus...

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Abstract

The invention discloses a kit for detecting free colorectal cancer markers in blood. The kit comprises the following components: a magnetic bead processing solution, magnetic beads, 4.0ml of a 10X buffering solution, 4.0ml of dNTPs (deoxyribonucleoside-5-triphosphate), 2.0ml of reverse transcriptase, 10.5mol of RNA enzyme inhibitor 40u / m, 25.0ml of 10X hot start PCR (Polymer China Reaction) mixed liquid, 13.0ml of PCR-dedicated ddH2O, and 4.0ml of primer. Compared with the traditional method, the kit disclosed by the invention has the capability of detecting colorectal cancer markers in peripheral blood more accurately and provides an important guiding significance for selection of colorectal cancer prognosis schemes and colorectal cancer treating schemes.

Description

technical field [0001] The present invention relates to a detection kit and composite detection technology, in particular to a high-accuracy detection technology and kit for detecting colorectal cancer cells (malignant tumor cells) from blood, especially capable of separating and extracting A detection kit for identifying free colorectal cancer cells remaining in the patient's blood after radiotherapy and chemotherapy treatment. Background technique [0002] As a malignant tumor, colorectal cancer has an increasing incidence rate year by year, and its harm to human beings is self-evident. The early detection and screening of colorectal cancer tumors and the analysis and prediction of prognosis and curative effect after chemotherapy have very important guiding significance for the treatment of colorectal cancer. [0003] Malignant tumor cells and cancer stem cells are a type of malignant tumor cells that can maintain and spread. Although many scientists believe that tumor c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/574
Inventor 牛刚谭焕然
Owner 牛刚
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